Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Attarian, Rodgoun
Doctor of Philosophy - PhD
University of British Columbia
Cryptococcus neoformans is a fungal pathogen and the causative agent of meningoencephalitis in immunocompromised and AIDS patients. Iron acquisition and the maintenance of iron homeostasis in C. neoformans are important aspects of virulence. Here, the monothiol glutaredoxin Grx4 was identified as a binding partner of Cir1, the master regulator of iron-responsive genes and virulence factor expression in C. neoformans. We confirmed that Grx4 binds Cir1 in vitro and in a yeast two-hybrid assay. RNA-Seq performed on the grx4 and cir1 mutants, and the WT strain, under low or high iron conditions identified genes involved in iron metabolism and expanded the concept that Grx4 plays a central role in iron homeostasis. A grx4 mutant, with deletion of the glutaredoxin domain, displayed iron-related phenotypes similar to those of a cir1 mutant, including elevated activity for cell surface reductases, sensitivity to high iron levels and increased susceptibility to phleomycin. Importantly it was shown that a grx4 mutant was avirulent in a mouse model of infection. We also observed that after 48 hour of starvation grx4 mutant had a growth defect in an iron-supplemented media. Interestingly, the growth defect of the grx4 mutant was rescued by exogenous GSH. We therefore further investigated the role of GSH in C. neoformans. Deletion of the GSH2 gene encoding the second enzyme in GSH biosynthesis resulted in a significant decrease in intracellular GSH levels in gsh2 mutants compared to the WT. This also resulted in loss of elaboration of major virulence factors including production of capsule and melanin, growth at host body temperature, and enhanced susceptibility to antifungal drugs. In conclusion GSH is required for expression of major virulence factors and defense against iron starvation-induced stress. GSH also influenced cell wall integrity. These results indicate that GSH plays a major role in the virulence of C. neoformans, and suggest a crosstalk between Grx4 and GSH as components of iron homeostasis and virulence expression. Overall, our findings provide further understanding of the regulation of virulence in C. neoformans and suggest novel drug targets for anticryptococcal therapy.
Analysis Â of Â the Â roles Â of Â a Â monothiol Â glutaredoxin Â and Â glutathione Â synthetase Â in Â the Â virulence Â of Â the Â AIDS-­âassociated Â pathogen Â Cryptococcus Â neoformans Â Â Â by Â Â Â Â Rodgoun Â Attarian Â Â Â Â M.Sc., Â The Â University Â of Â British Â Columbia, Â 2009 Â Â Â Â A Â THESIS Â SUBMITTED Â IN Â PARTIAL Â FULFILLMENT Â OF Â Â THE Â REQUIREMENTS Â FOR Â THE Â DEGREE Â OF Â Â Â Â DOCTOR Â OF Â PHILOSOPHY Â Â in Â Â THE Â FACULTY Â OF Â GRADUATE Â AND Â POSTDOCTORAL Â STUDIES Â Â (Microbiology Â and Â Immunology) Â Â Â Â THE Â UNIVERSITY Â OF Â BRITISH Â COLUMBIA Â Â (Vancouver) Â Â Â Â March Â 2016 Â Â Â Â © Â Rodgoun Â Attarian, Â 2016 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â ii Â Abstract Â Â Cryptococcus Â neoformans Â is Â a Â fungal Â pathogen Â and Â the Â causative Â agent Â of Â meningoencephalitis Â in Â immunocompromised Â and Â AIDS Â patients. Â Iron Â acquisition Â and Â the Â maintenance Â of Â iron Â homeostasis Â in Â C. Â neoformans Â are Â important Â aspects Â of Â virulence. Â Here, Â the Â monothiol Â glutaredoxin Â Grx4 Â was Â identified Â as Â a Â binding Â partner Â of Â Cir1, Â the Â master Â regulator Â of Â iron-­âresponsive Â genes Â and Â virulence Â factor Â expression Â in Â C. Â neoformans. Â Â We Â confirmed Â that Â Grx4 Â binds Â Cir1 Â in Â vitro Â and Â in Â a Â yeast Â two-­âhybrid Â assay. Â RNA-­âSeq Â performed Â on Â the Â grx4 Â and Â cir1 Â mutants, Â and Â the Â WT Â strain, Â under Â low Â or Â high Â iron Â conditions Â identified Â genes Â involved Â in Â iron Â metabolism Â and Â expanded Â the Â concept Â that Â Grx4 Â plays Â a Â central Â role Â in Â iron Â homeostasis. Â A Â grx4 Â mutant, Â with Â deletion Â of Â the Â glutaredoxin Â domain, Â displayed Â iron-­ârelated Â phenotypes Â similar Â to Â those Â of Â a Â cir1 Â mutant, Â including Â elevated Â activity Â for Â cell Â surface Â reductases, Â sensitivity Â to Â high Â iron Â levels Â and Â increased Â susceptibility Â to Â phleomycin. Â Importantly Â it Â was Â shown Â that Â a Â grx4 Â mutant Â was Â avirulent Â in Â a Â mouse Â model Â of Â infection. Â We Â also Â observed Â that Â after Â 48 Â hour Â of Â starvation Â grx4 Â mutant Â had Â a Â growth Â defect Â in Â an Â iron-­âsupplemented Â media. Â Interestingly, Â the Â growth Â defect Â of Â the Â grx4 Â mutant Â was Â rescued Â by Â exogenous Â GSH. Â We Â therefore Â further Â investigated Â the Â role Â of Â GSH Â in Â C. Â neoformans. Â Deletion Â of Â the Â GSH2 Â gene Â encoding Â the Â second Â enzyme Â in Â GSH Â biosynthesis Â resulted Â in Â a Â significant Â decrease Â in Â intracellular Â GSH Â levels Â in Â gsh2 Â mutants Â compared Â to Â the Â WT. Â This Â also Â resulted Â in Â loss Â of Â elaboration Â of Â major Â virulence Â factors Â including Â production Â of Â capsule Â and Â melanin, Â growth Â at Â host Â body Â temperature, Â and Â enhanced Â susceptibility Â to Â antifungal Â drugs. Â In Â conclusion Â GSH Â is Â required Â for Â expression Â of Â major Â virulence Â factors Â and Â defense Â against Â iron Â starvation-­âinduced Â stress. Â GSH Â also Â influenced Â cell Â wall Â integrity. Â These Â results Â indicate Â that Â GSH Â Â Â Â Â Â Â Â Â Â Â Â Â Â iii Â plays Â a Â major Â role Â in Â the Â virulence Â of Â C. Â neoformans, Â and Â suggest Â a Â crosstalk Â between Â Grx4 Â and Â GSH Â as Â components Â of Â iron Â homeostasis Â and Â virulence Â expression. Â Overall, Â our Â findings Â provide Â further Â understanding Â of Â the Â regulation Â of Â virulence Â in Â C. Â neoformans Â and Â suggest Â novel Â drug Â targets Â for Â anticryptococcal Â therapy. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â iv Â Preface Â Relative Â contributions Â of Â all Â collaborators Â Â The Â experimental Â design Â of Â the Â research Â outlined Â in Â this Â thesis Â was Â achieved Â through Â collaboration Â between Â Rodgoun Â Attarian Â and Â Dr. Â James Â W. Â Kronstad. Â R. Â Attarian Â conducted Â the Â experiments, Â and Â the Â people Â who Â have Â contributed Â to Â this Â work Â are Â identified Â below Â and Â acknowledged Â throughout Â the Â thesis. Â Â Â The Â work Â presented Â in Â Chapter Â 2 Â is Â in Â preparation Â for Â publication Â as Â a Â manuscript Â entitled Â âMonothiol Â Glutaredoxin-­â4 Â is Â required Â for Â iron Â homeostasis Â and Â virulence Â in Â the Â AIDS-­âassociated Â fungal Â pathogen Â Cryptococcus Â neoformansâ. Â The Â details Â of Â contributions Â to Â this Â work Â are Â as Â follows: Â Dr. Â Horacio Â Bach Â contributed Â to Â this Â study Â by Â identifying Â Grx4 Â by Â mass-­âspectrometry. Â Dr. Â Yossef Â Av-­âGay Â provided Â access Â to Â ALPHAScreen Â technology. Â Dr. Â Patricia Â Missall Â and Â Dr. Â Jennifer Â Lodge Â provided Â the Â grx4 Â mutant. Â Dr. Â Melissa Â Caza Â performed Â the Â virulence Â assays Â and Â determined Â the Â fungal Â loads Â in Â mice Â tissues. Â Dr. Â Daniel Â Croll Â performed Â gene Â ontology Â enrichment Â analyses Â on Â RNA-­âseq Â data. Â Dr. Â Guanggan Â Hu Â generated Â the Â CIR1-­â Â pDESTâ¢32 Â and Â GRX4-­âpDESTâ¢22 Â strains Â in Â E.coli Â for Â Y2H Â analysis, Â and Â the Â constructs Â for Â deletion Â of Â the Â entire Â GRX4 Â locus Â in Â the Â diploid Â strains. Â Dr. Â Alex Â Idnurm Â provided Â the Â diploid Â strain Â AI187. Â Dr. Â Alina Â Chan Â helped Â with Â spore Â isolation Â and Â Dr. Â Nigel Â OâNeil Â assisted Â in Â interpretation Â of Â data Â from Â sporulation Â progeny Â of Â grx4 Â mutant Â in Â the Â diploid Â background. Â A Â Work Â Learn Â student, Â Celine Â Chan, Â performed Â PCR Â on Â the Â generated Â progeny. Â Â Â The Â work Â presented Â in Â Chapter Â 3 Â is Â in Â preparation Â for Â publication Â in Â a Â manuscript Â entitled Â âGlutathione Â synthetase Â GSH2 Â is Â indispensable Â for Â growth Â under Â low Â iron Â condition Â and Â virulence Â in Â Cryptococcus Â neoformansâ. Â Dr. Â James Â W. Â Kronstad Â suggested Â Â Â Â Â Â Â Â Â Â Â Â Â Â v Â the Â idea Â to Â characterize Â the Â role Â of Â GSH Â synthesis. Â The Â work Â in Â Chapter Â 3 Â was Â performed Â by Â R. Â Attarian, Â and Â Dr. Â Guanggan Â Hu Â generated Â constructs Â for Â deletion Â of Â GSH2. Â Â Â Publications Â arising Â from Â graduate Â work Â Â -­â Â Attarian Â R, Â Hu Â G, Â Caza Â M, Â Croll Â D, Â Missall Â T, Â Lodge Â J, Â Kronstad Â J. Â (In Â preparation). Â Monothiol Â glutaredoxin-­â4 Â is Â required Â for Â iron Â homeostasis Â and Â virulence Â in Â the Â AIDS-­âassociated Â pathogen Â Cryptococcus Â neoformans. Â -­â Â Attarian Â R, Â Hu Â G, Â Kronstad Â J. Â (In Â preparation). Â Glutathione Â synthetase Â Gsh2 Â is Â indispensible Â for Â growth Â under Â low Â iron Â condition Â and Â virulence Â in Â Cryptococcus Â neoformans. Â Â Â University Â of Â British Â Columbia Â Ethics Â Board Â approval Â Â The Â protocol Â for Â the Â virulence Â assays Â was Â approved Â by Â the Â University Â of Â British Â Columbiaâs Â Committee Â on Â Animal Â Care Â (protocol Â A13-­â0093). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â vi Â Table Â of Â Contents Â Abstractâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..ii Â Preface Â ...................................................................................................................................................iv Â Table Â of Â Contents................................................................................................................................vi Â List Â of Â Tables......................................................................................................................................xiv Â List Â of Â Figures Â ....................................................................................................................................xvi Â List Â of Â Abbreviations.......................................................................................................................xix Â Acknowledgementsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦xxv Â Dedicationâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦. Â xxvi Â Chapter Â 1: Â Introduction Â ....................................................................................................................1 Â Â Â Â Â 1.1 Â Â Â Â Â Cryptococcus Â neoformans Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.......â¦â¦â¦â¦.....â¦1 Â Â Â Â Â Â Â Â Â Â 1.1.1 Â Â Â Â Â Cryptococcal Â disease Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.........â¦......â¦â¦â¦â¦â¦.â¦..1 Â Â Â Â Â Â Â Â Â Â 1.1.2 Â Â Â Â Â Cryptococcal Â meningoencephalitis Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦...â¦â¦â¦â¦â¦2 Â Â Â Â Â Â Â Â Â Â 1.1.3 Â Â Â Â Â The Â burden Â of Â cryptococcal Â infection Â and Â HIVâ¦...â¦â¦...â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦.â¦â¦.2 Â Â Â Â Â Â Â Â Â Â 1.1.4 Â Â Â Â Â Treatment Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦...â¦â¦â¦3 Â Â Â Â Â 1.2 Â Â Â Â Â Â Virulence Â features Â of Â C. Â neoformansâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦......â¦â¦â¦â¦â¦â¦â¦â¦3 Â Â Â Â Â Â Â Â Â Â 1.2.1 Â Â Â Â Â Growth Â at Â 37°C Â â¦â¦â¦........â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦..â¦...â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦..3 Â Â Â Â Â Â Â Â Â Â 1.2.2 Â Â Â Â Â Polysaccharide Â capsule Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦.â¦.....â¦â¦â¦â¦â¦â¦â¦â¦4 Â Â Â Â Â Â Â Â Â Â 1.2.3 Â Â Â Â Â Melanin Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦.....â¦â¦â¦6 Â Â Â Â Â Â Â Â Â Â 1.2.4 Â Â Â Â Â Other Â virulence Â traits Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦..7 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.2.4.1 Â Â Â Â Â Urease Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦7 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.2.4.2 Â Â Â Â Phospholipase Â â¦â¦â¦....â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦......â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦8 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â vii Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.2.4.3 Â Â Â Â Â Oxidative Â defensesâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦...â¦â¦â¦â¦..â¦â¦â¦â¦â¦...8 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.2.4.4 Â Â Â Â Â Secreted Â vesiclesâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦.â¦â¦..â¦.â¦â¦â¦â¦â¦..9 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.2.4.5 Â Â Â Â Â Nutrient Â acquisitionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦..â¦â¦..â¦â¦â¦â¦.10 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.2.4.5.1 Â Â Â Â Ironâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦...â¦...â¦...â¦â¦â¦..â¦â¦â¦.â¦â¦â¦â¦â¦11 Â Â Â Â Â 1.3 Â Â Â Â Â Â Iron Â homeostasis Â in Â eukaryotic Â models: Â S. Â cerevisiae Â and Â S. Â pombeâ¦â¦â¦...â¦.â¦â¦â¦..13 Â Â Â Â Â Â Â Â Â Â 1.3.1 Â Â Â Â Â Iron Â homeostasis Â in Â S. Â cerevisiaeâ¦â¦â¦....â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..â¦â¦â¦13 Â Â Â Â Â Â Â Â Â Â 1.3.2 Â Â Â Â Â Iron Â homeostasis Â in Â S. Â pombe Â â¦â¦â¦.............................................â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦15 Â Â Â Â Â 1.4 Â Â Â Â Â Monothiol Â glutaredoxins Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦...â¦â¦â¦16 Â Â Â Â Â Â Â Â Â Â 1.4.1 Â Â Â Â Â Roles Â of Â monothiol Â Grxs Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦..â¦â¦â¦18 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.4.1.1 Â Â Â Â Â Role Â of Â monothiol Â Grx3/4 Â in Â regulation Â of Â Aft1 Â in Â yeastâ¦â¦â¦â¦.â¦â¦.â¦â¦...18 Â Â Â Â 1.4.1.2 Â Â Â Â Role Â of Â monothiol Â Grx4 Â in Â the Â regulation Â of Â Fep1 Â in Â fission Â yeast..â¦â¦â¦...19 Â Â Â Â 1.4.1.3 Â Â Â Â Â Role Â of Â monothiol Â Grx4 Â in Â regulation Â of Â Php4 Â in Â fission Â yeastâ¦â¦â¦â¦â¦â¦20 Â Â Â Â 1.4.1.4 Â Â Â Â Â Monothiol Â CGFS Â Grxs Â contain Â a Â labile Â FeâS Â clusterâ¦â¦.â¦â¦â¦â¦.....â¦..â¦.â¦..20 Â Â Â Â Â 1.5 Â Â Â Â Â Glutathioneâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦21 Â Â Â Â Â Â Â Â Â Â 1.5.1 Â Â Â Â Â Roles Â of Â glutathione Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦22 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 1.5.1.1 Â Â Â Â Â A Â vital Â function Â of Â glutathione Â in Â iron Â homeostasisâ¦â¦â¦...â¦â¦â¦..â¦â¦.â¦â¦23 Â Â Â Â Â Â Â Â Â 1.5.1.1.1 Â Â Â Â Role Â of Â glutathione Â in Â iron Â homeostasis Â in Â fungiâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦24 Â Â Â Â Â Â 1.6 Â Â Â Â Â Iron Â uptake Â in Â C. Â neoformans Â â¦â¦â¦...â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦......â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦25 Â Â Â Â Â Â Â Â Â Â 1.6.1 Â Â Â Â Â The Â high-­âaffinity Â iron Â uptake Â pathway Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦26 Â Â Â Â Â Â Â Â Â Â 1.6.2 Â Â Â Â Â Low-­âaffinity Â iron Â uptake Â pathwayâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦26 Â Â Â Â Â Â Â Â Â Â 1.6.3 Â Â Â Â Â Siderophore-­âbound Â iron Â transportâ¦â¦â¦...â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦27 Â Â Â Â Â Â Â Â Â Â 1.6.4 Â Â Â Â Â Heme Â uptake Â â¦â¦â¦...â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦28 Â Â Â Â Â Â Â Â Â Â 1.6.5 Â Â Â Â Â Cir1: Â Master Â regulator Â of Â iron Â â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦28 Â Â Â Â Â Â Â Â Â Â Â Â Â Â viii Â Â Â Â Â Â 1.7 Â Â Â Â Â Rationale Â and Â aims Â of Â studyâ¦â¦â¦...â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦30 Â Â Â Â Â Â Â Â Â Â Â 1.7.1 Â Â Â Â Â Hypotheses Â â¦â¦â¦...â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦30 Â Â Â Â Â Â Â Â Â Â Â 1.7.2 Â Â Â Â Â Research Â objectives Â â¦â¦â¦...â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦32 Â Chapter Â 2: Â Â Monothiol Â glutaredoxin-­â4 Â is Â required Â for Â iron Â homeostasis Â and Â virulence Â in Â the Â AIDS-­âassociated Â pathogen Â Cryptococcus Â neoformansâ¦â¦â¦................â¦â¦â¦â¦â¦.â¦34 Â Â Â Â Â Â 2.1 Â Â Â Â Â Introduction Â Â â¦â¦â¦...â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦34 Â Â Â Â Â Â 2.2 Â Â Â Â Materials Â and Â methods Â â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦37 Â Â Â Â Â Â Â Â Â Â Â 2.2.1 Â Â Â Â Â Yeast Â strains, Â plasmids Â and Â growth Â mediaâ¦â¦â¦â¦...â¦â¦..â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦37 Â Â Â 2.2.2 Â Â Â Â Â Construction Â of Â strains Â â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦39 Â Â 2.2.2.1 Â Â Deletion Â of Â the Â conserved Â Grx Â domain Â to Â create Â a Â grx4 Â mutant Â and Â Â Â Â Â Â Â generation Â of Â grx4Î::GRX4 Â complemented Â strain Â â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦41 Â Â 2.2.2.2 Â Â Genetic Â analysis Â of Â GRX4 Â in Â a Â haploid Â and Â diploid Â C. Â neoformans Â Â Â backgroundâ¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦42 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.3 Â Â Â Â In Â silico Â protein Â analysis Â â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦......â¦â¦â¦â¦â¦â¦â¦â¦44 Â Â Â 2.2.4 Â Â Â Â Gene Â cloning, Â protein Â expression Â and Â purificationâ¦â¦...â¦..â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦44 Â Â 2.2.5 Â Â Â Â Proteinâprotein Â interactionâ¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦...â¦â¦...â¦â¦â¦45 Â Â Â Â Â Â Â 2.2.5.1 Â Â Â Â Â Â AlphaScreenâ¢ Â binding Â assays Â â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦......â¦.â¦â¦â¦45 Â Â Â Â 2.2.5.2 Â Â Â Â Â Â Â Yeast Â two-­âhybrid Â assayâ¦â¦...â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦........â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦46 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.6 Â Â Â Â Iron-­ârelated Â phenotypic Â analysisâ¦â¦.....â¦â¦â¦â¦..â¦â¦..â¦â¦â¦â¦â¦..........â¦â¦â¦â¦â¦â¦47 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.7 Â Â Â Â Virulence Â factor Â assays Â â¦â¦...â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦..â¦â¦47 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.8 Â Â Â Â Analysis Â of Â growth Â in Â liquid Â media.â¦â¦.â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦...â¦48 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.9 Â Â Â Â Virulence Â assayâ¦â¦...â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦49 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.10 Â Â Â Â Â Â Â Ethics Â statementâ¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦...â¦â¦..â¦â¦â¦â¦.â¦50 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â ix Â Â Â Â Â Â Â Â Â Â Â Â 2.2.11 Â Â Â Â Â Â Statistical Â analysis Â â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.....â¦â¦â¦â¦â¦â¦..â¦â¦â¦50 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.12 Â Â Â Â Â Â RNA-­âSeq Â library Â preparation Â and Â bioinformatics Â â¦â¦...â¦â¦.â¦..â¦â¦â¦â¦.â¦...â¦50 Â Â Â Â Â Â Â Â Â Â Â Â 2.2.13 Â Â Â Â Â Â Quantitative Â Real Â -­âTime Â PCR Â â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦...â¦â¦..â¦â¦..â¦..â¦52 Â Â Â Â Â Â 2.3 Â Â Â Â Resultsâ¦â¦...â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦.â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦53 Â Â Â Â Â Â Â Â Â Â Â Â 2.3.1 Â Â Â Â The Â monothiol Â glutaredoxin Â Grx4 Â binds Â Cir1.â¦â¦â¦â¦â¦â¦â¦.â¦â¦..â¦â¦â¦â¦â¦...â¦...53 Â Â Â Â 2.3.1.1 Â Â Â Â Â Grx4 Â was Â identified Â as Â a Â potential Â Cir1-­âassociated Â proteinâ¦â¦â¦â¦..â¦â¦â¦53 Â Â Â Â 2.3.1.2 Â Â Production Â of Â recombinant Â Grx4 Â and Â Cir1 Â in Â E. Â coli Â for Â interaction Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â studiesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦....â¦â¦.....â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦54 Â Â Â Â 2.3.1.3 Â Â Grx4 Â binds Â Cir1 Â in Â an Â ALPHAScreen® Â protein-­âprotein Â interaction Â Â Â Â Â Â Â Â Â Â Â Â assayâ¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦57 Â Â Â Â 2.3.1.4 Â Â Â Â Â Â Grx4 Â binds Â Cir1 Â in Â a Â yeast Â two-­âhybrid Â assayâ¦â¦â¦..â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦61 Â Â Â Â Â Â Â Â Â Â Â 2.3.2 Â Â Â Â Â Â A Â defect Â in Â Grx4 Â impacts Â iron Â homeostasis Â and Â virulenceâ¦â¦â¦â¦â¦..â¦â¦â¦...â¦.63 Â Â Â 2.3.2.1 Â Â Â Â Â Â Â Identification Â of Â a Â conserved Â C-­âterminal Â glutaredoxin Â domain Â in Â Grx4 Â in Â Â Â C. Â neoformans Â â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦...â¦â¦â¦â¦.â¦â¦â¦â¦63 Â Â Â Â Â 2.3.2.2 Â Â Â Â Â Â Â A Â grx4 Â deletion Â mutant Â has Â iron-­ârelated Â phenotypesâ¦â¦â¦â¦...â¦.â¦â¦â¦â¦..65 Â Â 2.3.2.3 Â Â Â Â Â Â Â Grx4 Â is Â required Â for Â inhibition Â of Â iron Â uptake Â in Â the Â presence Â of Â iron.â¦..71 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 2.3.2.4 Â Â Â Â Â Â Â Grx4 Â influences Â the Â virulence Â of Â C. Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦.â¦â¦74 Â Â Â Â Â Â Â 2.3.2.4.1 Â Â Â Â Â The Â grx4 Â mutant Â displays Â virulence-­ârelated Â phenotypesâ¦â¦â¦â¦â¦....74 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 2.3.2.4.2 Â Â Deletion Â of Â grx4 Â abrogates Â virulence Â in Â a Â mouse Â model Â of Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â cryptococcosis Â â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦.â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦.76 Â Â 2.3.3 Â Â Â Â Â Â A Â pilot Â transcriptomic Â analysis Â using Â RNA-­âseq Â provided Â further Â insight Â Â into Â role Â of Â Grx4 Â and Â also Â revealed Â complexities Â in Â the Â transcript Â structure Â of Â Â GRX4â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦.79 Â Â Â Â Â Â Â Â Â Â Â Â Â x Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 2.3.3.1 Â Â Â Â Â Â Grx4 Â is Â required Â for Â broad Â transcriptional Â remodeling Â in Â response Â to Â iron Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â in Â C. Â neoformans.â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦..â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦..â¦â¦â¦.79 Â Â Â Â Â Â Â Â Â 2.3.3.1.1 Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â Â Â Â Â Â Â Â Â Â Â Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â grown Â under Â the Â iron-­âdeplete Â conditionâ¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦.â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦80 Â Â Â Â Â Â Â Â Â 2.3.3.1.2 Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â Â Â Â Â Â Â Â Â Â Â grown Â under Â the Â iron-­âreplete Â conditionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..â¦â¦â¦â¦â¦â¦..â¦83 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 2.3.3.2 Â Â Â Â Analysis Â of Â the Â data Â from Â the Â pilot Â RNA-­âseq Â analysis Â identified Â several Â Â Â Â Â Â Â Â Â putative Â transcripts Â associated Â with Â the Â GRX4 Â locusâ¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦...â¦â¦â¦.â¦â¦85 Â Â Â 2.3.3.3 Â Â Â Â Â Â Â Analysis Â of Â GRX4 Â essentialityâ¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦87 Â Â Â Â Â Â Â 2.3.3.3.1 Â Â Â Deletion Â of Â the Â entire Â GRX4 Â including Â the Â N-­âterminal Â region Â in Â a Â Â Â Â Â Â Â stable Â diploid Â (strain Â AI187) Â background Â shows Â that Â the Â N-­âterminal Â region Â of Â Â Â Â Â Â GRX4 Â is Â not Â essentialâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦.87 Â Â Â Â Â Â Â Â Â 2.3.3.3.2 Â Â Â Genetic Â segregation Â analysis Â of Â heterozygous Â auxotrophic Â markers Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â in Â progeny Â isolated Â from Â a Â stable Â diploid Â with Â a Â confirmed Â deletion Â of Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â GRX4â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.91 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 2.4 Â Â Â Â Â Discussionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦...â¦â¦92 Â Â Â Â Â Â Â Â Â Â Â Â 2.4.1 Â Â Â Â The Â conserved Â C-­âterminal Â monothiol Â Grx Â domain Â of Â Grx4 Â physically Â binds Â to Â Â Â Â Â Â the Â N-­âterminal Â region Â of Â Cir1â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦93 Â Â Â Â Â Â Â Â Â Â Â Â 2.4.2 Â Â Â Â Â The Â grx4 Â mutant Â displays Â iron-­ârelated Â and Â virulence-­ârelated Â phenotypes.....96 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xi Â Â Â Â Â Â Â Â Â Â Â 2.4.3 Â Â Â Â Â Grx4 Â has Â a Â central Â role Â in Â maintenance Â of Â iron Â homeostasis Â in Â C. Â neoformans Â Â Â Â Â Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..98 Â Â Â Â Â Â Â Â Â Â Â 2.4.4 Â Â Â Â Grx4 Â is Â required Â for Â virulence Â in Â C. Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦.â¦â¦â¦â¦â¦â¦100 Â Â Â Â Â Â Â Â Â Â Â 2.4.5 Â Â Â Â The Â N-­âterminal Â of Â Grx4 Â is Â not Â essential Â and Â exists Â in Â some Â but Â not Â all Â transcript Â Â Â Â Â Â Â Â Â Â Â Â Â Â isoforms Â from Â the Â GRX4 Â locusâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦..â¦.â¦.101 Â Chapter Â 3: Â Analysis Â of Â the Â roles Â of Â glutathione Â synthetase Â in Â Cryptococcus Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦104 Â Â Â Â Â Â 3.1 Â Introduction Â â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦104 Â Â Â Â Â Â 3.2 Â Â Â Â Â Materials Â and Â methods Â â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦.â¦â¦â¦â¦..â¦â¦â¦â¦â¦.â¦â¦110 Â Â Â Â Â Â Â Â Â Â Â Â 3.2.1 Â Â Â Â In Â silico Â protein Â analysis Â â¦â¦...â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦110 Â Â Â Â Â Â Â Â Â Â Â Â 3.2.2 Â Â Â Yeast Â strains, Â plasmids Â and Â growth Â mediaâ¦..â¦â¦â¦â¦â¦â¦..â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦110 Â Â Â Â Â Â Â Â Â Â Â Â 3.2.3 Â Â Â Â Generation Â of Â gsh2 Â mutants Â and Â a Â gsh2Î::GSH2 Â complemented Â strain Â â¦â¦â¦111 Â Â Â Â Â Â Â Â Â Â Â Â 3.2.4 Â Â Â Â Phenotypic Â and Â biochemical Â analysesâ¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦.â¦.113 Â Â Â 3.2.4.1 Â Â Â Â Â Growth Â Assay Â â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦113 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 3.2.4.2 Â Â Â Â Â Quantification Â of Â the Â intracellular Â content Â of Â total Â (reduced Â and Â oxidized Â Â or Â GSHt) Â glutathioneâ¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦113 Â Â Â Â Â Â 3.2.4.3 Â Â Â Â Capsule Â formation, Â melanin Â production, Â and Â growth Â at Â 37°C Â â¦.â¦â¦......115 Â Â Â Â Â Â 3.2.4.4 Â Â Â Â Stress Â and Â drug Â response Â assaysâ¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦....116 Â Â Â Â Â Â 3.2.4.5 Â Â Â Â Sensitivity Â to Â inhibitors Â of Â the Â mitochondria Â electron Â transport Â chain.116 Â Â Â Â Â Â 3.3 Â Â Â Â Results Â â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦117 Â Â Â Â Â Â Â Â Â Â Â 3.3.1 Â Â Â Â GSH Â synthetase Â encoded Â by Â GSH2 Â in Â C. Â neoformans Â belongs Â to Â the Â eukaryotic Â Â Â Â Â Â Â Â Â Â Â Â Â glutathione Â synthetase Â (eu-­âGS) Â family Â in Â the Â ATP-­âgrasp Â superfamilyâ¦â¦â¦â¦â¦â¦..â¦.117 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xii Â Â Â Â Â Â Â Â Â Â Â 3.3.2 Â Â Â Â GSH Â synthetase Â (Gsh2) Â is Â dispensable Â for Â growth Â under Â normal Â conditions Â in Â Â Â Â Â Â Â Â Â Â Â Â Â Â C. Â neoformansâ¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦..119 Â Â Â Â Â Â Â Â Â Â Â 3.3.3 Â Â Â Â gsh2 Â mutants Â are Â sensitive Â to Â iron Â starvation Â and Â require Â exogenous Â GSH Â for Â Â Â Â Â Â Â Â Â Â Â Â Â growth Â on Â minimal Â or Â iron-­âreplete Â media Â after Â iron Â starvation Â â¦â¦â¦â¦â¦â¦.â¦..â¦â¦....119 Â Â Â Â Â Â Â Â Â Â Â Â 3.3.4 Â Â Â The Â gsh2 Â mutants Â have Â reduced Â intracellular Â total Â glutathione Â (GSHt) Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â compared Â to Â the Â WT Â strain Â â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦..123 Â Â Â Â Â Â Â Â Â Â Â Â 3.3.5 Â Â Â Â gsh2 Â is Â involved Â in Â multi-­âstress Â resistanceâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦....â¦â¦â¦â¦â¦â¦.126 Â Â 3.3.6 Â Â Â Â Lack Â of Â Gsh2 Â affects Â virulence Â factors Â in Â C. Â neoformansâ¦â¦â¦â¦â¦â¦.â¦â¦â¦...â¦â¦128 Â Â Â Â Â Â Â Â Â Â Â Â 3.3.7 Â Â Â Â gsh2 Â mutants Â display Â increased Â susceptibility Â to Â antifungal Â drugsâ¦â¦â¦.â¦â¦129 Â Â Â Â Â Â Â Â Â Â Â Â 3.3.8 Â Â Â Â gsh2 Â mutants Â display Â subtle Â susceptibility Â to Â mitochondria Â inhibitorsâ¦....â¦131 Â Â Â Â Â Â 3.4 Â Â Â Â Discussionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦132 Â Â Â Â Â Â Â Â Â Â Â 3.4.1 Â Â Â GSH Â synthetase Â is Â dispensable Â for Â growth Â under Â normal Â growth Â conditions.133 Â Â Â Â Â Â Â Â Â Â Â Â 3.4.2 Â Phenotypic Â analysis Â of Â gsh2 Â mutants Â suggest Â an Â interplay Â between Â GSH Â Â Â homeostasis Â and Â iron Â homeostasis Â in Â C. Â neoformans Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..134 Â Â Â Â Â Â Â Â Â Â Â 3.4.3 Â Â Â Â Phenotypic Â characterization Â of Â gsh2 Â mutants Â revealed Â that Â Gsh2 Â is Â required Â Â Â Â Â Â Â for Â a Â plethora Â of Â functions Â in Â C. Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦.137 Â Chapter Â 4: Â Discussionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦142 Â Â Â Â Â 4.1. Â Â Â Â Iron Â Homeostasis Â in Â C. Â neoformans Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦..142 Â Â Â Â Â 4.2. Â Â Monothiol Â Grx4 Â is Â required Â for Â iron Â homeostasis Â and Â virulence Â in Â C. Â neoformans Â Â Â Â Â Â Â Â Â Â Â Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦143 Â Â Â Â Â 4.3. Â Â Â Â GSH Â is Â involved Â in Â stress Â response Â and Â virulence Â in Â C. Â neoformans Â â¦â¦â¦.â¦â¦â¦.â¦148 Â Â Â Â Â 4.4. Â Â Â Â Conclusionâ¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦153 Â Â Â Â Â 4.5 Â Â Â Â Â Key Â areas Â for Â future Â directionsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..154 Â Â Â Â Â Â Â Â Â Â Â Â Â Â xiii Â Â Â Â Â Â Â Â Â Â Â 4.5.1 Â Â Â Â A Â Proposed Â model Â of Â Grx4-­âCir1 Â interaction Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦.154 Â Â Â Â Â Â Â Â Â Â Â 4.5.2 Â Â Â Â A Â proposed Â crosstalk Â model Â for Â GSH Â functions Â â¦â¦â¦â¦â¦..â¦â¦â¦.â¦..â¦â¦.â¦â¦â¦.156 Â Â Â Â Â 4.6 Â Â Â Â Â Â Future Â experimentsâ¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦161 Â Â Â Â Â Â Â Â Â Â Â 4.6.1 Â Â Â Â Studying Â the Â interaction Â of Â Grx4 Â with Â Cir1 Â in Â C. Â neoformans Â â¦â¦â¦â¦â¦..â¦â¦â¦.161 Â Â Â Â Â Â Â Â Â Â Â 4.6.2 Â Â Â Â Investigating Â Grx4 Â capacity Â to Â assemble Â Fe-­âS Â clustersâ¦â¦â¦â¦â¦.â¦..â¦â¦â¦â¦.â¦.161 Â Â Â Â Â Â Â Â Â Â Â 4.6.3 Â Â Â Â Further Â understanding Â of Â the Â transcript Â structure Â of Â the Â GRX4 Â locusâ¦â¦...â¦.162 Â Â Â Â Â Â Â Â Â Â Â 4.6.4 Â Â Â Â Investigation Â of Â reversible Â S-­âglutathionylation Â of Â Cir1â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦.163 Â Â Â Â Â Â Â Â Â Â Â 4.6.5 Â Â Construction Â and Â phenotypic Â characterization Â of Â GSH Â transporter Â deletion Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â mutants Â â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦164 Â Â Â Â Â Â Â Â Â Â Â 4.6.6 Â Â Â Â Investigation Â of Â Grx4 Â and Â GSH Â crosstalk Â in Â C. Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.164 Â Â Â Â Â Â Â Â Â Â Referencesâ¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦..166 Â Appendicesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..203 Â Appendix Â Aâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦203 Â Appendix Â Bâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦ Â 217 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xiv Â List Â of Â Tables Â Table Â 2.1: Â Â Strains Â used Â in Â Chapter Â 2â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.38 Â Table Â 2.2: Â Primers Â sequences Â for Â strain Â constructionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦39 Â Table Â 2.3: Â Primer Â sequences Â for Â quantitative Â qRT-­âPCRâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦53 Â Table Â 3.1: Â Strains Â used Â in Â Chapter Â 3â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦...â¦â¦â¦â¦â¦111 Â Table Â 3.2: Â Primer Â sequences Â for Â strain Â constructionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦111 Â Table Â A1: Â List Â of Â proteins Â identified Â in Â Cir1 Â binding Â assay Â identified Â by Â Mass-­âSpectrometry Â in Â an Â experiment Â performed Â by Â Dr. Â Horacio Â Bachâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦203 Â Table Â A2: Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â grown Â under Â the Â iron-­âdeplete Â conditionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦..206 Â Table Â A3: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â grown Â under Â iron-­âreplete Â conditionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦208 Â Table Â A4: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â similar Â patterns Â of Â expression Â in Â the Â grx4 Â and Â cir1 Â mutants Â compared Â to Â WT Â cells Â grown Â under Â iron-­âdeplete Â conditionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦210 Â Table Â A5: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â similar Â patterns Â of Â expression Â in Â the Â grx4 Â and Â cir1 Â mutants Â compared Â to Â WT Â cells Â grown Â under Â iron-­âreplete Â conditionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦213 Â Table Â A6: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â distinct Â patterns Â of Â expression Â in Â the Â grx4 Â or Â cir1 Â mutants Â compared Â to Â WT Â cells Â grown Â under Â iron-­âdeplete Â condition Â â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦..â¦â¦..â¦â¦215 Â Â Â Â Â Â Â Â Â Â Â Â Â Â xv Â Table Â A7: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â distinct Â patterns Â of Â expression Â in Â the Â grx4 Â or Â cir1 Â mutants Â compared Â to Â WT Â cells Â grown Â under Â iron-­âreplete Â conditionâ¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦216 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xvi Â List Â of Â Figures Â Figure Â 1.1: Â Â Microscopy Â image Â of Â India Â ink Â staining Â showing Â the Â characteristic Â halo Â caused Â by Â the Â presence Â of Â the Â polysaccharide Â capsule Â of Â C. Â neoformans Â â¦â¦â¦â¦â¦..â¦â¦...â¦â¦â¦â¦â¦..â¦5 Â Figure Â 1.2: Â The Â dark Â pigment Â melanin Â in Â the Â cell Â wall Â in Â presence Â of Â laccase Â substrates Â such Â as Â L-­â3,4-­âdihydroxyphenylalanine Â (L-­âDOPA)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..â¦.7 Â Figure Â 1.3: Â Reactive Â oxygen Â species Â (ROS) Â are Â generated Â in Â the Â Fenton Â reaction Â in Â the Â presence Â of Â iron Â and Â oxygenâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦11 Â Figure Â 1.4: Â Â Comparison Â of Â domain Â structures Â across Â species Â for Â monothiol Â Grxsâ¦â¦â¦.â¦17 Â Figure Â 1.5: Â Components Â of Â the Â iron Â network Â in Â C. Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦30 Â Figure Â 2.1: Â Construction Â of Â grx4 Â mutant Â and Â complemented Â grx4Î::GRX4 Â strain, Â and Â confirmation Â of Â grx4 Â mutant Â by Â Southern Â Hybridization Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦..43 Â Figure Â 2.2: Â Identified Â peptides Â obtained Â from Â MS Â analysisâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦54 Â Figure Â 2.3: Â Purification Â of Â the Â His6-­âtagged Â Grx4 Â polypeptideâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.. Â 55 Â Figure Â 2.4: Â Â Purification Â of Â Cir1 Â tagged Â with Â the Â maltose Â binding Â protein Â (MBP)â¦â¦â¦â¦â¦57 Â Figure Â 2.5: Â Overview Â of Â the Â Grx4-­âCir1 Â Alphascreen Â assayâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..58 Â Figure Â 2.6: Â Binding Â between Â Cir1 Â and Â Grx4 Â detected Â by Â ALPHAScreen Â analysisâ¦â¦â¦â¦â¦.60 Â Figure Â 2.7: Â Grx4 Â physically Â interacts Â with Â Cir1 Â in Â a Â yeast Â two-­âhybrid Â assayâ¦â¦â¦â¦â¦â¦...â¦62 Â Figure Â 2.8: Â Domain Â structure Â of Â C. Â neoformans Â Grx4â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦.64 Â Figure Â 2.9: Â A Â grx4 Â mutant Â displays Â iron-­ârelated Â phenotypes Â similar Â to Â the Â cir1 Â mutantâ¦.67 Â Figure Â 2.10: Â Grx4 Â is Â required Â for Â growth Â in Â iron-­âdepleted Â media Â supplemented Â with Â FeCl3â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦...â¦â¦â¦â¦..70Figure Â 2.11: Â Addition Â of Â Â GSH Â restored Â growth Â of Â grx4 Â mutant Â under Â iron-­âreplete Â conditions Â â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦..71 Â Â Â Â Â Â Â Â Â Â Â Â Â Â xvii Â Figure Â 2.12: Â Grx4 Â has Â a Â negative Â regulatory Â influence Â on Â the Â transcript Â levels Â for Â iron Â acquisition Â functionsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..73 Â Figure Â 2.13: Â Â A Â grx4 Â mutant Â does Â not Â express Â virulence-­ârelated Â traits.â¦â¦â¦â¦â¦â¦â¦...â¦..â¦75 Â Figure Â 2.14: Â Loss Â of Â GRX4 Â abolishes Â virulenceâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...76 Â Figure Â 2.15: Â Determination Â of Â fungal Â loads Â in Â mice Â tissueâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..â¦..78 Â Figure Â 2.16: Â Enrichment Â of Â genes Â differentially Â expressed Â in Â RNA-­âSeq Â analysis Â of Â the Â grx4 Â mutant Â and Â the Â WT Â strain Â under Â the Â iron-­âdeplete Â conditionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦....82 Â Figure Â 2. Â 17: Â Enrichment Â of Â genes Â differentially Â expressed Â in Â the Â RNA-­âSeq Â analysis Â of Â the Â grx4 Â mutant Â and Â the Â WT Â strain Â in Â the Â iron-­âreplete Â conditionâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦..â¦.84 Â Figure Â 2.18: Â Exonâintron Â organisation Â of Â C. Â neoformans Â GRX4 Â locusâ¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦....97 Â Figure Â 2.19: Â Full Â deletion Â of Â GRX4 Â in Â a Â stable Â H99 Â diploid Â backgroundâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.90 Â Figure Â 2.20: Â Analysis Â of Â progenyâ¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦..92 Â Figure Â 3.1: Â Structure Â of Â GSHâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦105 Â Figure Â 3.2: Â Biological Â functions Â associated Â with Â GSHâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.107 Â Figure Â 3.3: Â Enzymatic Â pathway Â for Â GSH Â synthesis Â in Â eukaryotesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...109 Â Figure Â 3.4: Â Domain Â structure Â and Â blastp Â analysis Â of Â Gsh2 Â in Â C. Â neoformans Â â¦â¦â¦â¦â¦â¦...118 Â Figure Â 3.5: Â Gsh2 Â is Â dispensable Â for Â growth Â under Â normal Â conditions Â in Â C. Â neoformans...119 Â Figure Â 3.6: Â Â Growth Â phenotypes Â of Â gsh2 Â mutants Â in Â YNB Â and Â iron-­âreplete Â media Â after Â iron Â starvationâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦122 Â Figure Â 3.7: Â Total Â glutathione Â (GSHt) Â levels Â in Â the Â gsh2 Â mutantsâ¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦..125 Â Figure Â 3.8: Â The Â gsh2 Â mutants Â have Â increased Â susceptibility Â to Â salt Â stress Â and Â thiol Â oxidizing Â agent Â diamideâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..127 Â Â Â Â Â Â Â Â Â Â Â Â Â Â xviii Â Figure Â 3.9: Â The Â gsh2 Â mutants Â are Â impaired Â in Â virulence-­ârelated Â traits Â â¦â¦â¦â¦â¦â¦â¦â¦â¦..129 Â Figure Â 3.10: Â Deletion Â of Â GSH2 Â Increases Â susceptibility Â to Â antifungal Â agentsâ¦â¦â¦â¦â¦â¦â¦130 Â Figure Â 3.11: Â Â gsh2 Â mutants Â display Â a Â subtle Â susceptibility Â to Â ETC Â inhibitorsâ¦â¦â¦...â¦â¦â¦131 Â Figure Â 4.1: Â A Â schematic Â model Â of Â Grx4-­âCir1 Â interaction Â â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...156 Â Figure Â 4.2: Â Schematics Â of Â the Â proposed Â functional Â crosstalk Â among Â GSH, Â iron Â homeostasis Â and Â cellular Â functions Â in Â C. Â neoformansâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..160 Â Figure Â A.1: Â Functional Â categorization Â of Â Cir1 Â potential Â binding Â partners.â¦â¦â¦..â¦.â¦â¦.â¦205 Â Figure Â B.1: Â Deletion Â of Â GSH2 Â in Â gDNA Â extracted Â from Â 3 Â individual Â gsh2Î Â mutants Â and Â re-­âintegration Â of Â GSH2 Â in Â locus Â in Â gDNA Â extracted Â from Â gsh2Î::GSH2 Â in Â C. Â neoformans Â H99 Â background Â was Â confirmed Â by Â PCRâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.217 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xix Â List Â of Â Abbreviations Â Â 3HAA Â Â Â 3-­âhydroxyanthranilic Â acid Â AAs Â Â amino Â acids Â AFT1 Â Â iron-­âregulated Â activator Â of Â transcription Â AIDS Â Â Â acquired Â immune Â deficiency Â syndrome Â ATP Â Â Â adenosine Â triphosphate Â BBB Â Â Â blood Â brain Â barrier Â BLAST Â Â basic Â local Â alignment Â search Â tool Â Â Â BPS Â Â bathophenanthroline Â disulfonate Â Â BSA Â Â Â bovine Â serum Â albumin Â Â cAMP Â Â Â cyclic Â adenosine Â monophosphate Â Â Â CCCP Â Â carbonyl Â cyanide Â m-­âchlorophenyl Â hydrazone Â Â cDNA Â Â complementary Â deoxyribonucleic Â acid Â Â Â Cfo1 Â Â Cryptococcus Â ferroxidase Â 1 Â Â Â Cft1 Â Â Â C. Â neoformans Â iron Â permease Â 1 Â Â CFUs Â Â colony Â forming Â units Â Â CGFS Â Â cysteine Â glycine Â phenylalanine Â serine Â Â Â CIG1 Â Â cytokine-­âinducing Â glycoprotein Â 1 Â Â Â Cir1 Â Â Â Cryptococcus Â iron Â regulator Â 1 Â Â CNS Â Â Â central Â nervous Â system Â Â Â Â C-­âterminal Â Â carboxyl-­âterminal Â Â Â Â CTAB Â Â Cetyl Â trimethylammonium Â bromide Â Â Da Â Â Daltons Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xx Â DEPC Â Â Â diethylpyrocarbonate Â Â DIC Â Â differential Â interference Â contrast Â Â DNA Â Â Â deoxyribonucleic Â acid Â Â DTT Â Â Â dithiothreitol Â Â EDTA Â Â Â ethylenediaminetetraacetic Â acid Â Â EtOH Â Â Â ethanol Â Â Fio1 Â Â iron Â transport Â multicopper Â oxidase Â Â FRA Â Â Fe Â repressor Â of Â activation Â Â Fre Â Â Â ferric Â iron Â reductases Â Â GalXM Â Â Â galactoxylomannan Â Â gDNA Â Â Â genomic Â deoxyribonucleic Â acid Â Â FET Â Â Ferous Â transporter Â Â FTR Â Â Fe Â transporter Â GFP Â Â Â green Â fluorescent Â protein Â Â GO Â Â Â gene Â ontology Â Â GPI Â Â Â glycophosphatidylinositol Â Â GPX Â Â glutathione Â peroxidase Â Â GRX Â Â glutaredoxin Â Â GSH Â Â glutathione Â Â GSSG Â Â glutathione Â disulphide Â Â GXM Â Â Â glucuronoxylomannan Â Â HAART Â highly Â active Â antiretroviral Â therapy Â Â HEPES Â Â 4-­â(2-­âhydroxyethyl)-­â1-­âpiperazineethanesulfonic Â acid Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xxi Â HIS Â Â Â histidine Â Â HIV Â Â Â human Â immunodeficiency Â virus Â Â HYG Â Â Â hygromycin Â Â iNOS Â Inducible Â nitric Â oxide Â synthase Â Â IPTG Â isopropyl Â Î²-­âD-­â1-­âthiogalactopyranoside Â Â ISC Â Fe-­âS Â cluster Â assembly Â machinery Â Â Kd Â Â dissociation Â constant Â L-­âDOPA Â Â L-­â3,4-­âdihydrophenylalanine Â Â LIM Â Â low Â iron Â medium Â Â MAPK Â mitogen Â activated Â protein Â kinase Â Â MBP Â Â maltose Â binding Â protein Â Â MIT Â Â Massachusetts Â Institute Â of Â Technology Â Â MP Â mannoprotein Â Â mRNA Â messenger ribonucleic acid NAT nourseothricin NEO neomycin Â NTA Â Â nitrilotriacetic Â acid Â Â N-­âterminal Â Â amino-­âterminal Â Â PBS Â Â phosphate-­âbuffered Â saline Â Â PCR Â Â polymerase Â chain Â reaction Â Â PKC Â protein Â kinase Â C Â Â PLB Â Â phospholipase Â B Â Â qRT-­âPCR Â Â quantitative Â real Â time Â polymerase Â chain Â reaction Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xxii Â RNA Â Â ribonucleic Â acid Â Â rRNA Â ribosomal Â ribonucleic Â acid Â Â RNA-­âseq Â Â ribonucleic Â acid Â sequencing Â Â RNI Â Reactive Â nitrogen Â intermediates Â Â ROS Â Reactive Â oxygen Â species Â Â rpm Â Â revolutions Â per Â minute Â Â SAGE Â Â serial Â analysis Â of Â gene Â expression Â Â SD Â Â standard Â deviation Â Â SDS Â sodium Â dodecyl Â sulfate Â Â SDS-­âPAGE Â sodium Â dodecyl Â sulfate Â polyacrylamide Â gel Â electrophoresis Â Â SHAM Â Â salicylhydroxamic Â acid Â Â Sit1 Â siderophore Â iron Â transporter Â Â Â Sod1 Â superoxide Â dismutase Â Â Â sp. Â species Â Â TCA Â trichloroacetic Â acid Â Â TRX Â thioredoxin Â Â TTC Â Â 2,3,5-­âTriphenyl Â tetrazolium Â chloride Â Â URA Â Â uracil Â Â UV Â Â Ultra Â Violet Â Â var. Â Â variety Â Â WT Â Â wild-­âtype Â Â YNB Â Â yeast Â nitrogen Â base Â Â Y2H Â Â yeast-­âtwo-­âhybrid Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xxiii Â YPD Â Â yeast Â extract Â peptone Â dextrose Â Â ZF Â Â zinc Â finger Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xxiv Â Acknowledgements Â Â First Â and Â foremost, Â I Â would Â like Â to Â extend Â my Â utmost Â and Â deepest Â gratitude Â to Â my Â supervisor Â Dr. Â James Â W. Â Kronstad, Â for Â providing Â me Â with Â the Â opportunity Â of Â pursuing Â my Â doctoral Â studies Â under Â his Â supervision Â and Â mentorship. Â Â This Â thesis Â would Â not Â have Â been Â possible Â without Â your Â guidance, Â support Â and Â supervision Â on Â every Â step Â of Â my Â progress. Â Your Â constructive Â criticism, Â encouragement, Â and Â patience Â with Â me Â throughout Â the Â course Â of Â my Â studies, Â have Â been Â pivotal Â in Â the Â success Â of Â my Â graduate Â studies Â and Â for Â the Â development Â of Â my Â critical Â thinking Â skills. Â Â Sincere Â and Â many Â thanks Â to Â my Â PhD Â supervisory Â committee Â members, Â Dr. Â Michael. Â Murphy, Â Dr. Â Brett Â Finlay Â and Â Dr. Â Charles Â Thompson, Â for Â their Â solid Â support Â and Â great Â suggestions Â throughout Â my Â studies. Â Your Â valuable Â input Â into Â my Â projects Â and Â feedback Â on Â my Â progress Â has Â been Â instrumental Â throughout Â my Â graduate Â program. Â I Â would Â also Â like Â to Â extend Â my Â appreciation Â to Â Theresa Â Charlton. Â Thanks Â to Â you Â I Â never Â had Â to Â be Â concerned Â with Â scheduling Â my Â committee Â meetings. Â I Â really Â appreciate Â your Â support Â Theresa Â throughout Â the Â years. Â Â Most Â special Â thanks Â to Â Dr. Â Guanggan Â Hu, Â for Â being Â so Â helpful, Â and Â for Â providing Â me Â with Â excellent Â scientific Â advice Â and Â encouragement Â towards Â success. Â I Â am Â always Â grateful Â to Â you Â for Â your Â tremendous Â amount Â of Â help Â and Â support. Â Â I would also like to thank Dr. Daniel Croll for sharing his vast amount of knowledge and expertise on RNA-seq data analysis and influential scientific discussions Â Next, Â I Â would Â like Â to Â thank Â members Â of Â Kronstad Â lab, Â Dr. Â Brigitte Â Cadieux, Â Dr. Â Sanjay Â Saikia, Â Dr. Â Melissa Â Caza, Â Dr. Â Emma Â Griffiths, Â Erik Â Nielson, Â Dr. Â Matthias Â Kretschmer, Â ScottLambie Â and Â Sean Â McCotter. Â I Â would Â also Â like Â to Â thank Â Megan Â Kofoed Â at Â Dr. Â Hieterâs Â lab Â Â Â Â Â Â Â Â Â Â Â Â Â Â xxv Â for Â treating Â me Â like Â a Â member Â of Â their Â own Â lab Â and Â trusting Â me Â with Â reagents Â at Â media Â room. Â Â Next, Â my Â sincere Â thanks Â go Â to Â the Â current Â and Â past Â members Â of Â Finlay Â lab Â and Â Piret Â lab, Â for Â their Â genuine Â friendship, Â caffeine Â breaks, Â fun Â times Â and Â good Â times. Â Â Â I Â extend Â my Â gratitude Â to Â the Â University Â of Â British Â Columbia Â and Â the Â department Â of Â Microbiology Â and Â Immunology Â for Â inspiration Â and Â financially Â supporting Â me Â with Â 4-­âYear Â Graduate Â Fellowship Â (4YF). Â Â Â I Â gratefully Â acknowledge Â the Â Canadian Â Institute Â of Â health Â and Â Research Â (CIHR) Â sources Â for Â further Â financial Â support Â of Â my Â doctoral Â studies. Â In Â addition Â to Â Dr. Â James Â Kroonstadâs Â NIH Â operating Â grant, Â I Â was Â funded Â by Â Frederick Â Banting Â and Â Charles Â Best Â Canada Â Graduate Â Scholarship Â (CGS) Â for Â 3 Â years, Â and Â I Â was Â honoured Â to Â be Â A Â CIHR Â trainee. Â Â Â Â I Â would Â like Â to Â thank Â my Â lovely Â parents Â Dania Â Babayan Â and Â Houshang Â Attarian Â for Â fueling Â me Â up Â with Â drive Â and Â courage. Â You Â are Â my Â pillars Â in Â life Â and Â rationale Â to Â pursue Â PhD Â studies. Â Â My Â wonderful Â husband Â Fabian Â Lemann Â for Â all Â the Â magic Â he Â has Â brought Â to Â my Â life Â since Â the Â Winter Â Olympics Â in Â Vancouver. Â Your Â enthusiasm Â for Â life Â is Â infectious, Â and Â I Â have Â learned Â so Â much Â at Â your Â adventurous Â school Â of Â life. Â Â My Â sincere Â hearty Â thanks Â to Â my Â one Â and Â only Â sister, Â Roxanne Â Attarian Â and Â her Â amazing Â husband Â Vafa Â Moghaddam Â for Â being Â the Â brother Â I Â never Â had. Â Â You Â have Â been Â and Â will Â always Â be Â there Â for Â me Â through Â it Â all. Â I Â would Â also Â like Â to Â thank Â my Â family Â in Â Switzerland, Â especially Â Martin Â Lemann Â and Â Suzette Â Gerber Â for Â your Â love Â and Â support. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â xxvi Â Dedication Â Â Dedication Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â To Â Dania, Â Houshang, Â Â Â Â Â Â Â Â Â Â Â & Â 20 Â years Â of Â Babken Â Avak Â Babayanâs Â solitude Â Â Â Â Â Â Â Â Â Â Â Â Â 1 Â Chapter Â 1: Â Â Introduction Â Â Â 1.1 Â Cryptococcus Â neoformans Â Â Cryptococcus Â neoformans Â is Â an Â opportunistic Â fungal Â pathogen Â that Â has Â been Â isolated Â from Â diverse Â environmental Â niches Â including Â several Â different Â tree Â species Â (e.g., Â eucalyptus Â trees, Â Douglas Â fir) Â and Â soil Â that Â is Â contaminated Â with Â the Â excreta Â of Â birds, Â particularly Â pigeons Â (J. Â A. Â Barnett, Â 2010, Â A. Â Botes Â et Â al., Â 2009, Â K. Â L. Â Buchanan Â and Â J. Â W. Â Murphy, Â 1998, Â N. Â P. Â Govender Â et Â al., Â 2011, Â H. Â C. Â Gugnani Â et Â al., Â 2005, Â S. Â E. Â Kidd Â et Â al., Â 2003, Â X. Â Lin Â et Â al. Â , Â 2006, Â A. Â Medeiros Â Ribeiro Â et Â al., Â 2006, Â K. Â Nielsen Â et Â al., Â 2007, Â N. Â Refojo Â et Â al., Â 2009). Â Â Importantly, Â C. Â neoformans, Â along Â with Â a Â related Â species Â C. Â gattii, Â is Â also Â the Â causative Â agent Â of Â cryptococcosis, Â an Â often-­âfatal Â fungal Â disease Â of Â humans Â and Â other Â animals Â (K. Â H. Â Bartlett Â et Â al., Â 2008, Â L. Â M. Â Hoang Â et Â al., Â 2004, Â N. Â Saul Â et Â al., Â 2008). Â 1.1.1 Â Â Cryptococcal Â disease Â Infection Â with Â C. Â neoformans Â is Â one Â of Â the Â leading Â causes Â of Â morbidity Â and Â mortality Â among Â patients Â afflicted Â with Â HIV/AIDS. Â Â Specifically, Â it Â has Â been Â estimated Â that Â cryptococcosis Â is Â responsible Â for Â an Â estimated Â 1 Â million Â cases Â of Â meningoencephalitis Â and Â over Â 600,000 Â deaths Â annually, Â mostly Â among Â HIV-­âinfected Â patients Â in Â sub-­âSaharan Â Africa Â (B. Â J. Â Park Â et Â al., Â 2009). Â Patients Â undergoing Â immunosuppressive Â therapy, Â such Â as Â transplant Â or Â cancer Â patients Â are Â also Â at Â risk Â for Â cryptococcal Â disease Â (P. Â G. Â Pappas, Â 2013, Â M. Â Revest Â et Â al., Â 2006). Â Individuals Â are Â usually Â colonized Â with Â C. Â neoformans Â through Â inhalation Â of Â either Â spores Â or Â desiccated Â yeast Â cells Â from Â the Â environment, Â although Â spores Â have Â yet Â to Â be Â observed Â outside Â the Â laboratory Â setting Â (R. Â Velagapudi Â et Â al., Â 2009). Â Infection Â with Â C. Â neoformans Â is Â most Â commonly Â initiated Â when Â fungal Â cells Â are Â inhaled Â and Â deposited Â into Â the Â alveoli Â (B. Â C. Â Fries Â et Â al., Â 2001). Â In Â immunocompetent Â individuals, Â the Â Â Â Â Â Â Â Â Â Â Â Â Â Â 2 Â infection Â is Â often Â cleared Â by Â the Â hostâs Â immune Â system Â or Â it Â can Â become Â latent Â upon Â containment Â by Â macrophages. Â In Â immunocompromised Â patients, Â however, Â the Â pathogen Â can Â proliferate, Â cross Â the Â blood-­âbrain Â barrier Â and Â progress Â to Â the Â central Â nervous Â system Â to Â cause Â meningoencephalitis Â (K. Â Vu Â et Â al., Â 2014). Â 1.1.2 Â Â Cryptococcal Â meningoencephalitis Â Â An Â important Â aspect Â of Â cryptococcal Â disease Â is Â the Â ability Â of Â this Â fungus Â to Â proliferate Â in Â the Â brain Â and Â cerebrospinal Â fluid Â leading Â to Â cryptococcal Â meningoencephalitis; Â this Â is Â the Â most Â frequent Â and Â fatal Â manifestation Â of Â cryptococcosis Â in Â HIV-­âinfected Â individuals Â (B. Â C. Â Fries Â et Â al., Â 2005, Â K. Â J. Â Kwon-­âChung Â et Â al., Â 2014, Â S. Â C. Â Lee Â et Â al., Â 1996, Â J. Â R. Â Perfect Â et Â al., Â 1998, Â A. Â Sudan Â et Â al., Â 2013). Â Patients Â have Â a Â variety Â of Â symptoms Â including Â headache, Â fever, Â malaise, Â altered Â mental Â status Â and, Â less Â frequently, Â a Â depressed Â level Â of Â consciousness. Â Furthermore, Â elevated Â intracranial Â pressure Â is Â a Â common Â complication, Â and Â this Â condition Â can Â result Â in Â loss Â of Â vision Â and Â hearing Â (T. Â Bicanic Â and Â T. Â S. Â Harrison, Â 2004). Â Â 1.1.3 Â Â The Â burden Â of Â cryptococcal Â infection Â and Â HIV Â Â As mentioned above, the worldwide burden of cryptococcosis is quite high with an estimate 1 million cases per annum in HIV/AIDS patients (B. J. Park, et al., 2009). The case fatality rate in patients with cryptococcal meningoencephalitis is between 35%-65% in sub-Saharan Africa (R. J. Lessells et al., 2011). This compares with a mortality rate of 10%-20% in most developed countries. The high mortality is mainly due to a delay in presentation of the disease, which leads to diagnosis only when meningoencephalitis is advanced and treatment is less effective. The high fatality rate is also partly due the poor availability, the high cost and the toxicity of the first-line anti-fungal treatments such as amphotericin B, as well as the frequent Â Â Â Â Â Â Â Â Â Â Â Â Â 3 Â complication of elevated intracranial pressure mentioned above (R. Rajasingham et al., 2012). The high burden of cryptococcosis demonstrates an urgent need for earlier diagnostic approaches, and improved treatment of cryptococcal disease, particularly in resource-poor settings such as Sub-Saharan Africa (B. J. Park, et al., 2009). Â 1.1.4 Â Â Treatment Â Â The Â patientâs Â underlying Â conditions, Â such Â as Â HIV Â status, Â or Â the Â occurrence Â of Â an Â organ Â transplant, Â are Â major Â determinants Â of Â the Â treatment Â guidelines Â for Â cryptococcal Â meningoencephalitis Â (J. Â R. Â Perfect Â et Â al., Â 2010). Â For Â instance, Â the Â primary Â therapy Â for Â an Â HIV-­âinfected Â patient Â is Â intravenous Â (IV) Â amphotericin Â B Â (AmB) Â deoxycholate Â (Ambd) Â at Â 0.7 Â -­â Â 1.0 Â mg/kg Â plus Â flucytosine Â at Â 100mg/kg Â for Â 4 Â days, Â followed Â by Â 2 Â weeks Â of Â fluconazole Â at Â 6 Â mg/kg Â (J. Â R. Â Perfect, Â et Â al., Â 2010, Â M. Â S. Â Saag Â et Â al., Â 2000, Â C. Â M. Â van Â der Â Horst Â et Â al., Â 1997). Â Â In Â some Â cases, Â a Â combination Â of Â antifungal Â drugs Â is Â used Â with Â immunomodulating Â agents Â such Â as Â monoclonal Â antibodies Â and Â gamma Â interferon Â as Â primary Â treatments Â (R. Â A. Â Larsen Â et Â al., Â 2005) Â (P. Â G. Â Pappas Â et Â al., Â 2004). Â 1.2 Â Â Virulence Â features Â of Â C. Â neoformans Â Â A Â combination Â of Â fungal Â virulence Â factors Â and Â host Â susceptibility Â factors Â enable Â C. Â neoformans Â to Â survive Â and Â proliferate Â within Â vertebrates Â (A. Â Casadevall Â and Â L. Â A. Â Pirofski, Â 1999, Â J. Â N. Â Steenbergen Â et Â al., Â 2003). Â Several Â major Â virulence Â traits Â have Â been Â identified Â and Â each Â plays Â a Â unique Â role Â in Â the Â pathogenesis Â of Â disease Â caused Â by Â C. Â neoformans. Â Â These Â traits Â are Â described Â in Â the Â following Â sections. Â 1.2.1 Â Â Growth Â at Â 37°C Â Â The Â ability Â to Â grow Â and Â proliferate Â at Â 37°C Â is Â an Â essential Â virulence Â attribute Â of Â pathogenic Â microorganisms Â that Â attack Â vertebrate Â hosts. Â Being Â an Â environmental Â yeast, Â a Â Â Â Â Â Â Â Â Â Â Â Â Â Â 4 Â key Â hallmark Â of Â C. Â neoformans Â is Â its Â ability Â to Â grow Â at Â the Â human Â body Â temperature Â of Â 37°C Â (J. Â A. Â Barnett, Â 2010, Â M. Â A. Â Garcia-­âSolache Â et Â al., Â 2013, Â L. Â R. Â Martinez Â et Â al., Â 2001, Â V. Â A. Â Robert Â and Â A. Â Casadevall, Â 2009). Â Â Although Â this Â trait Â is Â not Â traditionally Â considered Â to Â be Â a Â virulence Â factor, Â it Â has Â been Â proposed Â that Â any Â enzyme Â that Â confers Â fitness Â at Â high Â temperature Â would Â fit Â the Â definition Â of Â virulence Â factor Â (V. Â A. Â Robert Â and Â A. Â Casadevall, Â 2009). Â Â A Â number Â of Â proteins Â have Â been Â identified Â in Â C. Â neoformans Â that Â play Â a Â role Â in Â growth Â at Â high Â temperature. Â Â Some Â of Â the Â better Â characterized Â examples Â include Â Ras1, Â the Â p21-­âactivated Â kinase Â (PAK) Â kinase Â Ste20, Â the Â phospholipid-­âbinding Â protein Â Cts1, Â the Â vacuolar Â ATPase Â Vph1, Â the Â thiol Â peroxidase Â Tsa1, Â the Â cell Â integrity Â mitogen-­âactivated Â protein Â (MAP) Â kinase Â Mpk1, Â and Â the Â Ca2+/calmodulin-­âdependent Â phosphatase Â calcineurin Â (J. Â A. Â Alspaugh Â et Â al., Â 2000, Â T. Â Erickson Â et Â al., Â 2001, Â D. Â S. Â Fox Â et Â al., Â 2001, Â P. Â R. Â Kraus Â et Â al., Â 2003, Â T. Â A. Â Missall Â et Â al., Â 2004b, Â A. Â Odom Â et Â al., Â 1997, Â P. Â Wang Â et Â al., Â 2002). Â Deletion Â or Â disruption Â of Â a Â gene Â encoding Â one Â of Â these Â proteins Â results Â in Â either Â attenuation Â or Â complete Â loss Â of Â virulence Â in Â mammalian Â models Â of Â cryptococcosis Â (P. Â R. Â Kraus Â et Â al., Â 2004). Â Â A Â mouse Â model Â in Â which Â cryptococcal Â cells Â are Â introduced Â by Â inhalation Â is Â the Â most Â commonly Â used Â assay Â for Â differences Â in Â the Â virulence Â of Â strains Â and Â mutants. Â 1.2.2 Â Â Polysaccharide Â capsule Â Â A Â prominent Â and Â characteristic Â feature Â of Â C. Â neoformans Â is Â the Â presence Â of Â a Â polysaccharide Â capsule Â that Â surrounds Â the Â cells. Â The Â capsule Â is Â visible Â by Â India Â ink Â staining Â as Â a Â halo Â where Â the Â ink Â is Â excluded Â from Â contact Â with Â the Â cell Â surface Â by Â the Â presence Â of Â the Â polysaccharide Â (Figure Â 1.1) Â (O. Â Zaragoza Â et Â al., Â 2009). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 5 Â Â Figure Â 1.1: Â Microscopy Â image Â of Â India Â ink Â staining Â showing Â the Â characteristic Â halo Â caused Â by Â the Â presence Â of Â the Â polysaccharide Â capsule Â of Â C. Â neoformans Â strain Â H99 Â Â Â C. Â neoformans Â produces Â a Â complex Â polysaccharide Â made Â of Â glucuronoxylomannan Â (GXM) Â and Â galactoxylomannan Â (GalXM), Â and Â mannoproteins Â are Â also Â found Â associated Â with Â the Â capsule. Â The Â polysaccharides Â GXM Â and Â GalXM Â are Â attached Â to Â the Â cell Â surface Â as Â well Â as Â shed Â into Â culture Â medium; Â these Â polysaccharides Â can Â be Â isolated Â from Â culture Â supernatants Â and Â detected Â in Â the Â serum Â and Â cerebral Â spinal Â fluid Â of Â patients Â (M. Â Feldmesser Â and Â A. Â Casadevall, Â 1997, Â M. Â Feldmesser Â et Â al., Â 1996). Â Mannoproteins, Â minor Â component Â of Â the Â capsule, Â are Â heavily Â glycosylated Â that Â are Â shed Â during Â growth Â in Â vivo Â and Â in Â vitro. Â Â The Â functions Â of Â these Â proteins Â are Â poorly Â characterized Â with Â the Â exception Â that Â the Â mannoprotein Â Cig1 Â (the Â product Â of Â the Â CIG1 Â gene) Â participates Â in Â iron Â acquisition Â from Â heme Â at Â the Â cell Â surface Â (B. Â Cadieux Â et Â al., Â 2013). Â Â Mannoproteins Â are Â highly Â immunogenic Â and Â can Â elicit Â protective Â immune Â responses Â in Â mice Â (M. Â K. Â Mansour Â et Â al., Â 2004, Â D. Â Pietrella Â et Â al., Â 2001). Â In Â general, Â the Â capsule Â polysaccharides Â have Â unique Â physical Â and Â chemical Â characteristics Â compared Â with Â capsules Â from Â bacterial Â pathogens, Â although Â the Â synthesis, Â transport Â and Â attachment Â of Â the Â C. Â neoformans Â material Â are Â poorly Â characterized. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 6 Â It Â has Â been Â proposed Â that Â the Â capsule Â is Â antiphagocytic Â because Â it Â conceals Â cell-­âsurface Â pathogen-­âassociated Â molecular Â patterns, Â and Â capsule Â elaboration Â by Â cryptococcal Â cells Â leads Â to Â decrease Â in Â phagocytic Â uptake Â in Â the Â absence Â of Â opsonization Â (S. Â Shoham Â and Â S. Â M. Â Levitz, Â 2005). Â Â In Â addition Â to Â its Â antiphagocytic Â capacities, Â the Â capsule Â has Â many Â other Â proposed Â functions Â as Â a Â virulence Â factor. Â Â For Â example, Â it Â is Â proposed Â to Â protect Â C. Â neoformans Â from Â oxidative Â stress Â inside Â host Â cells Â by Â quenching Â reactive Â oxygen Â species Â (ROS) Â and Â other Â microbicidal Â molecules Â (P. Â K. Â Naslund Â et Â al., Â 1995, Â J. Â Rivera Â et Â al., Â 1998, Â O. Â Zaragoza, Â et Â al., Â 2009). Â Â The Â contribution Â of Â capsule Â to Â virulence Â in Â mouse Â models Â has Â clearly Â been Â demonstrated Â because Â C. Â neoformans Â acapsular Â mutants Â are Â avirulent Â (Y. Â C. Â Chang Â and Â K. Â J. Â Kwon-­âChung, Â 1994). Â However, Â one Â must Â consider Â and Â view Â every Â virulence Â factor Â in Â the Â context Â of Â the Â interaction Â with Â host Â factors. Â For Â example, Â the Â capsule Â is Â required Â for Â virulence Â in Â immunologically Â intact Â hosts, Â but Â acapsular Â strains Â can Â be Â pathogenic Â in Â mice Â lacking Â cell-­âmediated Â immunity Â (M. Â Del Â Poeta, Â 2004). Â 1.2.3 Â Â Melanin Â Â C. Â neoformans Â secretes Â the Â copper-­âdependent Â enzyme Â laccase Â to Â the Â cell Â wall Â and Â this Â enzyme Â catalyzes Â the Â oxidative Â polymerization Â of Â phenolic Â catecholamines Â that Â are Â deposited Â as Â a Â dark Â pigment Â melanin Â in Â the Â cell Â wall Â (Figure Â 1.2) Â (I. Â Polacheck, Â 1991, Â P. Â R. Â Williamson, Â 1997). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 7 Â Â Figure Â 1.2: Â The Â dark Â pigment Â melanin Â which Â is Â deposited Â in Â the Â cell Â wall Â in Â presence Â of Â laccase Â substrates Â such Â as Â L-­â3,4-­âdihydroxyphenylalanine Â (L-­âDOPA). Â Â Â Melanin Â is Â a Â negatively Â charged Â hydrophobic Â pigment Â with Â antioxidant Â properties. Â It Â provides Â resistance Â to Â reactive Â oxygen Â and Â nitrogen Â species Â generated Â by Â host Â phagocytes Â and Â protects Â C. Â neoformans Â from Â oxidative Â killing Â by Â these Â immune Â effector Â cells Â (A. Â Casadevall Â et Â al., Â 2000). Â Â In Â particular, Â it Â has Â been Â experimentally Â demonstrated Â that Â melanized Â cells Â are Â resistant Â to Â killing Â both Â by Â oxidants Â (H. Â S. Â Emery Â et Â al., Â 1994) Â and Â by Â antifungal Â drugs Â such Â as Â AmB Â (D. Â van Â Duin Â et Â al., Â 2002). Â Furthermore, Â melanin Â production Â is Â influenced Â by Â changes Â in Â chromosome Â copy Â number, Â as Â C. Â neoformans Â strains Â that Â were Â monosomic Â for Â chromosome Â 13 Â produced Â more Â melanin Â and Â were Â more Â virulent Â than Â non-­âmelanised Â strain Â disomic Â for Â chromosome Â 13 Â in Â a Â murine Â model Â of Â cryptococcosis Â (G. Â Hu Â et Â al., Â 2011). Â Â 1.2.4 Â Â Â Other Â virulence Â traits Â Â In Â addition Â to Â the Â major Â virulence Â factors Â described Â earlier, Â many Â other Â proteins Â and Â enzymes Â are Â associated Â with Â virulence Â in Â C. Â neoformans. Â Â These Â are Â briefly Â described Â in Â the Â following Â sections. Â 1.2.4.1 Â Â Urease Â Â Urease Â appears Â to Â have Â an Â important Â role Â in Â promoting Â fungal Â traversal Â of Â the Â blood-­â Â Â Â Â Â Â Â Â Â Â Â Â Â 8 Â brain Â barrier Â to Â promote Â brain Â invasion Â (M. Â A. Â Olszewski Â et Â al., Â 2004, Â M. Â Shi Â et Â al., Â 2010, Â A. Â Singh Â et Â al., Â 2013). Â Â Â However, Â the Â mechanisms Â by Â which Â C. Â neoformans Â traverses Â tissue Â barriers, Â and Â the Â contribution Â of Â urease Â to Â these Â processes Â are Â not Â clearly Â established. Â Â In Â general, Â C. Â neoformans Â appears Â to Â move Â from Â the Â microvasculature Â of Â the Â brain Â into Â the Â CNS Â either Â via Â a Â âTrojan Â horseâ Â mechanism Â involving Â macrophages Â or Â by Â direct Â transversal Â of Â free Â yeast Â cells Â (T. Â C. Â Sorrell Â et Â al., Â 2015, Â K. Â Vu, Â et Â al., Â 2014). Â 1.2.4.2 Â Â Phospholipase Â Â Phospholipases Â degrade Â phospholipids. Â It Â has Â been Â shown Â that Â phospholipase Â B Â (Plb1) Â is Â secreted Â during Â C. Â neoformans Â infection Â and Â leads Â to Â an Â increase Â in Â capsule Â growth Â as Â well Â as Â enhanced Â fungal Â survival Â in Â phagocytes Â (G. Â M. Â Cox Â et Â al., Â 2001). Â One Â proposed Â mechanism Â for Â the Â contribution Â of Â Plb1 Â is Â that Â it Â degrades Â host Â phospholipids Â as Â part Â of Â a Â role Â in Â nutrient Â acquisition, Â perhaps Â by Â damaging Â phagosomal Â membranes Â and Â allowing Â fungal Â access Â to Â cytoplasmic Â components Â (C. Â J. Â Chrisman Â et Â al., Â 2011). Â Â Plb1 Â might Â also Â play Â a Â role Â in Â extrapulmonary Â dissemination Â because Â Plb1-­âdeficient Â mutants Â have Â reduced Â virulence Â and Â decreased Â invasion Â of Â the Â brain Â (G. Â M. Â Cox, Â et Â al., Â 2001, Â R. Â Maruvada Â et Â al., Â 2012). Â Â A Â phospholipase Â C Â (Plc) Â was Â also Â recently Â shown Â to Â be Â involved Â in Â thermotolerance, Â capsule Â and Â cell Â wall Â synthesis, Â and Â virulence Â in Â C. Â neoformans Â (S. Â Lev Â et Â al., Â 2013). Â 1.2.4.3 Â Oxidative Â defenses Â Â Host Â immune Â cells Â secrete Â reactive Â oxygen Â and Â nitrogen Â species Â (ROS Â and Â RNS). Â These Â molecules Â are Â potent Â producers Â of Â free Â radicals Â and Â are Â powerful Â microbicides. Â Inducible Â Nitric Â Oxide Â Synthase Â (iNOS) Â also Â contributes Â to Â the Â production Â of Â nitrogen Â free Â radicals Â species Â (E. Â Novo Â and Â M. Â Parola, Â 2008). Â Â It Â has Â been Â shown Â that Â chemically-­âderived Â Â Â Â Â Â Â Â Â Â Â Â Â Â 9 Â NO Â is Â fungicidal Â (J. Â A. Â Alspaugh Â and Â D. Â L. Â Granger, Â 1991) Â and Â in Â vivo Â NO Â is Â a Â major Â contributor Â to Â microbicidal Â mechanisms Â (D. Â Goldman Â et Â al., Â 1996, Â A. Â Vazquez-­âTorres Â et Â al., Â 2008). Â Â Â Â C. Â neoformans Â employs Â numerous Â and Â redundant Â oxidative Â and Â nitrosative Â stress Â defense Â mechanisms, Â suggesting Â that Â this Â pathogen Â is Â well Â adapted Â to Â the Â oxidative Â defense Â environment Â in Â the Â host. Â Mouse Â infection Â studies Â revealed Â that Â C. Â neoformans Â mutants Â with Â defective Â nitrosative Â defenses Â are Â less Â virulent Â than Â the Â wild-­âtype Â strain Â (T. Â A. Â Missall Â et Â al., Â 2004a). Â Additionally, Â oxidative Â stress-­ârelated Â genes, Â for Â example Â encoding Â superoxide Â dismutases Â (SOD) Â and Â catalases, Â are Â upregulated Â in Â C. Â neoformans Â upon Â uptake Â by Â macrophages Â (F. Â A. Â Brandao Â et Â al., Â 2015). Â C. Â neoformans Â possesses Â two Â SOD Â isoforms Â and Â it Â was Â shown Â that Â an Â SOD1-­âdeficient Â mutant Â is Â more Â susceptible Â to Â macrophage Â killing Â and Â is Â slightly Â attenuated Â in Â mice Â (G. Â M. Â Cox Â et Â al., Â 2003). Â Â However, Â the Â SOD2 Â isoform Â is Â vital Â for Â aerobic Â growth Â at Â 37°C, Â and Â sod2 Â mutants Â are Â avirulent Â (S. Â S. Â Giles Â et Â al., Â 2005). Â Additional Â antioxidative Â enzymes Â in Â C. Â neoformans, Â such Â as Â thiol Â peroxidases, Â defend Â against Â both Â oxidative Â and Â nitrosative Â stresses Â and Â play Â a Â major Â role Â in Â cryptococcal Â virulence Â (T. Â A. Â Missall, Â et Â al., Â 2004a, Â T. Â A. Â Missall, Â et Â al., Â 2004b). Â Â 1.2.4.4 Â Secreted Â vesicles Â Â C. Â neoformans Â produces Â vesicles Â that Â can Â cross Â the Â cell Â wall Â through Â an Â as Â yet Â unknown Â mechanism Â to Â reach Â the Â extracellular Â environment Â (M. Â L. Â Rodrigues Â et Â al., Â 2008a, Â M. Â L. Â Rodrigues Â et Â al., Â 2007, Â A. Â Yoneda Â and Â T. Â L. Â Doering, Â 2006). Â The Â vesicles Â are Â known Â to Â contain Â and Â traffic Â capsular Â material Â across Â the Â cell Â wall Â via Â exocytosis Â (M. Â L. Â Rodrigues Â et Â al., Â 2008b). Â In Â addition Â to Â capsular Â materials, Â vesicles Â carry Â most Â of Â the Â virulence Â factors Â mentioned Â earlier Â such Â as Â laccase, Â urease, Â and Â superoxide Â dismutase Â (J. Â M. Â Wolf Â et Â al., Â Â Â Â Â Â Â Â Â Â Â Â Â Â 10 Â 2012). Â Â Â It Â has Â been Â suggested Â that Â C. Â neoformans Â may Â use Â the Â vesicles Â as Â part Â of Â an Â all-­âpurpose Â secretion Â mechanism Â for Â trans-­âcell Â wall Â transport Â and Â delivery Â of Â virulence Â cargo Â to Â the Â extracellular Â environment Â (M. Â L. Â Rodrigues, Â et Â al., Â 2008a). Â Â Furthermore, Â its Â been Â shown Â that Â vesicles Â can Â contribute Â to Â the Â pathogenesis Â of Â cryptococcal Â meningoencephalitis Â and Â cryptococcal Â progression Â in Â central Â nervous Â system Â by Â facilitating Â traversal Â across Â the Â Blood Â Brain Â Barrier Â (BBB) Â (K. Â Vu Â et Â al., Â 2009). Â It Â has Â also Â been Â shown Â that Â a Â Sec6-­âdependent Â pathway Â plays Â a Â major Â role Â in Â release Â of Â these Â so-­âcalled Â âvirulence Â delivery Â bagsâ Â because Â sec6 Â RNAi Â mutants Â of Â C. Â neoformans Â are Â defective Â in Â a Â number Â of Â virulence Â factors Â including Â laccase, Â urease, Â capsule Â and Â have Â attenuated Â virulence Â in Â mice Â (J. Â Panepinto Â et Â al., Â 2009). Â Â The Â exact Â mechanism Â by Â which Â the Â extracellular Â vesicles Â are Â produced, Â and Â how Â they Â cross Â the Â cell Â wall Â is Â largely Â unknown, Â but Â it Â has Â been Â suggested Â that Â they Â are Â potentially Â formed Â by Â a Â similar Â mechanism Â previously Â described Â for Â exosomes Â (C. Â V. Â Harding Â et Â al., Â 2013, Â R. Â M. Â Johnstone, Â 2005, Â J. Â D. Â Nosanchuk Â et Â al., Â 2008, Â M. Â L. Â Rodrigues, Â et Â al., Â 2008a). Â Briefly, Â it Â is Â suggested Â that Â C. Â neoformans Â multivesicular Â bodies Â fuse Â with Â the Â plasma Â membrane Â and Â release Â the Â extracellular Â vesicles; Â alternatively Â virulence Â factors Â may Â be Â secreted Â directly Â via Â exocytosis Â (T. Â L. Â Doering, Â 2009, Â J. Â D. Â Nosanchuk, Â et Â al., Â 2008, Â M. Â L. Â Rodrigues, Â et Â al., Â 2008a, Â A. Â Yoneda Â and Â T. Â L. Â Doering, Â 2009). Â 1.2.4.5 Â Nutrient Â acquisition Â Â The Â intracellular Â and Â extracellular Â growth Â of Â C. Â neoformans Â in Â the Â host Â and Â the Â biosynthesis Â of Â major Â virulence Â factors Â including Â melanin Â and Â capsule Â require Â fungal Â access Â to Â nutrients. Â Â Among Â these Â nutrients, Â metals Â such Â as Â copper Â and Â iron Â are Â of Â great Â Â Â Â Â Â Â Â Â Â Â Â Â Â 11 Â importance Â for Â C. Â neoformans Â pathogenesis, Â and Â so Â the Â ability Â to Â acquire Â these Â minerals Â under Â conditions Â of Â limited Â availability Â in Â the Â host Â is Â essential Â for Â cryptococcal Â survival Â and Â pathogenicity. Â The Â following Â sections Â summarize Â the Â mechanisms Â of Â acquisition Â for Â iron Â and Â copper, Â and Â the Â relevance Â of Â these Â metals Â to Â cryptococcal Â virulence. Â 1.2.4.5.1 Â Iron Â Â Both Â vertebrate Â hosts Â and Â microbial Â pathogens Â require Â iron Â as Â an Â essential Â element. Â Iron Â serves Â as Â a Â cofactor Â or Â a Â prosthetic Â group Â for Â essential Â enzymes Â in Â many Â basic Â cellular Â functions Â and Â metabolic Â pathways Â (J. Â Barasch Â and Â K. Â Mori, Â 2004, Â Y. Â Fu Â et Â al., Â 2004, Â M. Â Reyes Â and Â G. Â Imperatore, Â 2001, Â U. Â E. Â Schaible Â and Â S. Â H. Â Kaufmann, Â 2004, Â E. Â D. Â Weinberg, Â 1999). Â These Â functions Â and Â pathways Â include Â the Â synthesis Â of Â DNA Â as Â well Â as Â central Â energy Â metabolism, Â and Â iron Â is Â a Â critical Â co-­âfactor Â in Â various Â proteins Â that Â serve Â as Â oxygen Â carriers Â and Â electron Â transfer Â systems. Â Â Ferric Â iron Â (Fe3+) Â is Â almost Â insoluble Â under Â aerobic Â conditions, Â and Â free Â iron Â catalyzes Â the Â formation Â of Â oxygen Â radicals Â through Â the Â Fenton Â reaction Â (Figure. Â 1.3). Â These Â radicals Â are Â highly Â potent Â and Â damage Â cellular Â component Â including Â DNA, Â lipids, Â proteins Â and Â nucleic Â acids Â (J. Â A. Â Imlay Â et Â al., Â 1988). Â Â Â Figure Â 1.3: Â The Â Fenton Â reaction. Â Reactive Â oxygen Â species Â (ROS) Â are Â generated Â in Â the Â Fenton Â reaction Â in Â the Â presence Â of Â iron Â and Â oxygen Â Â In Â the Â host, Â iron Â is Â usually Â only Â available Â when Â it Â is Â bound Â to Â specific Â proteins Â such Â as Â transferrin Â (TF), Â lactoferrin Â (LF) Â and Â ferritin, Â or Â when Â it Â is Â complexed Â to Â heme Â within Â Â Â Â Â Â Â Â Â Â Â Â Â Â 12 Â hemoproteins Â (M. Â W. Â Hentze Â et Â al., Â 2004, Â U. Â E. Â Schaible Â and Â S. Â H. Â Kaufmann, Â 2004). Â Â The Â host Â deprives Â microbial Â pathogens Â of Â essential Â iron Â ions Â via Â a Â process Â called Â nutritional Â immunity Â (M. Â Nairz Â et Â al., Â 2014, Â N. Â L. Â Parrow Â et Â al., Â 2013, Â E. Â D. Â Weinberg, Â 2009). Â Â Â Iron Â availability Â in Â mammalian Â host Â fluids Â is Â maintained Â at Â extremely Â low Â levels Â (10-­â18 Â M) Â (J. Â Hegenauer Â and Â P. Â Saltman, Â 1975, Â C. Â Wandersman Â and Â P. Â Delepelaire, Â 2004, Â E. Â D. Â Weinberg, Â 1974) Â and Â pathogenic Â microbes Â require Â 10-­â6 Â to Â 10-­â7 Â M Â iron Â for Â growth Â in Â mammalian Â hosts Â (G. Â J. Â Anderson Â et Â al., Â 1994, Â G. Â J. Â Anderson Â and Â C. Â D. Â Vulpe, Â 2009, Â U. Â E. Â Schaible Â and Â S. Â H. Â Kaufmann, Â 2004). Â Â Therefore, Â pathogens Â have Â developed Â effective Â strategies Â to Â acquire Â iron Â from Â the Â limiting Â environment Â of Â the Â host. Â Â However, Â cellular Â iron Â levels Â are Â tightly Â regulated Â to Â avoid Â toxicity Â due Â to Â the Â formation Â of Â ROS. Â Â In Â general, Â iron Â homeostasis Â is Â considered Â as Â the Â balance Â between Â maintaining Â appropriate Â iron Â levels Â for Â cellular Â functions Â yet Â keeping Â levels Â sufficiently Â low Â or Â regulated Â to Â avoid Â iron-­âloading Â toxicity. Â Â Both Â host Â and Â pathogen Â cells Â have Â developed Â sophisticated Â systems Â for Â assuring Â a Â balanced Â cellular Â iron Â homeostasis Â (G. Â J. Â Anderson, Â et Â al., Â 1994, Â G. Â J. Â Anderson Â and Â C. Â D. Â Vulpe, Â 2009, Â Â Kaplan Â et Â al., Â 2006, Â S. Â Labbe Â et Â al., Â 2013, Â U. Â E. Â Schaible Â and Â S. Â H. Â Kaufmann, Â 2004). Â Â Specialized Â iron-­âacquiring Â systems Â in Â microbial Â pathogens Â consist Â of Â different Â molecules, Â which Â function Â in Â a Â highly Â interconnected Â fashion; Â these Â molecules Â include Â iron-­âbinding Â small Â molecules Â called Â siderophores, Â the Â cognate Â siderophore Â uptake Â functions Â and Â high Â affinity Â ferric Â iron Â uptake Â systems Â (S. Â C. Â Andrews Â et Â al., Â 2003, Â P. Â Canessa Â and Â L. Â F. Â Larrondo, Â 2013, Â M. Â Caza Â and Â J. Â W. Â Kronstad, Â 2013, Â W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2011, Â J. Â W. Â Kronstad Â et Â al., Â 2013, Â T. Â Lian Â et Â al., Â 2005). Â Â A Â thorough Â understanding Â of Â the Â mechanisms Â for Â regulating Â iron Â uptake Â and Â homeostasis Â is Â important Â for Â understanding Â the Â competition Â between Â host Â and Â pathogen, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 13 Â and Â could Â potentially Â identify Â new Â therapeutic Â targets Â for Â the Â treatment. Â Â Although Â much Â is Â known Â about Â iron Â acquisition Â by Â bacterial Â pathogens, Â much Â less Â is Â known Â about Â fungal Â pathogens. Â In Â this Â regard, Â Saccharomyces Â cerevisiae Â (bakerâs Â yeast) Â and Â Schizosaccharomyces Â pombe Â (fission Â yeast) Â have Â been Â valuable Â fungal Â models Â for Â studying Â molecular Â and Â cellular Â mechanisms Â of Â iron Â acquisition. Â Â Yeasts Â are Â one Â of Â the Â simplest Â eukaryotes Â with Â deciphered Â genomes, Â and Â they Â are Â also Â simple Â to Â manipulate Â genetically; Â they Â have Â also Â facilitated Â the Â identification Â of Â proteins Â required Â for Â iron Â uptake, Â intracellular Â iron Â trafficking, Â and Â Fe-­âS Â cluster Â biogenesis Â in Â C. Â neoformans Â and Â other Â eukaryotes Â (J. Â De Â Freitas Â et Â al., Â 2003, Â A. Â C. Â Dlouhy Â and Â C. Â E. Â Outten, Â 2013, Â J. Â Encinar Â del Â Dedo Â et Â al., Â 2015, Â P. Â A. Â Frey Â and Â C. Â E. Â Outten, Â 2011, Â A. Â Kumanovics Â et Â al., Â 2008, Â S. Â Labbe, Â 2010, Â S. Â Labbe Â et Â al., Â 2007, Â H. Â Li Â and Â C. Â E. Â Outten, Â 2012, Â A. Â Mercier Â and Â S. Â Labbe, Â 2010, Â C. Â E. Â Outten Â and Â A. Â N. Â Albetel, Â 2013, Â B. Â Pelletier Â et Â al., Â 2003, Â C. Â B. Â Poor Â et Â al., Â 2014, Â M. Â Schrettl Â et Â al., Â 2004). Â Â The Â key Â features Â of Â iron Â homeostasis Â for Â S. Â cerevisiae Â and Â S. Â pombe Â are Â presented Â below Â to Â provide Â context Â for Â the Â subsequent Â description Â of Â related Â functions Â in Â C. Â neoformans. Â 1.3. Â Â Iron Â homeostasis Â in Â eukaryotic Â models: Â S. Â cerevisiae Â and Â S. Â pombe Â 1.3.1 Â Â Iron Â homeostasis Â in Â S. Â cerevisiae Â Â Iron Â homeostasis Â in Â S. Â cerevisiae Â is Â mediated Â by Â the Â expression Â of Â iron Â uptake Â and Â storage Â functions Â that Â are Â regulated Â by Â the Â iron-­âresponsive Â transcription Â factor Â Aft1. Â Â Specifically, Â Aft1 Â regulates Â the Â transcription Â of Â a Â wide Â range Â of Â iron-­âresponsive Â genes Â in Â yeast, Â termed Â the Â iron Â regulon, Â by Â repressing Â expression Â in Â the Â presence Â of Â high Â available Â iron. Â Â The Â iron Â regulon Â includes Â genes Â encoding Â components Â of Â the Â iron Â transport Â systems Â on Â the Â plasma Â membrane Â and Â the Â vacuolar Â membrane, Â that Â is, Â ARN1-­â4, Â FRE1, Â FRE2, Â FET3, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 14 Â FTR1, Â FET5, Â FTH1 Â and Â SMF3 Â (C. Â Casas Â et Â al., Â 1997, Â R. Â F. Â Hassett Â et Â al., Â 1998a, Â R. Â F. Â Hassett Â et Â al., Â 1998b, Â A. Â Kumanovics, Â et Â al., Â 2008, Â R. Â Stearman Â et Â al., Â 1996, Â Y. Â Yamaguchi-­âIwai Â et Â al., Â 1995, Â Y. Â Yamaguchi-­âIwai Â et Â al., Â 1996, Â Y. Â Yamaguchi-­âIwai Â et Â al., Â 2002). Â The Â S. Â cerevisiae Â genome Â also Â encodes Â a Â paralogue Â of Â Aft1, Â namely Â Aft2, Â which Â is Â 39% Â identical Â to Â Aft1 Â in Â the Â N-­âterminal Â DNA-­âbinding Â domain Â (Kaplan Â et Â al., Â 2006). Â Both Â transcription Â factors Â activate Â gene Â expression Â in Â iron-­âdeficient Â conditions, Â presumably Â via Â a Â similar Â mechanism. Â The Â sets Â of Â genes Â controlled Â by Â Aft1 Â and Â Aft2 Â are Â partially Â overlapping, Â and Â the Â role Â of Â Aft2 Â seems Â to Â be Â more Â significant Â in Â the Â absence Â of Â Aft1 Â (P. Â L. Â Blaiseau Â et Â al., Â 2001, Â J. Â C. Â Rutherford Â et Â al., Â 2001). Â Â The Â Aft1 Â and Â Aft2 Â proteins Â are Â located Â in Â the Â cytosol Â under Â iron-­âreplete Â conditions Â and Â move Â to Â the Â nucleus Â under Â iron-­âdepleted Â conditions, Â where Â they Â activate Â the Â expression Â of Â the Â Fe-­âresponsive Â genes Â (M. Â Jbel Â et Â al., Â 2011, Â A. Â Mercier Â and Â S. Â Labbe, Â 2009). Â Â In Â addition Â to Â regulation Â at Â transcriptional Â level Â by Â Aft1/Aft1, Â iron Â homeostasis Â in Â yeast Â is Â also Â controlled Â by Â posttranscriptional Â mechanisms. Â Â Within Â the Â iron Â regulon Â mentioned Â earlier, Â there Â are Â genes Â encoding Â two Â mRNA-­âbinding Â (mRNA-­âdestabilizing) Â proteins, Â known Â as Â Cth1 Â and Â Cth2. Â Â These Â proteins Â are Â characterized Â by Â the Â presence Â of Â two Â zinc Â fingers Â (ZF). Â Â Cth1 Â is Â expressed Â in Â early Â stages Â of Â iron Â deficiency, Â whereas Â Cth2 Â is Â expressed Â during Â the Â persistence Â of Â iron Â limitation. Â Using Â their Â ZFs, Â Cth1 Â and Â Cth2 Â bind Â specific Â AU-­ârich Â elements Â (AREs) Â in Â the Â 3â²UTRs Â of Â mRNAs Â and Â modulate Â the Â 3â²-­âend Â processing Â of Â mRNAs Â to Â promote Â the Â degradation Â of Â target Â transcripts. Â Cth1 Â promotes Â the Â degradation Â of Â mRNAs Â encoding Â proteins Â that Â function Â in Â highly Â Fe-­âdependent Â processes, Â such Â as Â mitochondrial Â respiration, Â whereas Â Cth2 Â has Â a Â broader Â set Â of Â targets Â including Â functions Â of Â the Â tricarboxylic Â acid Â (TCA) Â cycle Â and Â respiration, Â lipid Â metabolism, Â heme Â synthesis, Â and Â amino Â acid Â biosynthesis Â (M. Â Martinez-­âPastor Â et Â al., Â 2013, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 15 Â S. Â Puig Â et Â al., Â 2008). Â In Â addition Â to Â transcriptional Â and Â post-­âtranscriptional Â levels Â mentioned Â above, Â iron Â homeostasis Â in Â yeast Â is Â further Â controlled Â at Â a Â post-­âtranslation Â level. Â It Â has Â been Â shown Â that Â the Â transcriptional Â activity Â of Â Aft1 Â is Â further Â regulated Â by Â and Â requires Â the Â two Â yeast Â cytosolic Â monothiol Â glutaredoxins Â (Grxs), Â ScGrx3 Â and Â ScGrx4. Â Â ScGrx3 Â and Â ScGrx4 Â maintain Â mitochondrial Â FeâS Â cluster Â biosynthesis Â via Â the Â aminopeptidase Â P-­âlike Â proteins Â Fra1 Â (Fe-­ârepressor Â of Â activation-­â1) Â and Â Fra2 Â (Fe-­ârepressor Â of Â activation-­â2 Â (A. Â Kumanovics, Â et Â al., Â 2008, Â L. Â Ojeda Â et Â al., Â 2006, Â N. Â Pujol-­âCarrion Â et Â al., Â 2006, Â J. Â C. Â Rutherford Â et Â al., Â 2005). Â Â Â 1.3.2 Â Â Iron Â homeostasis Â in Â S. Â pombe Â Â Â To Â achieve Â iron Â homeostasis Â in Â S. Â pombe, Â iron-­âresponsive Â gene Â expression Â is Â controlled Â by Â two Â repressor-­âtype Â regulators Â Fep1 Â and Â Php4. Â Specifically, Â Fep1 Â and Â Php4 Â are Â responsible Â for Â controlling Â iron Â acquisition Â and Â iron Â utilization, Â respectively Â (Kaplan Â et Â al., Â 2006, Â S. Â Labbe, Â et Â al., Â 2007, Â C. Â H. Â Lillig Â et Â al., Â 2008, Â P. Â A. Â Lindahl Â and Â G. Â P. Â Holmes-­âHampton, Â 2011). Â Fep1 Â is Â a Â GATA-­âtype Â transcription Â factor Â that Â binds Â to Â GATA-­âcontaining Â sequences Â in Â promoters Â of Â iron Â uptake Â and Â transport Â genes Â under Â iron-­âreplete Â conditions, Â repressing Â their Â expression Â to Â avoid Â iron Â overload. Â Â Â Fep1 Â orthologs Â are Â well Â conserved Â across Â various Â fungal Â species. Â Â Php4 Â is Â another Â arm Â of Â the Â maintenance Â system Â for Â iron Â homeostasis Â in Â S. Â pombe. Â Php4 Â is Â a Â transcriptional Â repressor Â and Â regulates Â expression Â of Â genes Â in Â iron-­âdependent Â metabolic Â pathways Â and Â forms Â a Â protein Â complex Â with Â Php2, Â Php3, Â and Â Php5 Â (M. Â G. Â Khan Â et Â al., Â 2014, Â A. Â Mercier Â and Â S. Â Labbe, Â 2009, Â A. Â Mercier Â et Â al., Â 2006, Â A. Â Mercier Â et Â al., Â 2008, Â P. Â Vachon Â et Â al., Â 2012). Â With Â sufficient Â levels Â of Â iron Â in Â the Â cell Â (i.e., Â iron-­âreplete Â conditions) Â Php4 Â is Â not Â expressed Â and Â the Â Php2/Php3/Php5 Â complex Â activates Â expression Â of Â its Â target Â Â Â Â Â Â Â Â Â Â Â Â Â Â 16 Â genes Â by Â binding Â to Â CCAAT Â sequences Â in Â their Â promoters. Â These Â target Â genes Â encode Â proteins Â involved Â in Â ironâsulfur Â cluster Â biogenesis, Â heme Â biosynthesis, Â the Â mitochondrial Â electron Â transport Â chain, Â and Â the Â TCA Â cycle. Â Under Â low-­âiron Â conditions, Â Php4 Â is Â expressed Â and Â binds Â to Â the Â Php2/Php3/Php5 Â complex, Â resulting Â in Â a Â switch Â of Â the Â complex Â from Â an Â activator Â to Â a Â repressor. Â Â This Â leads Â to Â down Â regulation Â of Â iron-­âutilizing Â pathways Â as Â an Â iron-­âsparing Â response Â to Â low Â levels Â of Â iron Â under Â iron-­âdepleted Â condition Â (A. Â Mercier Â and Â S. Â Labbe, Â 2009, Â A. Â Mercier, Â et Â al., Â 2006, Â A. Â Mercier, Â et Â al., Â 2008). Â 1.4 Â Â Monothiol Â glutaredoxins Â Â Â Monothiol Â Grxs Â with Â active-­âsite Â cysteine-­âglycine-­âphenylalanine-­âserine Â (CGFS) Â amino Â acid Â motifs Â are Â a Â family Â of Â proteins Â that Â are Â evolutionary Â conserved Â and Â found Â in Â organisms Â ranging Â from Â bacteria Â to Â humans. Â Monothiol Â Grxs Â possess Â specific Â structural Â domains Â as Â shown Â in Â Figure Â 1.4. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 17 Â Â Â Figure Â 1.4: Â Comparison Â of Â domain Â structures Â across Â species Â for Â monothiol Â glutaredoxins. Â Â The Â positions Â and Â sizes Â of Â glutaredoxin Â (red) Â and Â thioredoxin-­âlike Â (green) Â domains Â are Â indicated. Â Â Numbers Â represent Â the Â position Â of Â the Â cysteine Â residue Â in Â the Â active Â site Â CGFS Â motif, Â and Â the Â total Â lengths Â of Â the Â proteins Â (Adapted Â from Â Outten Â et Â al Â Biochemistry, Â 2012, Â 51 Â (22), Â pp Â 4377â4389). Â Â Â Â Â In Â general, Â this Â family Â of Â Grx Â proteins Â is Â divided Â into Â two Â subclasses. Â The Â first Â subclass Â includes Â those Â with Â a Â single Â Grx Â domain Â (e.g., Â E.coli Â (Ec) Â Grx4). Â The Â second Â subclass Â includes Â multi-­âdomain Â proteins Â comprised Â of Â an Â N-­âterminal Â thioredoxin-­âlike Â domain Â as Â well Â as Â one Â (Sc, Â Grx3 Â and Â Grx4), Â two Â (Hs, Â Grx3) Â or Â three Â (At, Â Grx3) Â Grx Â domains. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 18 Â Â Yeast Â Grx3 Â and Â Grx4 Â are Â highly Â homologous Â and Â are Â thought Â to Â possess Â redundant Â functions Â in Â the Â cell. Â Â A Â grx3Î Â grx4Î Â double Â mutant Â results Â in Â constitutive Â expression Â of Â the Â iron Â regulon Â and Â shows Â a Â significant Â growth Â defect Â (L. Â Ojeda, Â et Â al., Â 2006, Â N. Â Pujol-­âCarrion, Â et Â al., Â 2006). Â All Â bacterial Â monothiol Â CGFS Â Grxs Â are Â of Â the Â single Â Grx Â domain Â type, Â while Â in Â eukaryotes, Â both Â the Â single Â Grx Â domain Â and Â the Â multi-­âdomain Â Grxs Â are Â present Â (J. Â Couturier Â et Â al., Â 2009, Â E. Â Herrero Â and Â M. Â A. Â de Â la Â Torre-­âRuiz, Â 2007, Â F. Â Vilella Â et Â al., Â 2004). Â Â In Â addition Â to Â the Â three Â monothiol Â Grxs Â mentioned Â above, Â S. Â cerevisiae Â also Â possesses Â two Â other Â monothiol Â Grxs, Â ScGrx6 Â and Â ScGrx7. Â ScGrx6 Â is Â the Â first Â monothiol Â Grx Â shown Â to Â directly Â bind Â an Â iron-­âsulfur Â cluster Â that Â is Â stabilized Â by Â reduced Â glutathione Â (N. Â Mesecke Â et Â al., Â 2008). Â Â 1.4.1 Â Â Roles Â of Â monothiol Â Grxs Â Â Traditionally, Â Grxs Â function Â in Â the Â reduction Â of Â protein Â disulfides Â or Â the Â glutathionylation Â of Â proteins Â (J. Â Couturier, Â et Â al., Â 2009, Â E. Â Herrero Â and Â M. Â A. Â de Â la Â Torre-­âRuiz, Â 2007, Â C. Â H. Â Lillig, Â et Â al., Â 2008). Â Â However, Â recent Â biochemical Â characterization Â and Â in Â vivo Â studies Â indicate Â that Â monothiol Â CGFS Â Grxs Â have Â conserved Â roles Â in Â iron Â metabolism Â in Â multiple Â organisms, Â either Â through Â a Â role Â in Â the Â iron-­âsulphur Â cluster Â (ISC) Â assembly Â machinery Â or Â through Â the Â regulation Â of Â iron-­âresponsive Â transcription Â factors Â (F. Â Vilella, Â et Â al., Â 2004) Â (S. Â Bandyopadhyay Â et Â al., Â 2008a, Â Â Kaplan Â et Â al., Â 2006, Â H. Â Li Â and Â C. Â E. Â Outten, Â 2012, Â R. Â Lill Â et Â al., Â 2012, Â A. Â Mercier Â and Â S. Â Labbe, Â 2009, Â U. Â Muhlenhoff Â et Â al., Â 2010, Â N. Â Rouhier Â et Â al., Â 2010). Â Â 1.4.1.1 Â Role Â of Â monothiol Â Grx3/4 Â in Â regulation Â of Â Aft1 Â in Â yeast Â Â As Â mentioned Â above, Â under Â iron Â deficient Â conditions, Â Aft1 Â accumulates Â in Â the Â nucleus, Â and Â under Â iron-­âreplete Â conditions, Â Aft1 Â is Â shuttled Â to Â the Â cytosol Â by Â the Â nuclear Â Â Â Â Â Â Â Â Â Â Â Â Â Â 19 Â exportin Â Msn5 Â (R. Â Ueta Â et Â al., Â 2007, Â Y. Â Yamaguchi-­âIwai, Â et Â al., Â 1995, Â Y. Â Yamaguchi-­âIwai, Â et Â al., Â 2002). Â Â It Â is Â known Â that Â Fe-­âS-­âligated Â Grx3/4 Â promotes Â dissociation Â of Â Aft1 Â from Â its Â DNA Â target Â and Â Grx3/4 Â proteins Â are Â required Â to Â control Â the Â activity Â of Â Aft1 Â (R. Â Ueta Â et Â al., Â 2012). Â Â Briefly, Â Grx3 Â and Â Grx4 Â form Â [2Fe-­â2S] Â cluster-­âbinding Â complexes Â that, Â together Â with Â the Â cytosolic Â proteins Â Fra1 Â and Â Fra2, Â transmit Â an Â inhibitory Â signal Â to Â Aft1 Â that Â is Â dependent Â on Â the Â presence Â of Â glutathione Â and Â the Â synthesis Â of Â mitochondrial Â Fe-­âS Â clusters Â (A. Â Kumanovics, Â et Â al., Â 2008, Â H. Â Li Â et Â al., Â 2011, Â H. Â Li Â et Â al., Â 2009, Â H. Â Li Â and Â C. Â E. Â Outten, Â 2012, Â U. Â Muhlenhoff, Â et Â al., Â 2010, Â J. Â C. Â Rutherford, Â et Â al., Â 2005). Â Moreover, Â it Â has Â been Â shown Â that Â GSH Â depletion Â in Â S. Â cerevisiae Â leads Â to Â manifestation Â of Â similar Â phenotypes Â to Â those Â associated Â with Â loss Â of Â iron-­âdependent Â inhibition Â of Â Aft1/Aft2 Â (J. Â C. Â Rutherford, Â et Â al., Â 2005, Â K. Â Sipos Â et Â al., Â 2002). Â Â 1.4.1.2 Â Role Â of Â monothiol Â Grx4 Â in Â the Â regulation Â of Â Fep1 Â in Â fission Â yeast Â The Â fission Â yeast Â S. Â pombe Â contains Â two Â CGFS-­âtype Â monothiol Â glutaredoxins, Â Grx4 Â and Â Grx5, Â which Â are Â localized Â in Â the Â nucleus Â and Â mitochondria, Â respectively. Â Â Â Grx4 Â is Â required Â for Â iron Â limitation-­âdependent Â inhibition Â of Â Fep1 Â (M. Â Jbel, Â et Â al., Â 2011, Â K. Â D. Â Kim Â et Â al., Â 2011). Â Â Lack Â of Â Grx4 Â caused Â defects Â not Â only Â in Â growth Â but Â also Â in Â the Â expression Â of Â both Â iron-­âuptake Â and Â iron-­âutilizing Â genes Â regardless Â of Â iron Â availability. Â In Â order Â to Â identify Â the Â mechanisms Â by Â which Â Grx4 Â is Â involved Â in Â Fep1-­âmediated Â regulation, Â the Â protein-­âprotein Â interactions Â for Â these Â proteins Â were Â investigated. Â Co-­âimmunoprecipitation Â and Â bimolecular Â fluorescence Â complementation Â (BiFC) Â revealed Â that Â Grx4 Â physically Â interacts Â with Â Fep1 Â in Â vivo. Â Â BiFC Â identified Â punctate Â signals Â in Â the Â nucleus Â that Â were Â produced Â by Â the Â interaction Â of Â Grx4 Â with Â Fep1. Â Â Based Â on Â comparisons Â with Â S. Â cerevisiae, Â it Â has Â been Â suggested Â that Â Grx4 Â most Â likely Â binds Â Fe-­âS Â cofactors Â and Â could Â be Â critical Â in Â Â Â Â Â Â Â Â Â Â Â Â Â Â 20 Â interacting Â with Â Fep1 Â to Â modulate Â its Â activity. Â Â 1.4.1.3 Â Role Â of Â monothiol Â Grx4 Â in Â regulation Â of Â Php4 Â in Â fission Â yeast Â Â Monothiol Â Grx4 Â regulates Â the Â activity Â of Â Php4 Â in Â S. Â pombe Â through Â specific Â proteinâ Â protein Â interactions. Â Yeast Â two-­âhybrid Â and Â bimolecular Â fluorescence Â complementation Â experiments Â have Â shown Â that Â Grx4 Â physically Â interacts Â with Â Php4 Â regardless Â of Â cellular Â iron Â levels, Â and Â under Â iron-­âreplete Â conditions, Â Grx4 Â promotes Â the Â export Â of Â Php4 Â to Â the Â cytosol. Â Â It Â has Â been Â shown Â that Â deletion Â of Â GRX4 Â leads Â to Â constitutive Â nuclear Â localization Â of Â Php4 Â and Â constitutive Â repression Â of Â Php4-­âregulated Â genes Â (M. Â A. Â Huynen Â et Â al., Â 2005, Â M. Â Jbel, Â et Â al., Â 2011, Â K. Â D. Â Kim, Â et Â al., Â 2011). Â Similar Â to Â scenarios Â discussed Â above Â for Â Aft1 Â and Â Fep1, Â its Â been Â shown Â that Â Fe-­âS Â binding Â via Â multidomain Â CGFS Â Grx4 Â is Â an Â important Â factor Â required Â for Â inhibition Â of Â Php4, Â as Â mutation Â of Â the Â CGFS Â motif Â in Â Grx4 Â disrupts Â Fe-­âdependent Â inhibition Â of Â Php4. Â In Â addition, Â and Â similar Â to Â S. Â cerevisiae, Â depletion Â of Â glutathione Â leads Â to Â iron-­âdependent Â inhibition Â of Â Php4, Â most Â probably Â stemming Â from Â the Â requirement Â for Â GSH Â as Â a Â [2Fe-­â2S] Â cluster Â ligand Â in Â CGFS Â Grx Â homodimers Â (H. Â Li Â and Â C. Â E. Â Outten, Â 2012, Â R. Â Lill, Â et Â al., Â 2012). Â 1.4.1.4 Â Monothiol Â CGFS Â Grxs Â contain Â a Â labile Â FeâS Â cluster Â Fe-­âS Â clusters Â are Â cofactors Â that Â play Â important Â roles Â in Â cellular Â functions Â such Â as Â redox Â reactions, Â electron Â transport, Â and Â regulatory Â reactions. Â Â The Â synthesis Â of Â Fe-­âS Â clusters Â occurs Â exclusively Â at Â the Â mitochondrial Â matrix, Â independently Â of Â whether Â the Â final Â destination Â of Â the Â Fe-­âS Â clusters Â is Â to Â mitochondria, Â nuclear Â or Â cytosolic Â proteins. Â Â Â After Â synthesis, Â the Â clusters Â can Â be Â exported Â from Â the Â mitochondria, Â and Â additional Â iron-­âsulphur Â cluster Â assembly Â proteins Â (CIA Â machinery) Â are Â required Â to Â assemble Â Fe-­âS Â clusters Â into Â apoproteins Â in Â the Â cytosol Â or Â the Â nucleus Â (R. Â Lill Â et Â al., Â 2006, Â R. Â Lill Â and Â U. Â Muhlenhoff, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 21 Â 2006, Â 2005, Â T. Â A. Â Rouault Â and Â W. Â H. Â Tong, Â 2005). Â Â Recent Â studies Â indicate Â that Â the Â recombinant Â versions Â of Â bacterial, Â human, Â yeast Â and Â plant Â monothiol Â Grxs Â (produced Â in Â E. Â coli) Â are Â able Â to Â incorporate Â labile Â FeâS Â clusters Â (S. Â Bandyopadhyay Â et Â al., Â 2008b, Â M. Â M. Â Molina-­âNavarro Â et Â al., Â 2006, Â B. Â Zhang Â et Â al., Â 2013). Â Analytical Â and Â spectroscopic Â analyses Â of Â recombinant Â purified Â Grxs Â under Â anaerobic Â conditions Â demonstrated Â the Â incorporation Â of Â labile Â [Fe2S2]2+ Â clusters Â in Â the Â presence Â of Â glutathione Â (GSH) Â (S. Â Bandyopadhyay, Â et Â al., Â 2008b, Â T. Â Iwema Â et Â al., Â 2009, Â A. Â Picciocchi Â et Â al., Â 2007, Â N. Â Rouhier Â et Â al., Â 2007). Â Â As Â introduced Â above, Â the Â role Â of Â Grx3/4 Â in Â the Â regulation Â of Â Aft1 Â in Â yeast Â is Â mediated Â by Â the Â ability Â of Â monothiol Â Grxs Â to Â provide Â a Â bridging Â and Â glutathione-­âcontaining Â Fe/S Â center, Â that Â functions Â both Â as Â an Â iron Â sensor Â and Â in Â intracellular Â iron Â delivery Â (U. Â Muhlenhoff, Â et Â al., Â 2010, Â L. Â Ojeda, Â et Â al., Â 2006). Â It Â has Â also Â been Â shown Â that Â GSH Â depletion Â in Â yeast Â leads Â to Â constitutive Â activation Â of Â Aft1 Â and Â drastic Â intracellular Â iron Â accumulation Â which Â causes Â toxicity Â and Â cell Â death Â (J. Â C. Â Rutherford, Â et Â al., Â 2005). Â 1.5 Â Â Glutathione Â Â The Â tripeptide, Â Ï-­âL-­âGlutamyl-­âL-­âcysteinyl-­âglycine, Â or Â glutathione Â (GSH) Â is Â the Â major Â low-­âmolecular-­âweight Â thiol Â compound Â found Â in Â almost Â all Â organisms Â including Â plants, Â animals Â and Â most Â pathogens Â (A. Â Meister, Â 1984). Â GSH Â is Â synthesized Â from Â L-­âglutamate, Â L-­âcysteine, Â and Â glycine Â in Â two Â steps, Â catalyzed Â by Â Ï-­âglutamyl-­âcysteine Â synthase Â and Â glutathione Â synthase Â (S. Â C. Â Lu, Â 2009). Â The Â peptidic Â Ï-­âlinkage Â is Â known Â to Â protect Â GSH Â tripeptide Â against Â degradation Â by Â aminopeptidases. Â In Â addition Â to Â the Â reduced Â form, Â GSH Â is Â present Â in Â several Â other Â forms. Â Glutathione Â disulfide Â (GSSG) Â is Â formed Â upon Â oxidation, Â which Â is Â often Â referred Â to Â as Â âoxidized Â glutathione,â Â or Â âglutathione Â disulphideâ. Â Â Other Â Â Â Â Â Â Â Â Â Â Â Â Â Â 22 Â forms Â of Â disulfide Â are Â of Â the Â mixed Â type, Â for Â instance Â GSSR, Â is Â a Â major Â class Â of Â glutathione-­âcysteinyl Â disulfides Â on Â proteins. Â This Â form Â of Â protein Â is Â referred Â to Â as Â âglutathionylatedâ Â or Â âthiolatedâ Â (S. Â M. Â Beer Â et Â al., Â 2004, Â K. Â P. Â Huang Â and Â F. Â L. Â Huang, Â 2002, Â C. Â Luchese Â and Â C. Â W. Â Nogueira, Â 2010, Â Z. Â D. Â Zhou Â and Â T. Â M. Â Lim, Â 2010). Â Â 1.5.1 Â Â Roles Â of Â glutathione Â Â Â GSH Â has Â a Â high Â cellular Â abundance Â (3 Â -­â Â 10 Â mM) Â and Â a Â low Â redox Â potential Â of Â â240 Â mV. Â These Â properties Â give Â GSH Â the Â special Â attribute Â of Â serving Â as Â a Â redox Â buffer Â that Â is Â important Â for Â reducing Â oxidized Â cysteine Â residues Â and Â also Â for Â protecting Â them Â from Â irreversible Â oxidation Â (F. Â Q. Â Schafer Â and Â G. Â R. Â Buettner, Â 2001). Â Â All Â major Â cellular Â functions Â involve Â the Â thiol Â redox Â state Â (D. Â Trachootham Â et Â al., Â 2008). Â GSH Â also Â participates Â in Â scavenging Â various Â endogenous Â and Â exogenous Â electrophilic Â compounds Â (C. Â M. Â Grant Â et Â al., Â 1999, Â A. Â Meister Â and Â M. Â E. Â Anderson, Â 1983, Â M. Â D. Â Shelton Â and Â J. Â J. Â Mieyal, Â 2008). Â Â Â Since Â the Â discovery Â of Â GSH Â in Â 1889, Â the Â focus Â on Â GSH Â has Â increased Â in Â the Â last Â three Â decades Â and Â numerous Â functions Â of Â GSH Â have Â been Â elucidated Â (N. Â S. Â Kosower Â and Â E. Â M. Â Kosower, Â 1978). Â These Â include Â its Â biochemical Â properties, Â (H. Â F. Â Edelhauser Â et Â al., Â 1976, Â F. Â J. Â Giblin Â et Â al., Â 1976, Â W. Â A. Â Gunzler Â et Â al., Â 1974, Â J. Â Yam Â et Â al., Â 1976), Â and Â its Â significance Â in Â medical Â and Â clinical Â aspects, Â including Â carcinogenesis Â and Â drug Â resistance Â (A. Â P. Â Fernandes Â and Â A. Â Holmgren, Â 2004, Â J. Â D. Â Hayes Â and Â C. Â R. Â Wolf, Â 1990, Â S. Â Orrenius Â et Â al., Â 1983, Â B. Â Pool-­âZobel Â et Â al., Â 2005, Â J. Â Vina Â et Â al., Â 1995a, Â J. Â Vina Â et Â al., Â 1995b). Â Â Â Current Â active Â fields Â of Â research Â reveal Â roles Â of Â GSH Â in Â signal Â transduction, Â gene Â expression, Â apoptosis, Â glutathione-­âprotein Â interactions, Â and Â cell Â proliferation Â (A. Â P. Â Arrigo, Â 1999, Â I. Â A. Â Cotgreave Â and Â R. Â G. Â Gerdes, Â 1998, Â W. Â Droge Â et Â al., Â 1998, Â K. Â Gohil Â et Â al., Â 1999, Â C. Â Kretz-­âRemy Â and Â A. Â P. Â Arrigo, Â 2002, Â D. Â W. Â Voehringer, Â 1999). Â One Â recent Â topic Â in Â GSH Â Â Â Â Â Â Â Â Â Â Â Â Â Â 23 Â research, Â which Â is Â also Â developing Â at Â a Â fast Â pace, Â is Â âprotein Â glutathionylationâ Â which Â has Â been Â suggested Â to Â have Â a Â major Â role Â in Â control Â of Â the Â functions Â mentioned Â earlier. Â Â For Â instance, Â phosphorylase, Â carbonic Â anhydrase, Â glyceraldehyde Â 3-­âphosphate Â dehydrogenase Â (GAPDH), Â and Â other Â proteins Â have Â been Â shown Â to Â undergo Â glutathionylation Â (Y. Â C. Â Chai Â et Â al., Â 1991, Â R. Â M. Â Miller Â et Â al., Â 1990, Â J. Â A. Â Thomas Â et Â al., Â 1994). Â Â It Â has Â also Â been Â shown Â that Â Human Â immunodeficiency Â virus Â (HIV)-­â1 Â protease Â activity Â is Â regulated Â by Â reversible Â glutathionylation Â (D. Â A. Â Davis Â et Â al., Â 1996), Â and Â that Â GSH Â levels Â determines Â the Â outcome Â of Â the Â disease Â in Â HIV Â patients Â (L. Â A. Â Herzenberg Â et Â al., Â 1997, Â J. Â D. Â Peterson Â et Â al., Â 1998). Â 1.5.1.1 Â Â A Â vital Â function Â of Â glutathione Â in Â iron Â homeostasis Â One Â recent Â research Â area Â for Â GSH, Â which Â has Â expanded Â rapidly Â in Â the Â last Â 10 Â years, Â and Â is Â also Â challenging Â the Â traditional Â view Â of Â âGSHâ Â as Â the Â main Â player Â in Â thiol-­âredox Â control, Â is Â the Â critical Â role Â of Â GSH Â in Â iron Â homeostasis Â (C. Â Kumar Â et Â al., Â 2011). Â It Â has Â been Â shown Â that Â GSH Â serves Â as Â a Â point Â of Â intersection Â between Â iron Â metabolism Â and Â thiol-­âredox Â maintenance, Â and Â partners Â with Â monothiol Â Grxs Â to Â assemble Â FeâS Â clusters Â (H. Â Li, Â et Â al., Â 2011, Â H. Â Li, Â et Â al., Â 2009, Â H. Â Li Â and Â C. Â E. Â Outten, Â 2012, Â D. Â T. Â Mapolelo Â et Â al., Â 2013). Â GSH Â is Â also Â a Â major Â component Â of Â the Â labile Â iron Â pool Â (LIP) Â of Â available Â iron Â in Â cells, Â and Â this Â adds Â significance Â to Â the Â role Â of Â GSH Â in Â iron Â homeostasis Â (R. Â C. Â Hider Â and Â X. Â L. Â Kong, Â 2011). Â Â In Â this Â regard, Â GSH Â is Â a Â major Â cytosolic Â ligand Â for Â Fe Â (II). Â Â In Â eukaryotes, Â the Â majority Â of Â iron Â taken Â up Â by Â transporters Â and Â iron Â acquisition Â components Â enters Â a Â metabolically Â active Â pool Â in Â the Â cytosol Â known Â as Â the Â labile Â iron Â pool Â (LIP) Â (M. Â Shvartsman Â and Â Z. Â Ioav Â Cabantchik, Â 2012). Â Â The Â properties Â and Â features Â of Â this Â cytosolic Â pool Â are Â not Â completely Â understood, Â but Â the Â LIP Â is Â defined Â as Â the Â freely Â exchangeable Â iron Â that Â is Â loosely Â coordinated Â by Â water, Â small Â molecules, Â and Â proteins. Â Â Because Â the Â cytosolic Â LIP Â is Â Â Â Â Â Â Â Â Â Â Â Â Â Â 24 Â essentially Â present Â as Â Fe Â (II), Â the Â physiologic Â ligands Â for Â the Â cytosolic Â LIP Â must Â have Â affinity Â for Â this Â form Â of Â iron. Â Â Analysis Â of Â potential Â ligands Â in Â vitro Â suggests Â that Â FeâGSH Â complexes Â are Â likely Â the Â only Â species Â that Â forms Â at Â significant Â levels Â in Â cells, Â mainly Â due Â to Â the Â fact Â that Â GSH Â is Â present Â at Â relatively Â high Â levels Â in Â the Â cytosol Â and Â has Â moderate Â affinity Â (Kdâ¼8 Â μM) Â for Â Fe Â (II) Â (R. Â C. Â Hider Â and Â X. Â L. Â Kong, Â 2011, Â D. Â Kaur Â et Â al., Â 2009, Â C. Â Kumar, Â et Â al., Â 2011, Â R. Â Kumar, Â 1979). Â 1.5.1.1.1 Â Role Â of Â glutathione Â in Â iron Â homeostasis Â in Â fungi Â Â As Â mentioned Â in Â sections Â 1.4 Â and Â 1.5.1.1, Â the Â role Â of Â cytosolic Â monothiol Â glutaredoxins Â Grx3 Â and Â Grx4 Â in Â intracellular Â iron Â trafficking Â and Â sensing Â in Â yeast Â is Â well Â established Â and Â its Â been Â shown Â that Â depletion Â of Â Grx3/4 Â specifically Â impairs Â all Â iron-­ârequiring Â reactions Â in Â the Â cytosol, Â mitochondria, Â and Â nucleus, Â including Â the Â synthesis Â of Â Fe/S Â clusters Â and Â heme Â (U. Â Muhlenhoff, Â et Â al., Â 2010, Â Li Â et Â al., Â 2012). Â It Â has Â also Â been Â shown Â that Â GSH Â is Â required Â for Â maturation Â of Â cytosolic Â Fe-­âS Â clusters. Â (H. Â Li Â et Â al., Â 2012, Â K. Â Sipos, Â et Â al., Â 2002, Â U. Â Muhlenhoff, Â et Â al., Â 2010). Â Â Studies Â by Â Muhlenhoff Â et Â al., Â 2010 Â uncovered Â an Â important Â role Â of Â GSH Â in Â partnership Â with Â Grx3/4 Â in Â cellular Â iron Â metabolism Â in Â yeast. Â They Â showed Â that Â a Â crucial Â task Â of Â Grx3/4 Â is Â mediated Â by Â a Â bridging Â glutathione-­âcontaining Â Fe/S Â center Â that Â functions Â both Â as Â an Â iron Â sensor Â and Â in Â intracellular Â iron Â delivery. Â In Â fungi, Â recent Â studies Â have Â pr0-­âovided Â additional Â clues Â about Â the Â role Â of Â GSH Â (Rutherford Â et Â al., Â 2005 Â Mercier Â et Â al., Â 2008). Â Â It Â is Â known Â that Â the Â S. Â cerevisiae Â transcription Â factor Â Aft1 Â is Â activated Â by Â iron Â deficiency Â and Â inactivated Â by Â iron Â repletion, Â and Â it Â has Â been Â shown Â that Â the Â presence Â of Â GSH Â is Â required Â for Â iron-­âmediated Â inactivation Â of Â Aft1 Â function Â (J. Â C. Â Rutherford, Â et Â al., Â 2005). Â Â In Â fact, Â in Â GSH-­âdeficient Â cells, Â the Â S. Â cerevisiae Â iron Â regulon Â genes Â are Â constitutively Â upregulated Â and Â are Â less Â responsive Â to Â iron Â Â Â Â Â Â Â Â Â Â Â Â Â Â 25 Â repression Â (J. Â C. Â Rutherford, Â et Â al., Â 2005). Â Â Like Â Aft1, Â S. Â pombe Â Php4 Â is Â active Â during Â iron Â deficiency, Â except Â that Â it Â represses Â transcription. Â Â Â The Â significance Â of Â GSH Â in Â regulation Â of Â iron Â homeostasis Â is Â further Â underscored Â by Â the Â fact Â that Â Aft1 Â and Â Php4 Â are Â evolutionarily Â distant, Â but Â the Â presence Â of Â GSH Â is Â needed Â for Â iron-­âdependent Â inactivation Â of Â them Â both Â (W. Â H. Â Chung Â et Â al., Â 2005). Â Â As Â mentioned Â earlier, Â GSH Â is Â synthesized Â in Â two Â steps Â by Â Ï-­âglutamylcysteine Â synthetase Â (GCS) Â and Â glutathione Â synthetase. Â Null Â mutations Â of Â the Â genes Â encoding Â GCS Â in Â S. Â cerevisiae Â (GSH1) Â and Â S. Â pombe Â (gcs1) Â lead Â to Â GSH Â auxotrophy, Â indicating Â an Â essential Â requirement Â of Â GSH Â in Â these Â yeasts Â (B. Â Chaudhuri Â et Â al., Â 1997, Â C. Â M. Â Grant Â et Â al., Â 1996, Â D. Â Wu Â et Â al., Â 1994). Â It Â has Â also Â been Â shown Â that Â Ï-­âglutamylcysteine Â synthetase Â (GCS) Â is Â essential Â in Â Candida Â albicans, Â as Â the Â gcs1/gcs1 Â null Â mutants Â in Â this Â fungal Â pathogen Â also Â exhibit Â glutathione Â auxotrophy, Â which Â could Â be Â rescued Â by Â supplementing Â with Â reduced Â and Â oxidized Â glutathione Â and Â Ï Â âglutamylcysteine, Â indicating Â that Â glutathione Â biosynthesis Â (and Â glutathione Â itself) Â is Â essential Â C. Â albicans Â (Y. Â U. Â Baek Â et Â al., Â 2004). Â Â Additionally, Â dimorphic Â zoopathogenic Â fungi Â produce Â and Â secrete Â a Â family Â of Â iron Â reductases Â that Â use Â GSH Â as Â ligand Â and Â this Â complex Â is Â involved Â in Â the Â acquisition Â and Â utilization Â of Â iron. Â Briefly, Â it Â was Â shown Â that Â culture Â supernatants Â from Â Blastomyces Â dermatitidis, Â Histoplasma Â capsulatum, Â Paracoccidioides Â brasiliensis Â and Â Sporothrix Â schenckii Â strains Â grown Â in Â their Â yeast Â forms Â were Â able Â to Â reduce Â iron Â enzymatically, Â with Â GSH Â as Â a Â cofactor Â (R. Â Zarnowski Â and Â J. Â P. Â Woods, Â 2005) Â 1.6 Â Iron Â uptake Â in Â C. Â neoformans Â Â Similar Â to Â other Â fungi, Â including Â the Â model Â yeasts, Â filamentous Â fungi, Â and Â fungal Â pathogens, Â C. Â neoformans Â employs Â various Â strategies Â to Â acquire Â iron Â from Â the Â environment Â Â Â Â Â Â Â Â Â Â Â Â Â Â 26 Â or Â from Â the Â host. Â These Â strategies Â include Â cell Â surface Â reductases Â that Â reduce Â ferric Â iron Â to Â its Â ferrous Â state, Â a Â high-­âaffinity Â iron Â uptake Â pathway, Â a Â low-­âaffinity Â iron Â uptake Â pathway, Â the Â use Â of Â siderophore-­âbound Â iron Â and Â siderophore Â transporters, Â and Â the Â acquisition Â of Â iron Â from Â heme. Â C. Â neoformans Â also Â exports Â reductants Â such Â as Â 3-­âhydroxyanthranilic Â acid Â (Figure Â 1.5). Â 1.6.1 Â Â The Â high-­âaffinity Â iron Â uptake Â pathway Â Â Â The Â high-­âaffinity Â ferroxidase Â (Fet3)/permease Â (Ftr1) Â complex Â has Â been Â characterized Â in Â several Â fungi, Â including Â S. Â cerevisiae Â and Â C. Â albicans Â (R. Â Stearman, Â et Â al., Â 1996), Â (R. Â S. Â Almeida Â et Â al., Â 2008, Â J. Â Kaplan Â et Â al., Â 2006, Â S. Â A. Â Knight Â et Â al., Â 2005, Â D. Â J. Â Kosman, Â 2003, Â N. Â Ramanan Â and Â Y. Â Wang, Â 2000). Â Briefly, Â ferrous Â iron Â is Â first Â oxidized Â by Â the Â ferroxidase Â Fet3 Â before Â transport Â into Â the Â cytosol Â as Â ferric Â ion Â by Â the Â permease Â Ftr1. Â In Â C. Â neoformans, Â the Â Fet3 Â and Â Ftr1 Â orthologs Â Cfo1 Â and Â Cft1 Â are Â essential Â for Â ferric Â iron Â uptake, Â iron Â acquisition Â from Â transferrin, Â and Â full Â virulence Â in Â mice Â (W. Â H. Â Jung Â et Â al., Â 2008, Â W. Â H. Â Jung Â et Â al., Â 2009). Â Â Â It Â has Â also Â been Â shown Â that Â the Â iron Â permease/ferroxidase Â complex Â is Â induced Â by Â low Â levels Â of Â iron Â (T. Â Lian, Â et Â al., Â 2005) Â and Â is Â localized Â at Â the Â plasma Â membrane Â (E. Â S. Â Jacobson Â and Â J. Â D. Â Hong, Â 1997, Â K. Â J. Â Nyhus Â et Â al., Â 1997) Â (W. Â H. Â Jung, Â et Â al., Â 2008) Â (W. Â H. Â Jung, Â et Â al., Â 2009). Â 1.6.2 Â Â Low-­âaffinity Â iron Â uptake Â pathway Â Â As Â mentioned Â above, Â iron Â uptake Â relies Â on Â the Â high-­âaffinity Â uptake Â system Â in Â conditions Â of Â low Â iron Â concentration Â while Â low-­âaffinity Â uptake Â systems Â are Â used Â when Â substrate Â is Â present Â in Â high Â concentrations. Â Â A Â low-­âaffinity Â iron Â uptake Â pathway Â has Â been Â described Â in Â S. Â cerevisiae Â (C. Â C. Â Philpott, Â 2006, Â R. Â Sutak Â et Â al., Â 2008). Â Fet4 Â is Â described Â as Â a Â Â Â Â Â Â Â Â Â Â Â Â Â Â 27 Â non-­âATP-­âdependent Â trans-­âmembrane Â transporter, Â and Â the Â main Â component Â of Â the Â pathway Â (R. Â M. Â Dixon Â et Â al., Â 1994). Â The Â permease Â component Â of Â the Â low-­âaffinity Â iron Â uptake Â are Â not Â specific Â to Â iron Â and Â take Â up Â other Â metals Â such Â as Â copper Â and Â zinc Â (Kaplan Â et Â al., Â 2006). Â Â Similar Â to Â S. Â cerevisiae Â in Â the Â presence Â of Â sufficient Â levels Â of Â iron, Â iron Â uptake Â is Â mediated Â by Â the Â low-­âaffinity Â iron Â uptake Â pathway Â in Â C. Â neoformans Â (E. Â S. Â Jacobson Â et Â al., Â 1998). Â This Â system Â employs Â reductants, Â such Â as Â melanin, Â 3-­âhydroxyanthranilic Â acid Â (3HAA), Â and Â transporters. Â Â The Â exact Â mechanism Â of Â the Â iron Â uptake Â pathway Â is Â not Â fully Â known Â (Jacobson Â and Â Hong, Â 1997). Â 1.6.3 Â Â Siderophore-­âbound Â iron Â transport Â Â The Â siderophore Â non-­âreductive Â iron Â uptake Â pathway Â was Â first Â described Â in Â S. Â cerevisiae Â and Â several Â fungal Â species Â (C. Â C. Â Philpott, Â 2006) Â (P. Â Heymann Â et Â al., Â 2002) Â (E. Â Lesuisse Â et Â al., Â 2002). Â The Â siderophore-­âiron Â complexes Â are Â transported Â into Â the Â cell Â via Â siderophore Â transporters Â (Sit). Â S. Â cerevisiae Â possesses Â four Â siderophore Â transporters Â and Â each Â transports Â a Â specific Â kind Â of Â siderophore Â (D. Â J. Â Kosman, Â 2003). Â The Â mechanisms Â of Â iron Â acquisition Â are Â generally Â conserved Â among Â fungal Â pathogens Â although Â there Â is Â variability Â in Â the Â dependence Â on Â siderophores Â and Â the Â ability Â to Â use Â heme Â as Â an Â iron Â source Â (R. Â S. Â Almeida Â et Â al., Â 2009, Â H. Â Haas, Â 2012, Â D. Â Kornitzer, Â 2009). Â C. Â neoformans Â does Â not Â produce Â siderophores Â but Â it Â does Â possess Â several Â predicted Â siderophore Â transporters. Â Â Siderophore Â transporter Â 1 Â (Sit1) Â has Â been Â characterized Â as Â the Â specific Â transporter Â for Â the Â siderophore Â ferrioxamine Â B Â (K. Â L. Â Tangen Â et Â al., Â 2007). Â Â Loss Â of Â Sit1 Â results Â in Â poor Â growth Â under Â iron-­âlimiting Â conditions Â and Â increased Â melanin Â production; Â however, Â Sit1 Â is Â not Â required Â for Â virulence Â in Â a Â mouse Â model Â of Â cryptococcosis Â (K. Â L. Â Tangen, Â et Â al., Â 2007). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 28 Â 1.6.4 Â Â Heme Â uptake Â Â Â Pathogenic Â fungi Â utilize Â heme Â as Â an Â iron Â source Â (R. Â Santos Â et Â al., Â 2003) Â (L. Â A. Â Foster, Â 2002). Â The Â majority Â of Â iron Â in Â human Â body Â is Â present Â in Â heme Â and Â so Â this Â source Â may Â be Â particularly Â important Â for Â C. Â neoformans. Â It Â has Â been Â shown Â that Â C. Â neoformans Â can Â also Â utilize Â iron Â from Â heme Â (Jung Â et Â al., Â 2008), Â and Â that Â the Â ESCRT Â protein Â Vps23 Â plays Â a Â role Â in Â heme Â acquisition. Â Â Deletion Â of Â VPS23 Â resulted Â in Â a Â growth Â defect Â on Â heme, Â a Â capsule Â defect Â and Â attenuation Â of Â virulence Â in Â a Â mouse Â model Â (G. Â Hu Â et Â al., Â 2013). Â The Â mannoprotein Â Cig1 Â also Â plays Â an Â important Â role Â in Â iron Â acquisition Â from Â heme Â in Â C. Â neoformans. Â The Â transcript Â for Â the Â extracellular Â mannoprotein Â Cig1 Â is Â highly Â unregulated Â in Â low Â levels Â of Â iron Â and Â Cig1 Â is Â required Â for Â growth Â on Â heme, Â suggesting Â that Â Cig1 Â may Â function Â as Â a Â hemophore Â at Â the Â cell Â surface, Â suggesting Â that Â Cig1 Â may Â therefore Â function Â as Â a Â hemophore Â at Â the Â cell Â surface. Â Cig1 Â contributed Â to Â virulence Â in Â a Â mouse Â model Â of Â cryptococcosis Â but Â only Â in Â a Â mutant Â that Â also Â lacked Â the Â high-­âaffinity Â iron Â uptake Â system Â B. Â Cadieux, Â et Â al. Â (2013). Â 1.6.5 Â Â Cir1: Â Master Â regulator Â of Â iron Â Â Â The Â GATA-­âtype Â transcription Â factor Â Cryptococcus Â Iron Â Regulator Â 1 Â (Cir1) Â is Â a Â key Â regulator Â of Â the Â transcriptional Â response Â to Â iron Â in Â C. Â neoformans. Â Cir1 Â was Â identified Â based Â on Â sequence Â similarity Â to Â the Â iron Â regulators Â in Â other Â fungi Â (W. Â H. Â Jung Â et Â al., Â 2006). Â Urbs1 Â from Â Ustilago Â maydis, Â SRE Â in Â Neurospora Â crassa, Â and Â Fep1 Â in Â S. Â pombe Â had Â been Â identified Â and Â the Â list Â of Â related Â proteins Â now Â includes Â Sfu1 Â in Â C. Â albicans, Â SREB Â in Â H. Â capsulatum, Â and Â Sre1 Â in Â B. Â dermatitidis Â (Z. Â An Â et Â al., Â 1997b, Â L. Â Y. Â Chao Â et Â al., Â 2008, Â G. Â M. Â Gauthier Â et Â al., Â 2010, Â K. Â A. Â Harrison Â and Â G. Â A. Â Marzluf, Â 2002, Â C. Â Y. Â Lan Â et Â al., Â 2004, Â B. Â Pelletier Â et Â al., Â 2002). Â These Â transcription Â factors Â possess Â a Â conserved Â cysteine-­ârich Â region Â (CRR) Â flanked Â by Â two Â zinc Â finger Â motifs Â characteristic Â of Â GATA-­âtype Â transcription Â factors Â Â Â Â Â Â Â Â Â Â Â Â Â Â 29 Â in Â higher Â eukaryotes. Â For Â instance Â Fep1 Â and Â Urbs1 Â bind Â 5â-­âGATA-­â3â Â sequences Â in Â the Â promoters Â of Â genes Â encoding Â high-­âaffinity Â iron Â uptake Â system Â as Â well Â as Â siderophore Â transport Â functions Â (Z. Â An Â et Â al., Â 1997a, Â B. Â Pelletier, Â et Â al., Â 2003). Â The Â CRR Â has Â been Â demonstrated Â to Â coordinate Â iron Â binding, Â affecting, Â for Â example, Â the Â DNA Â binding Â affinity Â of Â the Â SRE1 Â transcription Â factor Â of Â H. Â capsulatum Â (L. Â Y. Â Chao, Â et Â al., Â 2008) Â and Â in Â S. Â pombe; Â mutation Â of Â CRR Â cysteine Â residues Â of Â the Â Fep1 Â protein Â leads Â to Â increased Â mRNA Â levels Â of Â its Â target Â gene, Â the Â multicopper Â oxidase Â Fio1 Â (B. Â Pelletier Â et Â al., Â 2005). Â Â Â Â The Â major Â impact Â of Â Cir1 Â was Â demonstrated Â by Â a Â comparison Â of Â the Â transcriptomes Â of Â the Â wild-­âtype Â (WT) Â strain Â and Â the Â cir1 Â mutant Â that Â revealed Â differential Â regulation Â by Â Cir1 Â of Â 2,311 Â genes Â under Â the Â iron-­âlimited Â condition Â and Â 1,623 Â genes Â upon Â iron Â repletion Â (out Â of Â ~6500 Â genes Â in Â the Â genome). Â Â For Â example, Â Cir1 Â positively Â regulated Â genes Â for Â siderophore Â transport Â and Â negatively Â regulated Â genes Â for Â reductive Â iron Â uptake Â and Â melanin Â synthesis. Â Â Loss Â of Â Cir1 Â attenuated Â virulence Â and Â influenced Â the Â elaboration Â of Â all Â of Â the Â major Â virulence Â factors, Â including Â derepression Â of Â melanin Â production, Â and Â reduced Â capsule Â production Â and Â growth Â at Â 37°C Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2011, Â W. Â H. Â Jung, Â et Â al., Â 2006). Â Â Â Overall, Â Cir1 Â regulates Â iron Â homeostasis Â and Â virulence Â factor Â expression Â (Jung Â et Â al., Â 2006). Â Â A Â recent Â study Â indicates Â that Â Cir1 Â also Â influences Â metabolic Â processes Â that Â require Â iron-­âdependent Â enzymes Â such Â as Â glycolysis, Â ergosterol Â biosynthesis, Â and Â inositol Â metabolism Â (J. Â N. Â Choi Â et Â al., Â 2012). Â Â Â It Â is Â not Â clear Â how Â many Â of Â these Â functions Â are Â directly Â regulated Â by Â Cir1 Â and Â which Â involve Â downstream Â transcriptional Â regulators. Â It Â has Â been Â suggested Â that Â there Â is Â potentially Â a Â crosstalk Â between Â these Â multiple Â processes Â and Â iron Â homeostasis. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 30 Â Â Â Figure Â 1.5: Â Components Â of Â iron Â network Â in Â C. Â neoformans. Â High Â affinity Â uptake Â pathway Â that Â depends Â on Â ferric Â reductase Â activity, Â the Â iron Â permease Â Cft1 Â and Â the Â ferroxidase Â Cfo1 Â is Â highlighted Â in Â light Â blue, Â iron Â reductases Â (Fre1â8), Â and Â cell Â wall Â melanin Â (produced Â by Â the Â laccases Â Lac1 Â and Â Lac2) Â have Â ferric Â reducing Â activities Â along Â with Â (3-­âHAA). Â Six Â siderophore Â transporters Â are Â predicted Â from Â the Â genome, Â heme Â uptake Â system Â involves Â the Â mannoprotein Â Cig1, Â and Â ESCRT Â protein Â Vps23. Â Transcription Â factors Â such Â as Â Cir Â and Â HapX Â control Â the Â expression Â of Â these Â components. Â Â (Figure Â adapted Â from Â Kronstad Â et Â al. Â (http://dx.doi.org/10.1016/j.tim.2013.05.00) Â 1.7 Â Rationale Â and Â aims Â of Â study Â Â As Â mentioned Â in Â section Â 1.6, Â iron Â acquisition Â and Â the Â maintenance Â of Â iron Â Â Â Â Â Â Â Â Â Â Â Â Â Â 31 Â homeostasis Â are Â critical Â for Â the Â pathogenesis Â of Â C. Â neoformans. Â Â The Â GATA-­âtype, Â zinc-­âfinger Â protein Â Cir1 Â controls Â the Â iron Â regulon Â and Â the Â known Â major Â virulence Â factors Â in Â C. Â neoformans. Â Thus Â understanding Â the Â mechanisms Â by Â which Â Cir1 Â senses Â iron Â availability Â and Â contributes Â to Â proliferation Â of Â the Â fungus Â in Â mammalian Â hosts Â is Â of Â great Â significance. Â Â The Â studies Â reported Â in Â this Â thesis Â aimed Â to Â identify Â additional Â components Â of Â iron Â acquisition Â functions Â that Â are Â essential Â for Â C. Â neoformans Â growth Â to Â add Â depth Â to Â our Â understanding Â of Â the Â complex Â iron Â regulatory Â network. Â Â Importantly, Â the Â identification Â and Â characterization Â of Â components Â of Â the Â iron Â regulatory Â and Â uptake Â network Â in Â C. Â neoformans Â could Â serve Â as Â new Â targets Â for Â the Â design Â and Â development Â of Â more Â efficient Â and Â less Â toxic Â novel Â therapies Â for Â cryptococcosis. Â Â 1.7.1 Â Â Hypotheses Â Â Â Initially, Â I embarked on a project in which potential binding partners of Cir1 were identified using a binding assay and a proteomic approach. This preliminary proteomic analysis of binding partners for Cir1 identified a monothiol glutaredoxin (Grx) encoded by the gene CNAG_02950). Based on the conserved evolutionary functions of monothiol Grxs in Fe-S cluster binding and regulation of iron homeostasis, as detailed in section 1.4, I hypothesized that Grx4 plays a role in iron homeostasis and contributes to the pathogenesis of C. neoformans. Importantly, a role for monothiol Grxs has not been explored previously in pathogenic fungi. Â Â Furthermore, Â an Â important Â observation Â in Â Chapter Â 2 Â was Â that Â after Â 48 Â hours Â of Â iron Â starvation, Â a Â grx4 Â mutant Â had Â a Â growth Â defect Â in Â the Â presence Â of Â iron Â compared Â to Â WT. Â Â I Â therefore Â tested Â the Â effect Â of Â exogenous Â GSH Â on Â the Â growth Â defect Â of Â grx4 Â in Â the Â presence Â of Â iron Â after Â a Â period Â of Â iron Â starvation. Â Â Interestingly, Â the Â growth Â defect Â of Â the Â grx4 Â mutant Â was Â rescued Â by Â exogenous Â GSH. Â As Â detailed Â in Â section Â 1.5 Â of Â this Â chapter, Â it Â is Â known Â that Â Â Â Â Â Â Â Â Â Â Â Â Â Â 32 Â glutathione Â protects Â against Â detrimental Â effects Â of Â iron-­âdeficiency Â and Â iron Â toxicity Â due Â to Â its Â antioxidant Â properties. Â Â Also Â monothiol Â glutaredoxins Â require Â GSH Â to Â exert Â their Â function Â in Â the Â maintenance Â of Â iron Â homeostasis, Â and Â GSH Â is Â known Â as Â the Â major Â and Â essential Â component Â of Â iron Â binding Â ligand Â in Â cytosol Â and Â affect Â intracellular Â distribution Â of Â iron. Â These Â observations, Â in Â addition Â to Â the Â findings Â on Â the Â effect Â of Â GSH Â on Â the Â grx4 Â mutant Â outlined Â in Â Chapter Â 2, Â prompted Â me Â to Â hypothesize Â that Â glutathione Â influences Â iron Â homeostasis Â in Â C. Â neoformans. Â I Â examined Â the Â impact Â of Â deletion Â of Â the Â gene Â encoding Â glutathione Â synthetase Â in Â Chapter Â 3. Â Â The Â physiological Â effect Â of Â GSH Â has Â not Â been Â investigated Â in Â C. Â neoformans Â prior Â to Â my Â study. Â 1.7.2 Â Â Research Â objectives Â Â Â The Â general Â objectives Â of Â my Â PhD Â studies Â were Â to Â elucidate Â the Â role Â of Â Grx4 Â and Â effect Â of Â GSH Â in Â iron Â homeostasis Â of Â C. Â neoformans. Â Â The Â main Â objective Â in Â Chapter Â 2 Â was Â to Â examine Â the Â role Â of Â the Â monothiol Â glutaredoxin Â Grx4 Â in Â the Â iron Â homeostasis Â and Â virulence Â in Â C. Â neoformans. Â Â The Â specific Â goals Â were Â to Â examine Â the Â Grx4-­âCir1 Â interaction, Â analyze Â the Â impact Â of Â a Â GRX4 Â deletion Â on Â iron Â and Â virulence-­ârelated Â phenotypes Â and Â finally Â evaluate Â the Â impact Â of Â GRX4 Â on Â expression Â of Â known Â targets Â that Â regulate Â iron Â homeostasis Â in Â C. Â neoformans. Â Â I Â continued Â the Â analysis Â on Â Grx4 Â by Â investigating Â transcript Â variants Â and Â the Â essentiality Â of Â the Â GRX4 Â locus. Â Â The Â specific Â goal Â of Â this Â follow-­âup Â study Â was Â to Â examine Â the Â transcript(s) Â structure Â of Â GRX4 Â locus Â with Â N-­âterminal Â variations Â by Â RNA-­âSeq Â analysis, Â and Â to Â test Â the Â essentiality Â of Â N-­âterminal Â region Â of Â GRX4 Â locus. Â Â Â Â My Â objective Â in Â Chapter Â 3 Â was Â to Â investigate Â the Â physiological Â role Â of Â glutathione Â in Â in Â C. Â neoformans. Â Â The Â specific Â goal Â of Â this Â section Â was Â initially Â the Â identification Â and Â Â Â Â Â Â Â Â Â Â Â Â Â Â 33 Â subsequent Â deletion Â of Â candidate Â targets Â in Â the Â GSH Â biosynthesis Â pathway Â in Â C. Â neoformans. Â Â Subsequently, Â I Â focused Â on Â the Â GSH2 Â gene Â encoding Â glutathione Â synthetase Â with Â a Â detailed Â analysis Â of Â phenotypic Â outcomes Â manifested Â upon Â deletion Â of Â GSH2. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 34 Â Chapter Â 2: Â Monothiol Â glutaredoxin-­â4 Â is Â required Â for Â iron Â homeostasis Â and Â virulence Â in Â the Â AIDS-­âassociated Â pathogen Â Cryptococcus Â neoformans Â 2.1 Â Introduction Â Â Â Â Pathogenic Â microorganisms Â need Â to Â overcome Â nutritional Â barriers Â to Â proliferate Â in Â the Â hostile Â host Â environment Â (K. Â Fischer Â et Â al., Â 2004). Â For Â example, Â both Â vertebrate Â hosts Â and Â microbial Â pathogens Â require Â iron Â as Â an Â essential Â cofactor Â or Â a Â prosthetic Â group, Â either Â alone Â or Â bound Â to Â Fe-­âS Â clusters, Â in Â a Â plethora Â of Â cellular Â and Â metabolic Â functions. Â The Â dependence Â of Â both Â host Â and Â pathogen Â on Â iron Â leads Â to Â a Â competition Â for Â this Â essential Â nutrient. Â The Â host Â actively Â restricts Â the Â bioavailability Â of Â iron Â to Â the Â pathogen Â by Â iron-­âwithholding Â mechanisms, Â which Â is Â countered Â by Â microbial Â strategies Â to Â effectively Â compete Â for Â iron Â during Â infection Â (S. Â Brill Â et Â al., Â 2000, Â M. Â Caza Â and Â J. Â W. Â Kronstad, Â 2013). Â Â Â Â Â Iron Â is Â essential Â for Â cell Â viability Â as Â an Â important Â cofactor Â in Â a Â variety Â of Â enzymes Â that Â are Â linked Â to Â essential Â cellular Â functions Â including Â DNA Â synthesis, Â mitochondrial Â respiratory Â pathway Â and Â TCA Â cycle, Â oxidative Â phosphorylation, Â and Â the Â biosynthesis Â of Â metabolites. Â Ironically Â however, Â the Â same Â redox-­âactive Â properties Â of Â this Â metal Â make Â it Â cytotoxic Â because Â iron Â undergoes Â Fenton-­âtype Â chemical Â reactions Â that Â result Â in Â the Â production Â of Â hydroxyl Â radicals Â that Â can Â lead Â to Â cell Â death Â (M. Â W. Â Hentze Â et Â al., Â 2010, Â C. Â C. Â Philpott Â et Â al., Â 2012) Â (B. Â Halliwell Â and Â J. Â M. Â Gutteridge, Â 1992, Â J. Â A. Â Imlay, Â et Â al., Â 1988, Â J. Â A. Â Imlay Â and Â S. Â Linn, Â 1988). Â Therefore, Â cells Â have Â developed Â homeostatic Â mechanisms Â to Â acquire Â adequate, Â but Â not Â excessive, Â concentrations Â of Â iron. Â Optimal Â intracellular Â levels Â of Â iron Â are Â therefore Â critical Â for Â cell Â survival Â and Â organisms Â have Â developed Â tightly Â regulated Â mechanisms Â to Â sense Â and Â maintain Â iron Â homeostasis. Â Â Â Competition Â for Â iron Â between Â microbes Â and Â mammalian Â hosts Â makes Â iron Â sensing Â Â Â Â Â Â Â Â Â Â Â Â Â Â 35 Â and Â acquisition Â key Â aspects Â of Â virulence Â for Â the Â pathogenic Â yeast Â C. Â neoformans Â (K. Â H. Â Bartlett, Â et Â al., Â 2008). Â Â As Â introduced Â in Â Chapter Â 1, Â C. Â neoformans Â causes Â invasive Â mycosis Â and Â severe Â meningoencephalitis Â in Â two Â immunocompromised Â risk Â groups: Â 1) Â individuals Â individuals Â infected Â with Â the Â human Â immunodeficiency Â virus Â (HIV), Â in Â particular Â those Â with Â acquired Â immunodeficiency Â syndrome Â (AIDS), Â and; Â 2) Â organ Â transplant Â recipients Â (T. Â G. Â Mitchell Â and Â J. Â R. Â Perfect, Â 1995). Â The Â widespread Â use Â of Â highly Â active Â antiretroviral Â therapy Â (HAART) Â has Â decreased Â the Â incidence Â of Â cryptococcosis Â in Â developed Â countries Â (R. Â Manfredi Â et Â al., Â 1999, Â D. Â W. Â Mackenzie, Â 1989). Â However, Â the Â mortality Â rate Â and Â incidence Â of Â infection Â remains Â high Â in Â areas Â where Â access Â to Â HAART Â is Â limited. Â The Â burden Â of Â cryptococcal Â disease Â underlines Â the Â urgent Â need Â for Â new Â drug Â and Â vaccine Â targets Â including, Â potentially, Â functions Â for Â iron Â homeostasis. Â Â Â Â Iron Â regulates Â the Â expression Â of Â the Â main Â virulence Â factors Â of Â C. Â neoformans Â including Â the Â production Â of Â a Â polysaccharide Â capsule Â and Â the Â deposition Â of Â melanin Â in Â the Â cell Â wall. Â Â C. Â neoformans Â is Â remarkably Â tuned Â to Â perceive Â iron Â as Â part Â of Â the Â disease Â process Â and Â employs Â various Â iron Â regulators Â and Â uptake Â mechanisms Â that Â contribute Â to Â virulence. Â These Â include Â high Â and Â low Â affinity Â iron Â uptake Â systems Â as Â well Â as Â heme Â uptake Â pathways Â (B. Â Cadieux, Â et Â al., Â 2013). Â High Â affinity Â uptake Â involves Â reduction Â of Â ferric Â iron Â (Fe3+) Â to Â ferrous Â form Â (Fe2+) Â by Â cell Â surface Â reductases, Â with Â subsequent Â transport Â by Â a Â permease Â (Cft1) Â and Â ferroxidase Â (Cfo1) Â (G. Â Hu, Â et Â al., Â 2013, Â E. Â S. Â Jacobson, Â et Â al., Â 1998, Â J. Â W. Â Kronstad Â et Â al., Â 2011). Â Its Â been Â shown Â that Â defects Â in Â iron Â acquisition Â attenuate Â virulence, Â and Â iron Â availability Â influences Â the Â expression Â of Â major Â virulence Â factors. Â Iron Â overload Â exacerbated Â manifestation Â of Â cryptococcal Â meningitis Â in Â a Â mouse Â model Â of Â infection Â and Â facilitated Â colonization Â of Â the Â brain Â (Jung Â et Â al., Â 2008; Â 2009). Â Â Â Â Â Â Â Â Â Â Â Â Â Â 36 Â Â The Â GATA-­âtype, Â zinc-­âfinger Â protein Â Cir1 Â regulates Â iron Â uptake, Â iron Â homeostasis Â and Â virulence Â factor Â expression Â in Â C. Â neoformans. Â Mutants Â defective Â in Â CIR1 Â exhibit Â iron-­ârelated Â phenotypes. Â Transcriptional Â profiling Â revealed Â that Â Cir1 Â regulates Â the Â majority Â of Â genes Â encoding Â iron Â uptake Â functions Â as Â well Â as Â genes Â for Â signal Â transduction, Â transcription, Â sterol Â biosynthesis, Â DNA Â replication, Â and Â cell Â wall Â biosynthesis Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â However, Â the Â mechanisms Â by Â which Â Cir1 Â senses Â iron Â availability Â are Â important Â aspects Â of Â the Â proliferation Â of Â the Â fungus Â in Â the Â host Â is Â not Â entirely Â known. Â Â So Â far Â it Â has Â been Â shown Â that Â iron Â influences Â abundance Â of Â Cir1 Â and Â that Â levels Â of Â Cir1 Â protein Â decreases Â upon Â iron Â deprivation. Â This Â destabilization Â is Â also Â shown Â to Â be Â influenced Â by Â reducing Â conditions Â and Â by Â inhibition Â of Â proteasome Â function; Â In Â addition, Â a Â post-­âtranslational Â mechanism Â for Â the Â control Â of Â Cir1 Â abundance Â in Â response Â to Â iron Â and Â redox Â status Â has Â been Â suggested Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2011). Â Given Â the Â importance Â of Â iron Â homeostasis Â in Â C. Â neoformans Â pathogenesis, Â there Â is Â a Â clear Â need Â to Â identify Â further Â major Â components Â that Â contribute Â to Â Cir Â function Â and Â iron Â regulation Â to Â fully Â understand Â the Â disease Â process. Â Â Â The Â mechanisms Â by Â which Â iron-­âresponsive Â transcription Â factors Â in Â fungi Â sense Â intracellular Â iron Â levels Â and Â regulate Â iron Â homeostasis Â are Â best Â understood Â in Â S. Â cerevisiae Â and Â S. Â pombe Â (M. Â Jbel, Â et Â al., Â 2011). Â In Â these Â fungi, Â the Â transcription Â factors Â interact Â with Â monothiol Â glutaredoxins Â (Grxs) Â that Â participate Â in Â iron Â sensing Â and Â regulation. Â Â Recent Â studies Â in Â S. Â cerevisiae Â and Â S. Â pombe Â have Â demonstrated Â essential Â roles Â for Â monothiol Â Grxs Â in Â intracellular Â iron Â homeostasis, Â iron Â trafficking Â and Â the Â maturation Â of Â Fe-­âS Â cluster Â proteins, Â and Â have Â established Â the Â proteins Â as Â novel Â Fe-­âS Â cluster-­âbinding Â regulatory Â partners. Â (M. Â Jbel, Â et Â al., Â 2011). Â Â In Â addition Â several Â studies Â point Â to Â critical Â roles Â for Â monothiol Â Grxs Â in Â the Â maintenance Â of Â cellular Â iron Â homeostasis Â (N. Â Rouhier, Â et Â al., Â 2010). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 37 Â In Â this Â chapter, Â a Â proteomic Â approach Â was Â initially Â taken Â by Â Dr. Â Horacio Â Bach Â to Â identify Â potential Â binding Â partners Â of Â Cir1 Â that Â may Â contribute Â to Â the Â complex Â and Â dynamic Â iron-­ârelated Â regulatory Â network Â of Â C. Â neoformans. Â Â The Â monothiol Â Grx4 Â was Â identified Â (gene Â ID: Â CNAG_02950) Â as Â a Â potential Â binding Â partner Â of Â Cir1 Â by Â mass-­âspectrometry. Â Â Given Â the Â conserved Â role Â of Â monothiol Â Grxs Â in Â iron Â homeostasis, Â I Â hypothesized Â that Â the Â monothiol Â Grx4 Â encoded Â by Â CNAG_02950 Â has Â a Â role Â in Â the Â iron Â network Â of Â C. Â neoformans. Â Â (N. Â Rouhier, Â et Â al., Â 2010). Â To Â test Â this Â hypothesis, Â I Â validated Â the Â Cir1-­âGrx4 Â interaction Â by Â protein-­âprotein Â interaction Â methods Â both Â in Â vitro Â and Â in Â vivo. Â Â I Â also Â tested Â the Â function Â of Â Grx4 Â in Â iron Â homeostasis Â by Â analyzing Â the Â phenotypes Â associated Â with Â deletion Â of Â the Â conserved Â Grx Â domain Â in Â Grx4. Â Growth Â of Â the Â mutant Â was Â tested Â in Â different Â concentrations Â of Â iron Â and Â the Â influence Â of Â Grx4 Â on Â expression Â of Â virulence Â factors Â was Â tested Â in Â vitro. Â To Â facilitate Â the Â analysis Â of Â genes Â and Â cellular Â functions Â that Â might Â be Â influenced Â by Â GRX4, Â I Â further Â explored Â the Â influence Â of Â Grx4 Â on Â the Â transcriptome Â of Â C. Â neoformans Â by Â RNA-­âSeq Â with Â subsequent Â validation Â of Â selected Â genes Â by Â quantitative Â Real-­âTime Â PCR Â (qRT-­âPCR). Â I Â continued Â our Â analysis Â on Â Grx4 Â by Â using Â the Â RNA-­âSeq Â data Â to Â characterize Â isoforms Â of Â the Â GRX4 Â transcript Â predicted Â from Â the Â genome Â annotation, Â and Â and Â used Â a Â genetic Â approach Â test Â whether Â larger Â deletions Â of Â GRX4 Â locus Â caused Â lethality. Â Â Overall, Â these Â studies Â reveal Â important Â roles Â for Â Grx4 Â in Â iron Â homeostasis Â and Â virulence Â in Â C. Â neoformans. Â Â 2.2 Â Materials Â and Â methods Â Â 2.2.1 Â Â Yeast Â strains, Â plasmids Â and Â growth Â media Â Â The Â serotype Â A Â strain Â H99 Â of Â C.âneoformans Â var. Â grubii Â was Â used Â as Â the Â WT Â strain Â along Â with Â the Â cir1 Â mutant, Â grx4 Â mutant, Â and Â the Â complemented Â strain Â grx4Î::GRX4. Â A Â Â Â Â Â Â Â Â Â Â Â Â Â Â 38 Â stable Â diploid Â C. Â neoformans Â strain Â AI187 Â was Â kindly Â provided Â by Â Dr. Â Alex Â Idnurm Â for Â progeny Â analysis. Â The Â strains Â were Â maintained Â on Â YPD Â medium Â (1% Â yeast Â extract, Â 2% Â peptone, Â 2% Â dextrose Â and Â 2% Â agar). Â Selectable Â markers Â for Â transformation Â were Â from Â plasmids Â pCH233 Â (nourseothricin Â resistance Â for Â grx4Î Â and Â cir1Î) Â and Â pJAF1 Â (neomycin Â resistance Â for Â the Â complemented Â strain). Â Â Nourseothricin Â was Â used Â for Â selection Â at Â 100Î¼gâmlâ1 Â and Â neomycin Â was Â used Â at Â 200Î¼gâmlâ1. Â Low Â iron Â medium Â (LIM) Â was Â prepared Â as Â described Â previously Â (S. Â E. Â Vartivarian Â et Â al., Â 1993). Â Briefly Â LIM Â was Â composed Â of Â 6.7g Â YNB Â plus Â 2% Â glucose Â in Â 1 Â L Â of Â iron-­âchelated Â H2O Â using Â BIORAD Â chelex-­â100 Â and Â supplemented Â with Â the Â membrane Â impermeable Â iron Â chelator Â bathophenanthrolinedisulfonic Â acid Â (BPS). Â The Â pH Â was Â adjusted Â to Â 7.2, Â and Â the Â medium Â was Â filter Â sterilized. Â To Â prepare Â iron-­âreplete Â media, Â FeCl3 Â was Â added Â to Â low-­âiron Â medium Â at Â 100 Â μM Â concentration. Â Cells Â were Â pre-­âcultured Â in Â YPD Â overnight Â at Â 30°C Â and Â starved Â in Â LIM Â for Â 48 Â hours, Â to Â make Â sure Â intracellular Â iron Â was Â depleted Â before Â transferring Â cells Â to Â LIM Â or Â iron-­âreplete Â media, Â as Â described Â previously Â (W. Â H. Â Jung, Â et Â al., Â 2009, Â W. Â H. Â Jung, Â et Â al., Â 2008). Â For Â growth Â assays Â on Â solid Â media, Â ten-­âfold Â serial Â dilutions Â of Â cells Â were Â spotted Â onto Â plates Â indicated Â in Â the Â text Â with Â or Â without Â the Â supplemented Â iron Â sources. Â Plates Â were Â incubated Â at Â 30°C Â for Â two Â days Â before Â being Â photographed. Â Â Table Â 2.1: Â Â Strains Â used Â in Â Chapter Â 2 Â Strain Â Description Â Provided/ Â Prepared Â by Â 6X Â His::Grx4 Â Â E. Â coli Â Â BL-­â21â¢ Â expressing Â His-­âtagged Â recombinant Â Grx4 Â Â Rodgoun Â Attarian Â MBP::Cir1 Â Â E. Â coli Â Â BL-­â21â¢ Â expressing Â MBP-­âtagged Â recombinant Â Cir1 Â Rodgoun Â Attarian Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 39 Â Â 2.2.2 Â Â Construction Â of Â strains Â Â Primers Â used Â for Â construction Â of Â strains Â are Â listed Â in Â Table Â 2.2. Â Table Â 2.2: Â Primer Â sequences Â for Â strain Â construction Â Allele Â constructed Â Â Primer Â identification Â Â Primer Â sequence Â Â Â 6X Â His::Grx4 Â NdeI-­âRA1-­âF Â Â AAACATATGATGAACTCTACACCTACGC Â Â RA1-­âR-­âHindIII Â AAAAAGCTTTTACTCCGTCTTGCCCTC Â MBP::Cir1 Â NdeI-­âRA3-­âF Â AAAAAAACATATGATGCCCGAAACGAATACCTCC Â Â RA1-­âR-­âEcoRV Â AAAGATATCCTAAGGAGAAGCACGGTGAGC Â Strain Â Description Â Provided/ Â Prepared Â by Â H99 Â C. Â neoformans Â wild-­âtype Â strain Â Dr. Â Joseph Â Heitman Â grx4Î Â grx4 Â disruption Â mutant Â in Â H99 Â background Â Dr. Â Patricia Â Missall Â grx4Î::GRX4 Â Â GRX4 Â complemented Â strain Â in Â the Â H99 Â strain Â background Â Rodgoun Â Attarian Â MaV203 Â S. Â cerevisiae Â competent Â strain Â ProQuestâ¢ Â Two-­âHybrid Â System Â Â CIR1-­â Â pDESTâ¢32 Â MaV203 Â harbouring Â bait Â Dr. Â Guanggan Â Hu Â Â GRX4-­âpDESTâ¢22 Â Â Â MaV203 Â harbouring Â prey Â Â Dr. Â Guanggan Â Hu Â Â AI187 Â C Â . Â neoformans Â diploid Â strain Â Â Dr. Â Alex Â Idnurm Â Â grx4âÎ Â Â grx4 Â full Â deletion Â mutant Â in Â the Â AI187 Â strain Â background Â Dr. Â Guanggan Â Hu Â Â Â Â Â Â Â Â Â Â Â Â Â Â 40 Â Allele Â constructed Â Â Primer Â identification Â Â Primer Â sequence Â Â grx4Î::GRX4 Â SS-­âA Â Â Â SS-­âB Â Â Â SS-­â7 Â Â SS-­âC Â Â SS-­â1 Â Â SS-­âD Â Â SS-­âE Â GTTTCTACATCTCTTCCGTGTTAATACAGAGACCTGACCCGATGACGCTGAAAAACGCACG Â Â CCGCGACGTGGTTCGGCTCTGGCTAATGAATGGGCGCGTGGTGGTGATTGTGATGGGGAT Â Â AGGACGTCACTCTCCGAGCTTAACAACC Â Â ATGGAATGCGTGAGATCG Â Â GCGAGATGGGCACATCCTTTATGTGACC Â Â CTGCGAGGATGTGAGCTG Â Â CAGCTCACATCCTCGCAG Â Â CIR1-­â Â pDESTâ¢32 Â Cir Â N-­âF Â Y2H Â Â Cir Â N-­âR-­âY2H Â Â ATGCCCGAAACGAATACCTCCGC Â ATCAAGAGGCGGTGCCACGTGAG Â GRX4-­âpDESTâ¢22 Â Y2H-­âGrx4-­âF Â Y2H-­âGrx4-­âR Â ATGAACTCTACACCTACGCCTAC Â TTACTCCGTCTTGCCCTCCTCGA Â grx4âÎ Â Â Â Â Â Â Â Â Â Grx4B-­â1 Â Â Grx4B-­â2 Â Â Â Grx4B-­â3 Â Â Â Grx4B-­â4 Â Â Â Grx4B-­â5 Â Â Â Grx4B-­â6 Â Â Grx4B-­â9PO Â Â Grx4B-­â10PO Â GGTAGTCAACATATTCGACGCCTGAG Â Â GCTAACTTTCTATATCCCATCGAAGCTGCGAGGATGTGAGCT Â Â Â AGCTCACATCCTCGCAGCTTCGATGGGATATAGAAAGTTAGC Â Â Â TAGTTTCTACATCTCTTCGCGAGCGCCTTGGAGCGGGAAAGGAGT Â Â Â ACTCCTTTCCCGCTCCAAGGCGCTCGCGAAGAGATGTAGAAACTA Â Â Â CCAGTATGCTTGGTGCTGTACGTCTC Â Â CGGTTGACACGAGACTTGATGTTGGA Â Â CCTCCCGTTCGCAAACCTAATCCT Â Â Â Â Â Â Â Â Â Â Â Â Â Â 41 Â Â Allele Â constructed Â Primer Â identification Â Primer Â sequence Â grx4âÎ Â (Continued) Â NAT3L-­âlong3 Â Â NAT5R-­âlong1 Â GCCACTCGAATCCTGCATGCTTATGTGAGT Â Â CAGCAACGCCGTTGAATCCTCAGGATCTTC Â Â 2.2.2.1 Â Deletion Â of Â the Â conserved Â Grx Â domain Â to Â create Â a Â grx4 Â mutant Â and Â generation Â of Â grx4Î::GRX4 Â complemented Â strain Â Â A Â grx4 Â disruption Â mutant Â was Â constructed Â by Â homologous Â recombination Â using Â a Â nourseothricin Â acetyltransferase Â (NAT) Â marker Â linked Â to Â 5â² Â and Â 3â² Â flanking Â sequences Â of Â GRX4 Â C-­âterminal Â domain Â by Â three-­âstep Â overlapping Â PCR Â (primer Â list Â 2.2). Â Â The Â overlap Â PCR Â product Â was Â biolistically Â transformed Â into Â the Â WT Â strain Â H99, Â and Â deletion Â was Â confirmed Â by Â PCR Â and Â Southern Â blot Â hybridization Â as Â previously Â described Â (R. Â C. Â Davidson Â et Â al., Â 2002, Â D. Â L. Â Toffaletti Â et Â al., Â 1993, Â J. Â H. Â Yu Â et Â al., Â 2004) Â (M. Â Chayakulkeeree Â et Â al., Â 2008). Â Briefly, Â genomic Â DNA Â for Â Southern Â blot Â analysis Â was Â prepared Â using Â cetyl Â trimethyl Â ammonium Â bromide Â (CTAB) Â phenol-­âchloroform Â extraction Â (Figure Â 2.1). Â Â To Â reconstitute Â the Â deleted Â region Â of Â GRX4 Â in Â the Â mutant Â strain Â in Â locus, Â a Â genomic Â DNA Â fragment Â containing Â 1.1 Â Kb Â of Â upstream Â promoter Â region Â and Â a Â 1.7 Â Kb Â region Â carrying Â the Â deleted Â portion Â of Â GRX4 Â gene Â was Â amplified Â by Â PCR. Â Â This Â PCR Â fragment Â was Â fused Â with Â the Â neomycin Â (NEOr) Â selectable Â marker Â (1.9 Â Kb) Â at Â its Â C Â terminus Â in Â an Â overlap Â PCR Â reaction. Â Â The Â overlap Â PCR Â product Â was Â introduced Â into Â the Â grx4 Â mutant Â by Â biolistic Â transformation. Â Targeted Â integration Â was Â confirmed Â by Â PCR Â and Â phenotypic Â overlay Â assays Â with Â TTC Â were Â performed Â to Â confirm Â restoration Â of Â the Â grx4 Â mutant Â phenotypes. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 42 Â 2.2.2.2 Â Genetic Â analysis Â of Â GRX4 Â in Â a Â haploid Â and Â a Â diploid Â C. Â neoformans Â background Â Â A Â deletion Â mutant Â lacking Â the Â complete Â GRX4 Â open Â reading Â frame Â was Â constructed Â by Â homologous Â recombination Â using Â a Â neomycin Â (NEO) Â marker Â linked Â to Â 5â² Â and Â 3â² Â flanking Â sequences Â of Â GRX4 Â by Â three-­âstep Â overlapping Â PCR. Â Â Â The Â overlap Â PCR Â product Â was Â biolistically Â transformed Â into Â haploid Â H99 Â strain, Â or Â diploid Â strain Â AI187 Â (a Â kind Â gift Â provided Â by Â Dr. Â Idnurm Â School Â of Â BioSciences, Â University Â of Â Melbourne). Â Deletion Â of Â full-­âlength Â GRX4 Â in Â the Â haploid Â H99 Â background Â was Â assessed Â by Â PCR, Â growth Â on Â YPD Â supplemented Â with Â 100 Â μg/ml Â neomycin, Â and Â a Â TTC Â overlay Â assay Â to Â evaluate Â cell Â surface Â reductase Â activity Â (R. Â Hassett Â and Â D. Â J. Â Kosman, Â 1995, Â M. Â Ogur Â et Â al., Â 1957). Â Sporulation Â of Â the Â transformed Â diploid Â strains Â was Â performed Â on Â V8 Â mating Â medium Â in Â the Â dark Â at Â room Â temperature Â for Â 5 Â days Â as Â described Â previously Â (R. Â A. Â Sia Â et Â al., Â 2000). Â Â The Â appearance Â of Â haploid Â meiotic Â progeny Â was Â evaluated Â by Â observing Â the Â peripheries Â of Â test Â spots Â on Â V8 Â plates Â and Â spores Â were Â collected Â from Â prominently Â formed Â hyphae Â followed Â by Â random Â spore Â analysis. Â To Â test Â for Â full Â deletion Â of Â GRX4, Â basidiospores Â were Â incubated Â on Â YDP Â at Â 30â°C Â and Â transferred Â to Â YPD Â plate Â containing Â NEO. Â Deletion Â was Â further Â analyzed Â by Â growth Â in Â presence Â of Â NEO, Â by Â the Â TTC Â assay Â and Â by Â PCR. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 43 Â Â Â Figure Â 2.1: Â Construction Â of Â grx4 Â mutant Â and Â complemented Â grx4Î::GRX4 Â strain, Â and Â confirmation Â of Â grx4 Â mutant Â by Â Southern Â Hybridization. Â Schematic Â representation Â of Â the Â WT Â loci Â for Â GRX4 Â open Â reading Â frame Â (A), Â nourseothricin Â resistance Â gene Â used Â to Â replace Â the Â 1kB Â of Â C-­âterminal Â Grx Â domain Â (B) Â and Â construct Â used Â to Â generate Â grx4Î::GRX4 Â reconstituted Â strain Â (D). Â The Â blue Â box Â represents Â the Â GRX4 Â reading Â frame. Â NAT Â and Â NEO Â represent Â the Â nourseothricin Â and Â neomycin Â resistance Â cassettes, Â respectively. Â The Â grx4Î Â strain Â contains Â deletion Â of Â C-­âterminal Â Grx Â domain Â in Â a Â haploid Â WT-­âH99 Â background Â (B). Â In Â panel Â (A) Â WT-­âH99 Â genomic Â DNA Â was Â digested Â with Â BglII Â and Â XbaI Â restriction Â enzymes. Â In Â panel Â (D), Â genomic Â DNA Â from Â grx4 Â mutant Â was Â digested Â with Â the Â same Â combination Â of Â restriction Â enzymes. Â The Â digested Â DNA Â was Â hybridized Â with Â a Â probe Â amplified Â from Â the Â Â Â Â Â Â Â Â Â Â Â Â Â Â 44 Â DNA Â sequence Â upstream Â of Â the Â GRX4 Â open Â reading Â frame Â (A Â and Â B). Â Panel Â C Â illustrates Â genomic Â hybridization Â results Â with Â the Â probes Â indicated Â in Â panels Â A Â and Â B Â and Â confirmed Â disruption Â of Â GRX4 Â and Â deletion Â of Â C-­âterminal Â conserved Â Grx Â domain. Â Neomycin Â resistant Â gene Â was Â used Â for Â complementation Â of Â grx4 Â mutant Â reconstitution Â of Â Grx Â deleted Â domain Â back Â in Â locus Â by Â biolistic Â transformation Â (D). Â 2.2.3 Â Â In Â silico Â protein Â analysis Â Â Â The Â Grx4 Â amino Â acid Â sequence Â from Â the Â C. Â neoformans Â serotype Â A Â strain Â H99 Â was Â used Â to Â search Â the Â C. Â neoformans Â serotype Â A Â genome Â sequence Â of Â the Â H99 Â strain Â (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans, Â 2009) Â as Â well Â as Â other Â serotypes Â of Â C. Â neoformans, Â fungal Â species Â and Â eukaryotes. Â (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). Â Multiple Â sequence Â alignments Â were Â performed Â using Â Clustal Â W Â (http://www.ebi.ac.uk/clustalw/). Â Â 2.2.4 Â Â Gene Â cloning, Â protein Â expression Â and Â purification Â Â Coding Â sequence Â region Â of Â Grx4 Â (1-­â1275 Â bp) Â and Â N-­âterminal Â region Â of Â Cir1 Â (1-­â1224 Â bp) Â from Â C. Â neoformans Â H99 Â strain Â (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans, Â 2009) Â were Â amplified Â from Â cDNA Â template Â using Â oligonucleotides Â detailed Â in Â Table Â 2.2. Â Â Â GRX4 Â cDNA Â was Â purified Â and Â subsequently Â cloned Â into Â pET-­â28b Â (Invitrogen) Â using Â NdeI Â and Â HindIII Â restriction Â enzymes. Â Â The Â Grx4/pET-­â28b Â plasmid Â was Â then Â transformed Â into Â Escherichia Â coli Â DH5α Â cells Â and Â once Â the Â identity Â of Â the Â gene Â was Â confirmed Â by Â sequencing, Â it Â was Â transformed Â into Â E. Â coli Â BL-­â21 Â (DE3) Â cells Â for Â expression Â of Â the Â His6x-­âtagged Â protein Â as Â described Â previously Â (R. Â Attarian Â et Â al., Â 2009). Â The Â N-­âterminal Â coding Â sequence Â region Â of Â Cir1 Â was Â re-­âamplified Â from Â a Â previously Â sequenced Â CIR1 Â full-­âlength Â Â Â Â Â Â Â Â Â Â Â Â Â Â 45 Â cDNA Â clone Â in Â pET-­â32a Â and Â subcloned Â into Â pMAL-­âc5X Â using Â Nde1 Â and Â EcoRV Â restriction Â enzymes Â and Â Cir1 Â produced Â as Â a Â maltose-­âbinding-­âprotein-­âtagged Â protein Â in Â E. Â coli Â BL-­â21 Â (DE3). Â Â LuriaâBertani Â medium Â supplemented Â with Â 100 Â μg Â of Â ampicillin Â was Â used Â for Â bacterial Â cultures. Â Â Overnight Â cultures Â were Â prepared Â by Â picking Â a Â single Â colony Â for Â the Â E. Â coli Â BL-­â21 Â (DE3) Â strains Â harboring Â the Â Grx4/pET-­â28b Â or Â Cir1/pMAL-­âc5X Â plasmids, Â and Â inoculating Â liquid Â medium Â for Â growth Â at Â 37°C. Â Â The Â next Â day, Â the Â cultures Â were Â diluted Â 1:100 Â and Â the Â cells Â were Â induced Â with Â 0.3 Â mM Â IPTG Â (isopropyl Â Î²-­âD-­âthiogalactoside) Â at Â an Â OD600 Â of Â 0.6â0.8. Â Â Induced Â cells Â were Â shaken Â overnight Â at Â room Â temperature Â (25â¦C), Â harvested Â and Â stored Â at Â â20â¦C Â until Â further Â processing. Â Â The Â N-­âterminal Â MBP-­âtagged Â Cir-­â1 Â was Â purified Â by Â affinity Â chromatography Â using Â maltose Â resin Â (New Â England Â Biolabs) Â and Â His-­â6x-­âtagged Â Grx4 Â was Â purified Â using Â a Â Ni2+-­âNTA Â (Ni2+-­ânitrilotriacetate) Â resin Â (Qiagen) Â following Â the Â manufacturerâs Â instructions. Â Proteins Â were Â separated Â by Â SDS-­âPAGE Â (12%) Â and Â visualized Â with Â Coomassie Â staining. Â Â 2.2.5 Â Proteinâprotein Â interaction Â 2.2.5.1 Â AlphaScreenâ¢ Â binding Â assays: Â Â Â AlphaScreenâ¢ Â assay Â (Amplified Â Luminescent Â Proximity Â Homogeneous Â Assay, Â PerkinElmer Â Life Â Sciences) Â is Â a Â bead-­âbased Â technology Â that Â was Â designed Â to Â measure Â the Â proximity Â of Â donor Â and Â acceptor Â beads Â conjugated Â to Â analytes Â of Â interest Â (E. Â F. Â Ullman Â et Â al., Â 1994). Â Â The Â binding Â between Â Grx4 Â and Â Cir1 Â was Â measured Â using Â an Â AlphaScreen Â histidine Â (nickel Â chelate) Â detection Â kit Â (6760619M) Â and Â AlphaScreen Â assay Â buffer Â containing Â 25 Â mM Â HEPES Â (pH Â 7.5), Â 100 Â mM Â NaCl, Â and Â 0.1% Â BSA. Â Â Specifically, Â purified Â N-­âterminal Â MBP-­âtagged Â Cir1 Â was Â first Â biotinylated Â using Â the Â EZ-­âlink Â Biotinylation Â Kit Â (Pierce). Â Â Then Â the Â biotinylated Â MBP-­âtagged Â Cir1 Â or Â biotinylated Â purified Â MBP Â tag Â (as Â Â Â Â Â Â Â Â Â Â Â Â Â Â 46 Â control) Â were Â serial Â diluted Â in Â assay Â buffer Â in Â white Â opaque Â 384-­âwell Â microplates Â (PerkinElmer), Â followed Â by Â addition Â of Â streptavidin-­âcoated Â donor Â beads. Â The Â reaction Â mixes Â were Â incubated Â for Â 30 Â minutes Â at Â room Â temperature. Â Â In Â the Â next Â step, Â purified Â 6x Â His-­âtagged Â recombinant Â Grx4 Â was Â added Â to Â the Â reaction Â mix Â followed Â by Â addition Â of Â nickel-­âchelated Â acceptor Â beads, Â which Â bind Â the Â 6x Â His Â tag Â fused Â to Â the Â analyte. Â An Â unrelated Â purified Â protein Â from Â Mycobacterium Â tuberculosis, Â His-­âtagged Â Rv0323c, Â was Â used Â as Â a Â control. Â Â The Â reactions Â were Â incubated Â for Â 1 Â hour Â at Â room Â temperature. Â Â The Â reaction Â kinetics Â was Â then Â monitored Â on Â the Â Fusion-­âÎ±-­âHT Â Multimode Â Microplate Â Reader Â (PerkinElmer) Â for Â luminescence Â signal Â generated Â from Â protein-­âprotein Â interaction Â (counts Â per Â seconds). Â When Â the Â donor Â and Â acceptor Â beads Â were Â brought Â into Â proximity Â by Â the Â interaction Â between Â N-­âterminal Â MBP-­â Â Cir1 Â and Â 6x Â His-­âtagged Â Grx4, Â and Â the Â sample Â was Â excited Â with Â a Â laser Â at Â 680 Â nm Â to Â release Â singlet Â oxygen Â molecules. Â Within Â its Â 4-­âmillisecond Â half-­âlife, Â singlet Â oxygen Â can Â diffuse Â approximately Â 200 Â nm Â in Â solution. Â Â This Â enables Â the Â excitation Â of Â the Â Alpha Â acceptor Â beads, Â which Â then Â elicit Â a Â strong Â emission Â of Â light Â at Â a Â shorter Â wavelength Â (520-­â620 Â nm) Â to Â be Â counted Â and Â registered Â by Â FusionAlpha Â plate Â reader Â (PerkinElmer), Â as Â counts Â per Â second. Â Â To Â determine Â the Â approximate Â dissociation Â constant Â values, Â the Â data Â were Â fitted Â to Â a Â hyperbolic Â function Â using Â Graphpad®. Â 2.2.5.2 Â Yeast Â two-­âhybrid Â assay Â Â Â The Â assays Â were Â performed Â using Â the Â ProQuest Â Two Â hybrid Â system Â with Â Gateway Â Technology Â Manual, Â Invitrogen Â Life Â Technologies Â Inc. Â according Â to Â manufacturerâs Â protocols Â as Â previously Â described Â (G. Â Hu Â et Â al., Â 2015). Â Briefly, Â GRX4 Â (1-­â1648 Â bp) Â and Â the Â N-­âterminal Â region Â of Â CIR1 Â (1-­â1203 Â bp) Â were Â PCR Â amplified Â from Â C. Â neoformans Â cDNA Â using Â the Â primers Â listed Â in Â Table Â 2.2, Â cloned Â into Â pDEST-­â22 Â (GAL4 Â activting Â domain) Â and Â pDEST-­â Â Â Â Â Â Â Â Â Â Â Â Â Â 47 Â 32 Â (GAL4 Â DNA Â binding Â domain) Â vectors, Â respectively Â and Â co-­âtransformed Â into Â MaV203 Â yeast Â competent Â cells. Â Â Â Â The Â growth Â of Â MaV203 Â yeast Â expressing Â both Â bait Â and Â prey Â vectors Â was Â tested Â on Â synthetic Â complete Â medium Â (0.7% Â Yeast Â nitrogen Â base Â without Â amino Â acids Â (Difco), Â 2.0% Â glucose, Â 0.07% Â synthetic Â complete Â selection Â medium Â mix Â (Sigma), Â 1.7% Â bacto Â agar Â (Difco), Â pH Â 5.6) Â lacking Â leucine Â and Â tryptophan Â to Â select Â for Â each Â vector, Â and Â histidine Â and Â uracil Â to Â test Â for Â an Â interaction. Â Â Empty Â pDEST-­â32 Â and Â pDEST-­â22 Â vectors Â were Â used Â as Â negative Â controls. Â The Â physical Â interaction Â between Â the Â encoded Â proteins Â in Â these Â plasmids Â was Â tested Â by Â assessing Â restoration Â of Â uracil Â and Â histidine Â prototrophy. Â Â 2.2.6 Â Â Iron-­ârelated Â phenotypic Â analysis Â Â An Â overlay Â assay Â to Â examine Â the Â reduction Â of Â TTC Â was Â performed Â to Â evaluate Â cell Â surface Â reductase Â activity Â as Â described Â previously Â (R. Â Hassett Â and Â D. Â J. Â Kosman, Â 1995, Â M. Â Ogur, Â et Â al., Â 1957). Â Â Briefly, Â cells Â grown Â in Â YPD Â were Â harvested Â and Â diluted Â with Â sterile Â ddH2O. Â 1.0 Â Ã Â 106 Â cells Â were Â spotted Â on Â agar Â medium Â and Â incubated Â at Â 30°C Â for Â 1 Â day. Â Â The Â colonies Â were Â then Â overlaid Â with Â agarose Â (1.5% Â in Â PBS) Â containing Â 0.1% Â TTC. Â Â The Â development Â of Â red Â color Â on Â the Â colony Â spots Â was Â monitored Â and Â photographed. Â Â Phleomycin Â sensitivity Â was Â tested Â by Â spotting Â 1.0 Â Ã Â 106 Â cells Â onto Â YPD Â medium Â containing Â 0.25 Â μg/ml Â phleomycin Â with Â subsequent Â incubation Â at Â 30°C Â for Â 1 Â to Â 2 Â d. Â To Â determine Â sensitivity Â to Â iron-­ârestricted Â or Â to Â iron-­âoverload Â conditions, Â 106 Â and Â 10-­âfold Â serial Â dilution Â cells Â were Â spotted Â onto Â YPD Â medium Â (containing Â 400 Â mM Â BPS) Â with Â or Â without Â 250 Â μM Â FeCl3 Â and Â were Â grown Â at Â 30°C Â for Â 2 Â days. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 48 Â 2.2.7 Â Â Virulence Â factor Â assays Â Â Â Capsule Â formation Â was Â examined Â by Â DIC. Â Â For Â this Â assay, Â cells Â were Â pre-­âgrown Â in Â YPD, Â washed Â twice Â with Â low Â iron Â water Â (Li-­âH2O), Â diluted Â to Â 106 Â cells/ml Â in Â Capsule Â Inducing Â media Â (CIM), Â and Â grown Â for Â 24 Â hours Â at Â 30°C. Â Â CIM Â was Â prepared Â by Â dissolving Â 5g Â glucose, Â 5g Â asparagine, Â 4.78g Â HEPES, Â 0.4g Â K2HPO4, Â 0.25g Â CaCl2.2H2O, Â 0.08g Â MgSO4.7H2O, Â 1.85g Â NaHCO3, Â add Â 1ml Â of Â 1000X Â salt Â stock Â solution Â in Â 1L Â of Â Li-­âH2O, Â pH Â adjusted Â to Â 7.2 Â using Â 1 Â M Â MOPS Â made Â with Â low Â iron Â water, Â sterilized Â and Â 100ul Â sterile Â thiamine Â (4 Â mg/ml) Â added. Â Â The Â cells Â were Â then Â resuspended Â in Â 1/10th Â volume Â of Â CIM, Â mixed Â 1:1 Â in Â India Â ink Â and Â visualized Â by Â negative Â staining Â on Â a Â Zeiss Â Axioplan Â 2 Â Imaging Â microscope Â by Â DIC. Â Melanin Â production Â was Â examined Â on Â l-­â3,4-­âdihydroxyphenylalanine Â (l-­âDOPA) Â agar Â containing Â 0.1% Â glucose. Â Â Cells Â were Â pre Â grown Â in Â YPD, Â washed Â once, Â counted, Â and Â adjusted Â to Â 2 Â Ã Â 106 Â cells Â mlâ1 Â in Â YPD. Â Â Serial Â dilutions Â were Â performed, Â and Â 5 Â μl Â of Â each Â dilution Â was Â spotted Â onto Â agar Â medium. Â Â Plates Â were Â incubated Â at Â 30°C. Â Â The Â ablity Â of Â the Â grx4 Â mutant Â to Â grow Â at Â host Â temperature Â was Â tested Â by Â plating Â on Â solid Â media Â at Â 37°C. Â 2.2.8 Â Â Analysis Â of Â growth Â in Â liquid Â media Â Â Â For Â growth Â assays Â in Â liquid Â media, Â a Â final Â concentration Â of Â 5 Â Ã Â 104 Â cells Â mlâ1 Â was Â inoculated Â in Â the Â medium Â with Â or Â without Â supplemented Â iron Â sources. Â Â Briefly, Â strains Â (WT, Â grx4 Â mutant, Â grx4Î::GRX4 Â and Â cir1 Â mutant) Â were Â pre-­âgrown Â in Â triplicate Â cultures Â of Â 5ml Â of Â YPD Â overnight Â at Â 30°C, Â the Â cells Â were Â washed Â twice Â with Â low Â iron Â water, Â diluted Â 1:100, Â and Â incubated Â in Â low Â iron Â YNB Â at Â 30°C Â for Â an Â additional Â 48 Â h Â to Â eliminate Â any Â iron Â carryover Â from Â the Â rich Â medium. Â Â The Â cells Â were Â then Â harvested Â and Â washed Â twice Â with Â low Â iron Â water. Â Â The Â cells Â were Â counted Â and Â 104 Â cells Â were Â transferred Â to Â LIM Â alone Â or Â LIM Â supplemented Â with Â 100 Â µM Â FeCl3 Â (Sigma). Â Growth Â of Â the Â strains Â was Â also Â assessed Â by Â a Â Â Â Â Â Â Â Â Â Â Â Â Â Â 49 Â high-­âthroughput Â 96-­âwell Â microplate Â assay Â using Â the Â Infinite Â M200 Â PRO Â plate Â reader Â (Tecan, Â Austria). Â For Â this Â assay, Â iron-­âstarved Â cells Â were Â inoculated Â in Â 200 Â μl Â of Â YNB-­âLIM Â with Â or Â without Â supplemented Â iron Â sources Â for Â 3 Â to Â 4 Â days Â at Â 30°C Â (final Â inoculum Â of Â 104 Â cells). Â In Â addition, Â the Â same Â experiment Â was Â performed Â by Â measurement Â of Â the Â optical Â density Â of Â high Â volume Â cultures Â (50 Â ml) Â at Â 600 Â nm Â using Â a Â DU530 Â Life Â Science Â UV/Vis Â spectrophotometer Â (Beckman). Â In Â a Â separate Â experimental Â set Â up Â to Â test Â the Â influence Â exogenous Â GSH Â on Â growth Â phenotype Â of Â strains Â in Â presence Â of Â iron, Â iron-­âstarved Â strains Â (WT, Â grx4 Â mutant, Â grx4Î::GRX4 Â and Â cir1 Â mutant) Â were Â pre-­âgrown Â in Â the Â presence Â of Â iron Â to Â an Â O.D.600 Â of Â 0.3. Â These Â pre-­âtreated Â cells Â were Â diluted, Â and Â 104 Â cells Â were Â re-­âintroduced Â to Â fresh Â LIM Â supplemented Â with Â 100 Â µM Â FeCl3 Â in Â with/without Â 5 Â mM Â final Â concentration Â of Â glutathione. Â 2.2.9 Â Â Virulence Â assay Â Â For Â virulence Â assays, Â female Â BALB/c Â mice Â (4â6 Â weeks Â old) Â were Â obtained Â from Â Charles Â River Â Laboratories Â (Ontario, Â Canada). Â Â The Â WT, Â grx4 Â mutant Â and Â grx4Î::GRX Â cells Â were Â grown Â in Â YPD Â overnight Â at Â 30°C, Â washed Â in Â PBS Â and Â re-­âsuspended Â at Â 1.0âÃâ106âcellsâmlâ1 Â in Â PBS. Â Â Inoculation Â was Â by Â intranasal Â instillation Â with Â 50âÎ¼l Â of Â cell Â suspension Â (inoculum Â of Â 2.0âÃâ105 Â cells Â per Â mouse). Â Â Groups Â of Â 10 Â mice Â were Â inoculated Â for Â each Â strain. Â Â The Â status Â of Â the Â mice Â was Â monitored Â twice, Â daily Â post Â inoculation. Â Â For Â the Â determination Â of Â fungal Â burdens Â in Â organs, Â infected Â mice Â were Â euthanized Â by Â CO2 Â inhalation Â and Â organs Â were Â excised, Â weighed Â and Â homogenized Â in Â 1âml Â of Â PBS Â using Â a Â MixerMill Â (Retsch). Â Serial Â dilutions Â of Â the Â homogenates Â were Â plated Â on Â YPD Â agar Â plates Â containing Â 35âÎ¼gâmlâ1 Â chloramphenicol Â and Â CFUs Â were Â counted Â after Â incubation Â for Â 48âh Â at Â 30°C. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 50 Â Furthermore, Â fungal Â load Â distribution Â in Â different Â tissues Â of Â the Â infected Â mice Â was Â determined. Â Mice Â reaching Â the Â endpoint Â were Â euthanized Â by Â CO2 Â asphyxiation, Â and Â fungal Â loads Â in Â different Â tissues Â of Â the Â mice Â including Â the Â lungs, Â kidney, Â liver, Â spleen, Â brain Â and Â blood Â were Â determined. Â Tissues Â were Â aseptically Â removed Â and Â immersed Â in Â PBS. Â Organs Â were Â homogenized Â using Â an Â automated Â tissue Â homogenizer. Â The Â samples Â were Â serially Â diluted Â in Â PBS Â and Â plated Â on Â YPD Â supplemented Â with Â 35 Â μg/mL Â chloramphenicol. Â After Â two Â days Â of Â incubation Â at Â 30° Â C, Â the Â colony Â forming Â units Â (CFUs) Â were Â counted Â manually. Â 2.2.10 Â Â Ethics Â statement Â Â All Â experiments Â with Â mice Â were Â conducted Â in Â full Â compliance Â with Â the Â guidelines Â of Â the Â Canadian Â Council Â on Â Animal Â Care Â and Â approved Â by Â the Â University Â of Â British Â Columbia's Â Committee Â on Â Animal Â Care Â (protocol Â A13-­â0093). Â Â 2.2.11 Â Â Â Statistical Â analysis Â Â Â Â Statistical Â significance Â of Â mouse Â survival Â times Â was Â assessed Â using Â a Â single-­âfactor Â ANOVA Â variance Â test. Â Significant Â differences Â in Â fungal Â loads Â were Â determined Â by Â a Â two-­âtailed Â unpaired Â Student Â t Â test, Â using Â GraphPad Â Prism Â 6.0 Â for Â MAC Â OS Â X Â (Graph-­âPad Â Software, Â San Â Diego, Â CA, Â USA). Â Â The Â virulence Â data Â were Â analyzed Â with Â the Â log Â rank Â test Â for Â statistical Â differences. Â 2.2.12 Â Â RNA-­âSeq Â library Â preparation Â and Â bioinformatics Â Â Â Comparative Â transcriptional Â profiling Â of Â the Â WT, Â grx4 Â mutant Â and Â cir1 Â mutant Â strains Â in Â both Â iron-­âdeplete Â and Â iron-­âreplete Â condition Â was Â performed Â by Â RNA Â sequencing. Â Â Each Â strain Â was Â grown Â in Â 25 Â ml Â of Â YPD Â overnight Â at Â 30°C. Â Â Cells Â were Â washed Â twice Â with Â low Â iron Â medium Â (LIM) Â followed Â by Â growth Â in Â LIM Â at Â 30°C Â for Â an Â additional Â 48h Â to Â eliminate Â any Â iron Â carryover Â from Â the Â rich Â medium. Â Â Cells Â were Â then Â harvested Â and Â washed Â twice Â Â Â Â Â Â Â Â Â Â Â Â Â Â 51 Â with Â LIM. Â The Â cells Â were Â counted Â and Â transferred Â to Â 50 Â ml Â LIM Â or Â LIM+100 Â μM Â of Â FeCl3 Â final Â density Â of Â 2x105 Â cells/ml). Â Â The Â cells Â were Â then Â grown Â at Â 30°C Â for Â another Â 6 Â h Â and Â harvested Â for Â RNA Â extractions. Â Â RNA Â was Â purified Â with Â the Â RNeasy Â kit Â (Qiagen) Â and Â treated Â with Â DNase Â (Qiagen) Â following Â the Â manufacturerâs Â recommendations. Â The Â quality Â of Â RNA Â was Â analyzed Â with Â an Â Agilent Â 2100 Â Bioanalyzer Â and Â stored Â at Â -­â80°C. Â A Â total Â of Â 10 Â µg Â RNA Â was Â used Â for Â the Â TruSeq Â RNA Â Sample Â Prep Â Kit Â v2 Â (Illumina, Â Inc., Â CA). Â In Â order Â to Â construct Â libraries, Â the Â libraries Â were Â sequenced Â on Â a Â HiSeq Â 2000 Â (Illumina, Â Inc., Â CA) Â using Â 100 Â bp Â in Â paired-­âend Â mode. Â Â Raw Â read Â data Â was Â quality-­âchecked Â and Â trimmed Â using Â FastQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Â Â Trimmed Â reads Â were Â aligned Â to Â the Â C. Â neoformans Â serotype Â A Â strain Â H99 Â reference Â genome Â obtained Â from Â the Â Broad Â Institute Â [Cryptococcus Â neoformans Â Sequencing Â Project, Â Broad Â Institute Â of Â Harvard Â and Â MIT Â (http://www.broadinstitute.org). Â For Â aligning Â reads, Â TopHat Â 2.0.9 Â (C. Â Trapnell Â et Â al., Â 2009) Â was Â used Â with Â default Â settings Â except Â for Â adjusting Â the Â expected Â minimum Â and Â maximum Â intron Â lengths Â to Â 10 Â bp Â and Â 5000 Â bp, Â respectively. Â Â Aligned Â reads Â were Â assembled Â into Â transcripts Â and Â compared Â to Â the Â reference Â genome Â annotation Â with Â Cufflinks Â 2.1.1 Â (C. Â Trapnell Â et Â al., Â 2010). Â Â Cufflinks Â was Â further Â used Â for Â de Â novo Â assembly Â of Â predicted Â and Â unannotaded Â transcript Â variants Â of Â GRX4. Â The Â R Â packages Â cummeRbund Â version Â 0.1.3 Â (C. Â Trapnell, Â et Â al., Â 2010), Â ggplot2 Â version Â 0.9.3 Â and Â custom Â scripts Â were Â used Â to Â analyze Â and Â visualize Â the Â transcript Â data. Â The Â merged Â data Â was Â collected Â into Â a Â consensus Â GTF Â file Â using Â Cuffmerge Â (version Â 2.02). Â For Â Gene Â Ontology Â analyses, Â transcript Â sequences Â with Â an Â absolute Â fold Â change Â of Â 3 Â or Â more Â with Â a Â p-­âvalue Â of Â less Â than Â p<0.05 Â between Â WT Â and Â grx4 Â mutant Â under Â low Â iron Â and Â iron Â replete Â conditions Â were Â analysed Â using Â Gene Â Ontology Â (GO) Â terms Â available Â on Â Â Â Â Â Â Â Â Â Â Â Â Â Â 52 Â locally Â installed Â Blast2GO Â (Conesa Â et Â al., Â 2005). Â Â The Â C. Â neoformans Â H99 Â reference Â genome Â was Â used Â for Â gene Â annotation Â [Cryptococcus Â neoformans Â Sequencing Â Project, Â Broad Â Institute Â of Â Harvard Â and Â MIT Â (http://www.broadinstitute.org Â May Â 2014). Â GO Â term Â enrichment Â datasets Â were Â visualized Â using Â the Â R Â package Â ggplot2. Â 2.2.13 Â Â Quantitative Â Real-­âTime Â PCR Â Â To Â examine Â gene Â expression, Â three Â biological Â replicates Â of Â the Â WT Â and Â grx4 Â mutant Â and Â cir1 Â mutant Â strains Â were Â grown Â in Â 5 Â ml Â of Â YPD Â medium Â overnight Â at Â 30°C. Â Cells Â were Â washed Â twice Â in Â chelex-­âtreated Â water Â followed Â by Â growth Â in Â YNB-­âLIM Â for Â two Â days Â to Â starve Â the Â cells Â for Â iron. Â Cells Â were Â then Â washed Â and Â grown Â either Â in Â 5 Â ml Â YNB-­âLIM Â (with Â or Â without Â 100 Â μ Â M Â FeCl3 Â for Â six Â hours Â at Â 30°C. Â Â Samples Â were Â collected Â in Â triplicate Â and Â flash Â frozen Â in Â liquid Â N2, Â and Â stored Â at Â -­â80°C. Â Total Â RNA Â was Â extracted Â using Â the Â QIAGEN Â RNeasy Â Mini Â Kit Â and Â subsequently Â treated Â with Â RNase-­âfree Â DNase. Â Â cDNA Â was Â synthesized Â using Â oligo(dT) Â primer Â and Â Applied Â BiosystemsTM Â High-­âCapacity Â cDNA Â Reverse Â Transcription Â Kit. Â Â The Â resulting Â cDNA Â was Â used Â as Â template Â for Â quantitative Â real-­âtime Â PCR Â using Â iQ Â SYBR Â Green Â Supermix Â (Bio-­âRad) Â according Â to Â the Â manufacturer's Â specifications. Â The Â iCycler Â iQ Â multicolor Â real-­âtime Â detection Â system Â was Â used Â as Â the Â fluorescence Â detector. Â Â The Â relative Â gene Â expression Â was Â quantified Â based Â on Â the Â 2-­âÎÎC Â method Â with Â 18S Â rRNA Â as Â an Â internal Â control Â for Â data Â normalization. Â Â Primers Â (Table Â 2.3) Â were Â designed Â using Â Primer3 Â v.4.0 Â (http://bioinfo.ut.ee/primer3-­â0.4.0) Â and Â targeted Â to Â the Â 3âregions Â of Â transcripts Â Relative Â gene Â expression Â was Â quantified Â using Â the Â Applied Â BiosystemsTM Â 7500 Â Fast Â Real-­âtime Â PCR Â system Â and Â fold Â changes Â were Â determined Â based Â on Â comparison Â of Â gene Â expression Â to Â the Â WT-­âH99 Â strain Â Â Â Â Â Â Â Â Â Â Â Â Â Â 53 Â Table Â 2.3: Â Primer Â sequences Â for Â quantitative Â qRT-­âPCR Â Primer Â identification Â Â Primer Â sequence Â Â Target Â Gene Â Â sit1-­âf1 Â sit1-­âr1 Â GCACGGAGGAGGTCGTTGTA Â GCTCAGCGAGGTGTGCAAA Â Â SIT1 Â (CNAG_00815) Â str3-­âf1 Â str3-­âr1 Â TCGCCGCTTCCTCCAA Â GCTTCGTGTTGACCGGTAAGA Â Â STR3 Â (CNAG_07387) Â Â mirB-­âf1 Â mirB-­âr1 Â GCTCAGCGAGGTGTGCAAA Â CGACATTCTCGACCAAGTCAAA Â Â MIRB Â (CNAG_07751) Â cfo1-­âf1 Â cfo1-­âr1 Â GGACCTTGGCCGCTCAA Â CAAGCGCGCCAATCG Â Â CFO1 Â (CNAG_02958) Â cft1-­âf1 Â cft1-­âr1 Â TCGCACCGGCTCTACCA Â GGAAAGAGGTGGAACCGATGA Â Â CFT1 Â (CNAG_02959) Â cat3-­âf1 Â cat3-­âr1 Â GCGACGAGTCAAAGGAACGT Â CGCTTGCAGGTCGACATGT Â Â catalase Â (CNAG_05015) Â cat4-­âf1 Â cat4-­âR1 Â CGCAATGGTTGGTTGAGACA Â GCCATTCTCTTGCACCACACT Â Â catalase Â (CNAG_00575) Â 18S-­âRT-­âF Â 18S-­âRT-­âR Â AACAGGTCTGTGATGCCCTTAGA Â ACTCGCTGGCTCAGTCAGTGT Â 18S Â rRNA Â (CNAG_03246) Â Â 2.3 Â Â Results Â 2.3.1 Â Â The Â monothiol Â glutaredoxin Â Grx4 Â binds Â Cir1 Â Â 2.3.1.1 Â Grx4 Â was Â identified Â as Â a Â potential Â Cir1-­âassociated Â protein Â Â Â Â The Â monothiol Â glutaredoxin Â Grx4 Â encoded Â by Â CNAG_02950 Â was Â originally Â identified Â by Â mass Â spectrometry Â (MALDI-­âTOF, Â UBC) Â in Â a Â preliminary Â experiment Â performed Â by Â Dr. Â Horacio Â Bach Â to Â identify Â Cir1-­âassociated Â proteins. Â Â The Â proteins Â identified Â by Â Dr. Â Horacio Â Bach Â in Â this Â experiment Â are Â listed Â in Â Appendix Â A: Â Table Â A1, Â and Â Figure Â A1.). Â The Â identified Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 54 Â Â Â peptides Â that Â matched Â the Â CNAG_02950 Â polypeptide Â predicted Â from Â the Â C. Â neoformans Â serotype Â A Â genome Â sequence Â (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans). Â Â Database Â version Â 2009, Â are Â highlighted Â in Â Figure Â 2.2. Â The Â putative Â interaction Â of Â Grx4 Â with Â Cir1 Â observed Â in Â this Â preliminary Â experiment Â was Â intriguing Â given Â the Â known Â interactions Â of Â monothiol Â glutaredoxins Â with Â iron Â regulatory Â factors Â in Â S. Â cerevisiae Â and Â S. Â pombe Â (Chapter Â 1). Â Â Â Â Â Figure Â 2.2: Â Identified Â peptides Â obtained Â from Â MS Â analysis. Â Peptides Â that Â matched Â the Â CNAG_02950 Â polypeptide Â are Â highlighted Â in Â yellow. Â Â 2.3.1.2 Â Production Â of Â recombinant Â Grx4 Â and Â Cir1 Â in Â E. Â coli Â for Â interaction Â studies Â Â As Â an Â initial Â step Â to Â validate Â the Â interaction Â between Â Grx4 Â and Â Cir1, Â each Â of Â the Â proteins Â were Â expressed Â in Â E. Â coli Â and Â purified Â by Â affinity Â chromatography. Â As Â a Â first Â step, Â cDNA Â from Â the Â WT Â strain Â H99 Â was Â produced Â as Â described Â in Â the Â Materials Â and Â Methods Â (section Â 2.2.13.1). Â The Â Grx4 Â full-­âlength Â cDNA Â was Â then Â obtained Â by Â PCR Â and Â cloned Â into Â the Â expression Â vector Â pET-­â28b. Â Â Following Â overexpression Â in Â E. Â coli Â BL-­â21, Â the Â protein Â was Â MNSTPTPTPQPDTDNPHSNDANHADDRRPLAAHIPDYAGKSPFPAPPSRFTPRTPLQFLSNSPTNTDASAISCPSYILPTLPSSAASLPSLDPPSSNDRLAMINRIKSLGSYTNSPATVKIPLPTPSPSVSLSLSSRGPATPSSSIIRNKPHMPSPLNKLDYLYDIPPSPPISADADPGQLLVMPRMPNPDVESPIASPFVNLNLQTSTSSNRKPGGHTLLARHSGADASLLRSLLTQHASPSAPLSTSSAQPQAPAAAQRPRTEAEIVARCHELMNKHKVVLFMKGNPTAPKCGFSRQTVGLLREQGVEFAWFDIFSDEDVRQGLKKVNDWPTFPQIIVNGELVGGLDILREMIENGEWQELMDSIEEGKA Â Â Â Â Â Â Â Â Â Â Â Â Â Â 55 Â purified Â as Â a Â recombinant Â 6x Â His-­âtagged Â polypeptide Â by Â Ni-­âNTA Â affinity Â chromatography Â (Figure Â 2.3.). Â (See Â Material Â and Â Methods, Â section Â 2.2.4). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Figure Â 2.3: Â Purification Â of Â the Â His6-­âtagged Â Grx4 Â polypeptide. Â The Â 6x Â His-­âtagged Â recombinant Â Grx4 Â polypeptide Â was Â resolved Â by Â 12% Â SDS-­âPAGE Â and Â detected Â with Â Î±-­âHis Â antibody. Â The Â Precision Â Plus Â Proteinâ¢ Â Dual Â Color Â Standards Â (#161-­â0374EDU) Â are Â indicated Â on Â the Â right Â side Â (B). Â Â Â The Â Cir Â polypeptide Â is Â 952 Â amino Â acids Â long Â and Â contains Â a Â cysteine-­ârich Â domain Â (amino Â acids Â 162-­â189) Â and Â a Â conserved Â zinc-­âfinger Â domain Â (amino Â acids Â 307-­â359) Â in Â the Â Â Â Â Â Â Â Â Â Â Â Â Â Â 56 Â N-­âterminal Â region Â (amino Â acids Â 1-­â408). Â Â It Â has Â been Â shown Â previously Â that Â the Â zinc-­âfinger Â domain Â in Â the Â N-­âterminal Â region Â of Â Cir1 Â is Â critical Â for Â function Â and Â required Â for Â the Â stability Â of Â Cir1. Â Â Of Â note, Â the Â contribution Â of Â the Â C-­âterminal Â region Â to Â the Â function Â of Â Cir1 Â is Â not Â known Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2011). Â In Â this Â study, Â several Â attempts Â to Â produce Â the Â full-­âlength Â version Â of Â Cir1 Â were Â not Â successful; Â however, Â I Â was Â able Â to Â successfully Â produce Â the Â N-­âterminal Â version Â of Â Cir1 Â tagged Â with Â the Â maltose Â binding Â protein Â (MBP) Â (See Â Materials Â and Â Methods, Â section Â 2.2.4). Â Â As Â shown Â in Â Figure Â 2.4, Â it Â was Â necessary Â to Â concentrate Â the Â fusion Â protein Â with Â an Â Amicon Â filter Â to Â separate Â it Â from Â contaminating Â proteins Â or Â degradation Â products Â of Â lower Â mass. Â Â Together, Â the Â expression Â approaches Â for Â Grx4 Â and Â Cir1 Â provided Â the Â proteins Â necessary Â for Â subsequent Â validation Â of Â interaction. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 57 Â Â Figure Â 2.4: Â Â Purification Â of Â Cir1 Â tagged Â with Â the Â maltose Â binding Â protein Â (MBP). Â Â Lane Â 1 Â contains Â the Â eluate Â of Â the Â MBP-­âtagged Â recombinant Â N-­âterminal Â region Â of Â Cir1 Â following Â maltose Â affinity Â purification. Â Lane Â 2 Â contains Â the Â eluate Â of Â the Â MBP-­âCir1 Â fusion Â protein Â concentrated Â on Â Amicon Â Ultra Â 50 Â K Â (Milipore) Â before Â SDS-­âPAGE. Â Â The Â lane Â labeled Â M Â contains Â the Â Protein Â Ladder Â Precision Â Plus Â Proteinâ¢ Â Dual Â Color Â Standards Â (#1610374EDU). Â Â Â 2.3.1.3 Â Â Grx4 Â binds Â Cir1 Â in Â an Â ALPHAScreen® Â protein-­âprotein Â interaction Â assay Â Â As Â mentioned Â in Â section Â 2.3.1.1, Â Grx4 Â was Â initially Â pulled Â down Â by Â Cir1 Â in Â a Â binding Â assay Â and Â identified Â by Â mass Â spectrometry. Â Â I Â sought Â to Â further Â validate Â this Â interaction Â by Â using Â the Â AlphaScreen® Â technique, Â an Â in Â vitro Â method Â for Â detecting Â and Â characterizing Â Â Â Â Â Â Â Â Â Â Â Â Â Â 58 Â protein-­âprotein Â interactions. Â ALPHAScreen® Â employs Â photoactive Â donor Â and Â acceptor Â beads Â that Â recognize Â specific Â tags Â on Â candidate Â interacting Â proteins. Â Â In Â my Â assay, Â the Â biotinylated Â MBP-­âtagged Â N-­âterminal Â region Â of Â Cir1 Â (amino Â acids Â 1-­â408) Â and Â recombinant Â 6X Â His-­âtagged Â Grx4 Â (amino Â acids Â 1-­â425) Â were Â used Â to Â examine Â the Â potential Â interaction. Â An Â overview Â of Â this Â assay Â is Â illustrated Â in Â Figure Â 2.5. Â Â Figure Â 2.5: Â Overview Â of Â the Â Grx4-­âCir1 Â Alphascreen Â assay. Â The Â 6x Â Hisâtagged Â Grx4 Â protein Â (black) Â was Â mixed Â with Â biotinylated Â Cir1 Â (purple) Â at Â a Â range Â of Â concentrations. Â Â Ni-­âchelate Â acceptor Â (yellow) Â and Â streptavidin Â donor Â AlphaScreen Â beads Â (blue) Â were Â then Â added. Â Upon Â irradiation Â with Â 680 Â nm Â laser Â light, Â the Â donor Â beads Â produce Â singlet Â oxygen Â (1ÎO2) Â that Â diffuses Â Â ~200 Â nm. Â Â Acceptor Â beads Â that Â are Â potentially Â in Â close Â proximity Â because Â of Â interactions Â of Â the Â Grx4 Â and Â Cir1 Â proteins Â on Â their Â surface Â may Â react Â with Â the Â 1ÎO2 Â and Â emit Â light Â between Â 520-­â620 Â nm. Â Â Emission Â is Â measured Â using Â a Â Fusion® Â αHT Â apparatus Â (PerkinElmer). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 59 Â Â The Â AlphaScreen Â analysis Â demonstrated Â direct Â binding Â of Â Cir1 Â and Â Grx4 Â in Â reciprocal Â reactions Â in Â vitro Â (Figure Â 2.6). Â Â A Â hyperbolic Â curve Â plotting Â emission Â at Â 520-­â620 Â nm Â as Â a Â function Â of Â Cir1 Â concentration Â generated Â a Â curve Â fitting Â the Â 1:1 Â Langmuir Â binding Â model, Â predicted Â a Â Cir1-­âGrx4 Â dissociation Â constant Â (Kd) Â of Â 2x10-­â6M. Â The Â purified Â MBP Â did Â not Â bind Â His6-­âtagged Â Grx4 Â in Â the Â control Â experiment Â (Figure Â 2.6 Â A). Â Â The Â reciprocal Â experiment Â with Â increasing Â amounts Â of Â Grx4 Â yielded Â similar Â results. Â A Â hyperbolic Â curve Â fitting Â the Â 1:1 Â Langmuir Â binding Â model Â and Â a Â dissociation Â constant Â (Kd) Â of Â 2.x Â 10-­â6 Â M Â confirmed Â binding Â between Â Cir1 Â and Â Grx4. Â Â Â I Â did Â not Â detect Â an Â interaction Â between Â MBP-­âtagged Â Cir1 Â and Â a Â recombinant Â protein Â from Â Mycobacterium Â tuberculosis Â (His6-­âtagged Â Rv0323c Â overexpressed Â in Â E. Â coli) Â in Â a Â control Â experiment Â Â (Figure Â 2.6 Â B). Â Â Â Overall, Â these Â results Â validated Â the Â initial Â interaction Â detected Â by Â mass Â spectrometry Â and Â indicated Â that Â Grx4 Â binds Â Cir1. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 60 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Figure Â 2.6: Â Binding Â between Â Cir1 Â and Â Grx4 Â detected Â by Â ALPHAScreen Â analysis. Â (A) Â Recombinant Â Cir1 Â was Â subjected Â to Â the Â ALPHAScreen Â assay Â with Â increasing Â concentrations Â of Â Grx4. Â Â The Â MBP Â served Â as Â a Â negative Â control. Â (B) Â A Â reciprocal Â experiment Â was Â performed Â with Â increasing Â concentrations Â of Â Cir1. Â His6-­âtagged Â Rv0323c Â protein Â was Â used Â as Â a Â negative Â control. Â A Â 0 1 2 3 4010000200003000040000[Grx4] µMALPHAScreen Signal (cps)MBP-Cir1MBPB Â 0 1 2 3 4010000200003000040000[Cir1] µMALPHAScreen Signal (cps) His-Grx4His-Rv0323c Â Â Â Â Â Â Â Â Â Â Â Â Â 61 Â 2.3.1.4 Â Grx4 Â binds Â Cir Â in Â a Â yeast Â two-­âhybrid Â assay Â Â I Â further Â sought Â to Â confirm Â the Â interaction Â between Â Grx4 Â and Â Cir1 Â in Â vivo. Â The Â yeast Â two-­âhybrid Â system Â was Â employed Â for Â this Â approach. Â Â The Â cDNAs Â for Â Grx4 Â and Â the Â N-­âterminal Â region Â of Â Cir1 Â were Â fused Â to Â the Â GAL4 Â activating Â domain Â (AD) Â in Â pDEST-­â22 Â (prey Â plasmid) Â and Â the Â GAL4 Â DNA Â binding Â domain Â (BD) Â in Â pDEST-­â32 Â (bait Â plasmid), Â respectively. Â These Â vectors Â were Â co-­âtransformed Â into Â competent Â yeast Â cells Â (strain Â MaV203). Â Â Yeast Â cells Â that Â harbored Â both Â AD-­âGrx4 Â and Â BD-­âCir1 Â were Â selected Â based Â on Â growth Â in Â the Â absence Â of Â leucine Â and Â tryptophan. Â Â The Â ability Â of Â these Â transformants Â to Â grow Â without Â uracil Â or Â histidine Â was Â then Â tested Â and Â growth Â used Â as Â an Â indication Â of Â a Â positive Â interaction Â between Â Grx4 Â and Â Cir1 Â (Figure Â 2.7). Â Â Overall, Â the Â yeast Â two-­âhybrid Â assay Â confirmed Â the Â findings Â from Â mass Â spectrometry Â and Â the Â AlphaScreen Â assay Â in Â further Â support Â of Â the Â conclusion Â that Â Grx4 Â interacts Â with Â Cir1. Â Â As Â mentioned, Â this Â interaction Â is Â consistent Â with Â observed Â interactions Â between Â monothiol Â glutaredoxins Â and Â iron Â regulators Â in Â other Â fungi, Â including Â Grx4 Â with Â Fep1 Â in Â S. Â pombe Â and Â Grx3/4 Â with Â Aft1 Â in Â S. Â cerevisiae Â (M. Â Jbel, Â et Â al., Â 2011, Â L. Â Ojeda, Â et Â al., Â 2006). Â Â The Â interaction Â of Â Grx4 Â with Â Cir1, Â the Â main Â iron Â regulatory Â factor Â in Â C. Â neoformans, Â raised Â the Â possibility Â that Â loss Â of Â Grx4 Â might Â lead Â to Â shared Â phenotypes Â with Â mutants Â lacking Â Cir1. Â Â Therefore, Â I Â continued Â to Â further Â investigate Â the Â association Â between Â Grx4 Â and Â Cir1 Â by Â phenotypic Â analysis Â of Â grx4 Â and Â cir1 Â mutants, Â as Â described Â below. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 62 Â Â Â Figure Â 2.7: Â Grx4 Â physically Â interacts Â with Â Cir1 Â in Â a Â yeast Â two-­âhybrid Â assay. Â Â The Â constructs Â with Â BD Â and Â AD Â indicate Â the Â GAL4 Â DNA Â binding Â and Â activation Â domains Â fused Â to Â Grx4 Â and Â Cir1, Â respectively. Â Â The Â vector Â designation Â indicates Â the Â empty Â vector Â control. Â (A) Â All Â combinations Â of Â transformants Â grew Â in Â the Â absence Â of Â leucine Â (Leu) Â and Â tryptophan Â (Trp), Â confirming Â the Â presence Â of Â the Â plasmids Â in Â the Â strains. Â (B) Â Yeast Â cells Â co-­âtransformed Â with Â plasmids Â containing Â AD-­âGrx4 Â and Â BD-­âCir1 Â grew Â in Â the Â absence Â of Â uracil Â (Ura) Â or Â histidine Â (His), Â confirming Â an Â interaction Â that Â allowed Â expression Â of Â URA3 Â and Â HIS3. Â As Â part Â of Â the Â detection Â of Â HIS3 Â expression, Â 3-­âamino-­â1,2,4-­âtrazole Â was Â used Â at Â different Â concentrations. Â A Â strain Â harboring Â plasmids Â expressing Â the Â known Â interacting Â proteins Â Rim20 Â and Â Snf7 Â was Â used Â as Â a Â positive Â control Â (G. Â Hu, Â et Â al., Â 2015). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 63 Â 2.3.2 Â Â Â A Â defect Â in Â Grx4 Â impacts Â iron Â homeostasis Â and Â virulence Â 2.3.2.1 Â Identification Â of Â a Â conserved Â C-­âterminal Â glutaredoxin Â domain Â in Â GRX4 Â in Â C. Â neoformans Â Â Â An Â analysis Â of Â the Â amino Â acid Â sequence Â similarity Â of Â Grx4 Â with Â other Â members Â of Â the Â monothiol Â glutaredoxin Â class Â of Â proteins Â revealed Â that Â Grx4 Â has Â a Â conserved Â C-­âterminal Â glutaredoxin Â (Grx) Â domain Â with Â a Â signature Â âCGFSâ Â motif Â in Â the Â predicted Â active Â site Â (Figure Â 2.8 Â A). Â A Â Clustal Â Omega Â alignment Â of Â the Â amino Â acid Â sequence Â of Â the Â C-­âterminal Â Grx Â domain Â of Â the Â C. Â neoformans Â protein Â with Â four Â selected Â Grx Â sequences Â from Â other Â organisms Â is Â shown Â in Â Figure Â 2.8 Â B. Â According Â to Â a Â Blastp Â analysis Â (https://blast.ncbi.nlm.nih.gov/Blast.cgi), Â the Â C-­âterminal Â domain Â in Â Grx4 Â in Â C. Â neoformans Â shares Â 44% Â amino Â acid Â sequence Â identity Â with Â C-­âterminal Â region Â in Â Grx4 Â from Â S. Â pombe, Â 41% Â with Â Grx4 Â from Â S. Â cerevisiae, Â 60% Â with Â Grx3 Â from Â S. Â pombe, Â and Â 51% Â identity Â with Â Grx3 Â from Â Homo Â sapiens Â (Figure Â 2.8 Â C). Â Â Of Â note, Â the Â monothiol Â Grx Â domain Â with Â the Â CGFS Â active Â site Â motif Â is Â highly Â conserved Â and Â is Â known Â to Â be Â required Â for Â Fe-­âS Â cluster Â binding Â and Â trafficking, Â and Â the Â regulation Â of Â iron Â homeostasis Â in Â fungi Â ((J. Â Encinar Â del Â Dedo, Â et Â al., Â 2015, Â H. Â Li Â and Â C. Â E. Â Outten, Â 2012). Â Â Combined Â with Â its Â interaction Â with Â the Â iron Â regulator Â Cir1, Â the Â sequence Â analysis Â supports Â a Â possible Â role Â for Â Grx4 Â in Â iron-­ârelated Â processes Â in Â C. Â neoformans. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 64 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Figure Â 2.8: Â Domain Â structure Â of Â C. Â neoformans Â Grx4. Â Â (A) Â The Â N-­âterminal Â Trx Â domain Â consisting Â of Â amino Â acids Â 193-­â313 Â and Â the Â Grx Â domain Â consisting Â of Â amino Â acids Â 332-­â399 Â are Â separated Â by Â a Â linker Â region. Â Â The Â active Â site Â motif Â of Â the Â Trx-­âlike Â domain Â (WAxxC) Â and Â Grx-­âlike Â domain Â (CGFS) Â are Â indicated Â at Â the Â top Â of Â the Â figure. Â Â (B) Â Sequence Â alignment Â of Â the Â C-­âterminal Â Grx Â domain Â of Â monothiol Â Grxs Â in Â model Â yeasts Â and Â human. Â Â The Â conserved Â active Â site Â motif Â (CGFS) Â is Â highlighted Â in Â magenta Â and Â the Â Â (*) Â and Â (:) Â symbols Â indicate Â A Â B Â HsGrx3 LPSANEHLKEDLNLRLKKLTHAAPCMLFMKGTPQEPRCGFSKQMVEILHKHNIQFSSFDI CnGrx4 AAAQRPRTEAEIVARCHELMNKHKVVLFMKGNPTAPKCGFSRQTVGLLREQGVEFAWFDI SpGrx4 TSKAPNGLDSELNERLSTLTNAHNVMLFLKGTPSEPACGFSRKLVGLLREQNVQYGFFNI ScGrx3 DEEEEEETEEQINARLTKLVNAAPVMLFMKGSPSEPKCGFSRQLVGILREHQVRFGFFDI ScGrx4 SDDEEDETEEEINARLVKLVQAAPVMLFMKGSPSEPKCGFSRQLVGILREHQIRFGFFDI . :: * * . :**:**.* * ****:: * :*::: :.:. *:* HsGrx3 FSDEEVRQGLKAYSSWPTYPQLYVSGELIGGLDIIKELEAS CnGrx4 FSDEDVRQGLKKVNDWPTFPQIIVNGELVGGLDILREMIEN SpGrx4 LADDSVRQGLKVFSDWPTFPQLYIKGEFVGGLDIVSEMIEN ScGrx3 LRDESVRQNLKKFSEWPTFPQLYINGEFQGGLDIIKESLEE ScGrx4 LRDENVRQSLKKFSDWPTFPQLYINGEFQGGLDIIKESIEE : *:.*** ** ..***:**: :.**: *****: * . C Â Â Â Â Â Â Â Â Â Â Â Â Â Â 65 Â identical Â and Â similar Â amino Â acids, Â respectively. Â Â Sequences Â were Â aligned Â using Â the Â CLUSTAL Â Omega Â (1.2.1) Â multiple Â sequence Â alignment Â tool Â (http://www.ebi.ac.uk/Tools/msa/clustalo/). Â Â Abbreviations Â are Â as Â follows: Â Cn= Â (C. Â neoformans Â var. Â grubii Â strain Â H99), Â Hs= Â (H. Â sapiens), Â Sc= Â (S. Â cerevisiae), Â Scp= Â (Schizosaccharomyces Â pombe). Â (C) Â A Â high Â percentage Â of Â identity Â in Â amino Â acid Â sequences Â in Â the Â C-­â Â terminal Â Grx Â domain Â across Â the Â selected Â species Â as Â determined Â by Â Blastp Â analysis Â is Â shown. Â Â 2.3.2.2 Â A Â grx4 Â deletion Â mutant Â has Â iron-­ârelated Â phenotypes Â Â Next Â I Â sought Â to Â gain Â insights Â into Â the Â function Â of Â Grx4 Â in Â C. Â neoformans Â by Â phenotypic Â characterization Â of Â a Â grx4 Â mutant Â in Â which Â the Â C-­âterminal Â conserved Â Grx Â domain Â was Â deleted Â and Â replaced Â by Â a Â NAT Â resistance Â marker. Â Â This Â grx4 Â mutant Â was Â kindly Â provided Â by Â our Â collaborator Â Dr. Â J Â Lodge Â (Washington Â University, Â St. Â Louis, Â MO, Â USA). Â Â I Â confirmed Â the Â deletion Â by Â Southern Â hybridization, Â and Â also Â complemented Â the Â mutation Â by Â inserting Â the Â WT Â GRX4 Â gene Â at Â its Â original Â locus Â to Â generate Â a Â grx4D::GRX4 Â strain Â (see Â Material Â and Â Methods, Â section Â 2.2.2.1 Â ). Â Â Â Â The Â grx4 Â mutant Â was Â tested Â for Â iron-­ârelated Â phenotypes Â including Â growth Â in Â the Â presence Â of Â elevated Â iron Â levels, Â cell Â surface Â reductase Â activity Â and Â susceptibility Â to Â the Â iron-­âdependent Â inhibitor Â phleomycin. Â Each Â assay Â was Â performed Â at Â least Â 3 Â times Â and Â representative Â results Â are Â presented Â in Â Fig. Â 2.9. Â Â A Â cir1 Â mutant Â was Â included Â throughout Â the Â experiments Â for Â comparison. Â Â Initially, Â the Â growth Â phenotype Â of Â the Â grx4 Â mutant Â in Â rich Â YPD Â medium Â was Â tested Â and Â it Â was Â found Â that Â the Â mutant Â exhibits Â equivalent Â growth Â to Â the Â WT Â strain Â in Â YPD Â in Â both Â plate Â assays Â and Â liquid Â culture. Â Â Â Therefore, Â YPD Â was Â used Â in Â Â Â Â Â Â Â Â Â Â Â Â Â Â 66 Â subsequent Â plate Â assays Â as Â the Â standard Â medium. Â Â Also, Â it Â has Â been Â shown Â that Â loss Â of Â Cir1 Â causes Â sensitivity Â to Â elevated Â iron Â levels Â ((W. Â H. Â Jung, Â et Â al., Â 2006). Â Â Â We Â therefore Â tested Â the Â growth Â phenotype Â of Â grx4 Â mutant Â in Â presence Â of Â elevated Â iron Â levels Â in Â both Â absence Â or Â presence Â of Â the Â iron Â chelator Â BPS. Â Â Ten Â fold Â serial Â dilutions Â of Â the Â WT Â strain Â (H99), Â the Â grx4 Â mutant, Â the Â grx4Î::GRX4 Â complemented Â strain Â and Â the Â cir1 Â mutant Â were Â grown Â in Â YPD Â at Â 30°C Â and Â spotted Â onto Â YPD+ Â 250 Â μM Â FeCl3. Â Â Similar Â to Â the Â cir1 Â mutant, Â the Â grx4 Â mutant Â displayed Â slower Â growth Â in Â the Â presence Â of Â iron Â (Figure Â 2.9). Â Â We Â further Â tested Â this Â phenotype Â in Â the Â grx4 Â mutant Â by Â first Â chelating Â available Â iron Â in Â YPD Â by Â addition Â of Â 400 Â mM Â of Â the Â iron Â chelator Â BPS. Â Â All Â four Â strains, Â including Â the Â cir1 Â and Â grx4 Â mutants, Â the Â WT Â strain Â and Â the Â complemented Â strain, Â have Â an Â impaired Â growth Â phenotype Â in Â the Â iron-­ârestricted Â condition Â in Â the Â presence Â of Â BPS. Â However, Â the Â growth Â of Â all Â of Â the Â strains, Â including Â the Â grx4 Â and Â cir1 Â mutants, Â was Â improved Â upon Â addition Â of Â iron Â thus Â suggesting Â that Â iron Â uptake Â is Â not Â impaired Â in Â both Â mutants. Â Â Â Â It Â has Â been Â shown Â that Â cir1 Â mutants Â have Â increased Â cell Â surface Â reductase Â activity, Â which Â appears Â as Â a Â red Â colony Â color Â on Â media Â with Â TTC Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â This Â phenotype Â is Â consistent Â with Â the Â derepression Â of Â transcript Â levels Â for Â ferric Â reductase Â genes Â in Â the Â cir1 Â mutant Â (W. Â H. Â Jung Â et Â al., Â 2010). Â Â As Â expected, Â colonies Â of Â the Â grx4 Â mutant Â showed Â an Â enhanced Â red Â color Â compared Â with Â the Â WT Â strain Â indicating Â higher Â cell Â surface Â reductase Â activity. Â Â I Â speculate Â that Â this Â phenotype Â is Â due Â to Â derepression Â of Â reductase Â gene Â transcription Â in Â the Â grx4 Â mutant Â similar Â to Â the Â cir1 Â mutant. Â Â Complementation Â of Â the Â grx4 Â deletion Â with Â GRX4 Â resulted Â in Â WT Â phenotypes Â in Â these Â assays Â indicating Â that Â the Â observed Â phenotypes Â are Â due Â to Â deletion Â of Â GRX4. Â Â Â Excess Â iron Â is Â damaging Â due Â to Â formation Â of Â reactive Â oxygen Â species Â via Â the Â Haber-­â Â Â Â Â Â Â Â Â Â Â Â Â Â 67 Â Weiss/Fenton Â reaction Â (J. Â A. Â Imlay, Â 2008, Â J. Â A. Â Imlay, Â et Â al., Â 1988). Â Â In Â this Â context, Â phleomycin Â is Â a Â glycopeptide Â antibiotic, Â which Â causes Â DNA Â damage Â in Â the Â presence Â of Â ferrous Â iron Â and Â oxygen Â due Â to Â the Â production Â of Â reactive Â oxygen Â species Â (L. Â F. Â Povirk Â et Â al., Â 1981). Â Â It Â was Â also Â shown Â previously Â that Â a Â cir1 Â mutant Â displays Â susceptibility Â to Â phleomycin Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Â Similar Â to Â cir1 Â mutant, Â we Â found Â that Â the Â grx4 Â mutant Â exhibits Â enhanced Â sensitivity Â to Â phleomycin Â relative Â to Â the Â WT Â and Â complemented Â strains. Â Â Overall, Â the Â accumulated Â iron-­ârelated Â phenotypes Â observed Â in Â the Â grx4 Â mutant Â indicated Â that Â Grx4 Â is Â required Â for Â growth Â in Â the Â presence Â of Â iron Â and Â overall Â maintenance Â of Â iron Â homeostasis Â in Â C. Â neoformans. Â Â Â Figure Â 2.9: Â A Â grx4 Â mutant Â displays Â iron-­ârelated Â phenotypes Â similar Â to Â the Â cir1 Â mutant. Â Â Spot Â assays Â starting Â with Â a Â culture Â of Â 2x108 Â cells Â per Â ml Â (106 Â cells Â in Â 5μl Â spotted) Â and Â 10-­âfold Â dilutions Â from Â left Â to Â right Â were Â performed Â on Â YPD Â media Â containing Â the Â various Â conditions Â indicated Â below Â the Â panels. Â Â These Â conditions Â include: Â (A) Â YPD Â without Â additions Â to Â demonstrate Â similar Â growth Â of Â the Â grx4 Â and Â cir1 Â mutants; Â (B) Â elevated Â iron Â levels Â (YPD+ Â 250 Â μM Â FeCl3) Â in Â which Â the Â grx4 Â and Â cir1 Â mutants Â show Â sensitivity; Â (C) Â an Â iron Â restricted Â condition Â (-­âFe, Â 400 Â μM Â BPS) Â in Â which Â both Â mutants Â have Â impaired Â growth; Â (D) Â an Â elevated Â iron Â level Â (+Fe, Â 400 Â μM Â BPS Â + Â 250 Â μM Â FeCl3) Â in Â grx4 Â and Â cir1 Â mutants Â Â Â Â Â Â Â Â Â Â Â Â Â Â 68 Â display Â sensitivity; Â (E) Â cell Â surface Â reductase Â activity Â as Â indicated Â by Â the Â red Â colony Â color Â in Â the Â presence Â of Â TTC, Â in Â which Â the Â grx4 Â and Â cir1 Â mutants Â display Â increased Â activity; Â and Â (F) Â susceptibility Â to Â phleomycin Â (0.25 Â g/ml) Â for Â the Â grx4 Â and Â cir1 Â mutants. Â Â I Â further Â analyzed Â the Â growth Â phenotypes Â of Â the Â grx4 Â and Â cir1 Â mutants, Â along Â with Â the Â WT Â strain Â and Â the Â grx4Î::GRX4 Â complemented Â strain Â in Â the Â iron-­ârestricted Â condition Â in Â liquid Â assays. Â Â The Â strains Â were Â first Â grown Â for Â two Â days Â in Â low-­âiron Â medium Â (LIM-­âYNB Â containing Â 150 Â μM Â of Â the Â iron Â chelator Â BPS) Â to Â exhaust Â intracellular Â iron Â stores. Â Supplementation Â of Â this Â medium Â with Â 25 Â μM, Â 50 Â μM Â or Â 100 Â μM Â ferric Â iron Â generated Â iron-­âreplete Â conditions. Â Â After Â iron Â starvation, Â I Â tested Â the Â growth Â of Â all Â of Â the Â strains Â in Â YNB-­âLIM Â without Â or Â with Â the Â various Â levels Â of Â FeCl3. Â Â The Â growth Â assays Â revealed Â that Â the Â grx4 Â mutant Â does Â not Â respond Â to Â the Â presence Â of Â iron Â at Â lower Â concentrations Â (25 Â μM, Â 50 Â μM) Â (Figure Â 2.10 Â A). Â Analysis Â of Â the Â growth Â of Â the Â grx4 Â mutant Â in Â the Â presence Â of Â 100 Â μM Â FeCl3 Â showed Â that Â this Â concentration Â of Â iron Â in Â liquid Â culture Â supports Â growth. Â Â However, Â I Â did Â observe Â that Â the Â grx4 Â mutant Â had Â a Â prominent Â growth Â defect Â compared Â with Â the Â parental Â WT Â strain, Â the Â cir1 Â mutant Â and Â the Â grx4Î::GRX4 Â complemented Â strain Â (Figure Â 2.10). Â Â As Â expected, Â none Â of Â the Â strains Â grew Â in Â LIM Â after Â 48 Â hours Â of Â pre-­âstarvation Â for Â iron Â (Figure Â 2.10). Â Â Overall, Â these Â results Â indicated Â that Â Grx4 Â is Â required Â for Â robust Â growth Â in Â the Â presence Â of Â iron Â and Â further Â highlighted Â the Â contribution Â of Â Grx4 Â to Â iron Â homeostasis Â in Â C. Â neoformans. Â Â Based Â on Â the Â iron-­ârelated Â phenotypes, Â I Â speculated Â that Â grx4 Â mutant Â might Â be Â particularly Â susceptible Â to Â oxidative Â stress Â upon Â iron Â starvation Â and/or Â iron Â repletion Â leading Â to Â impaired Â growth. Â It Â has Â been Â shown Â that Â GSH Â protects Â against Â detrimental Â effects Â of Â iron-­âdeficiency Â and Â iron Â toxicity Â due Â to Â its Â antioxidant Â properties Â (L. Â M. Â Milchak Â Â Â Â Â Â Â Â Â Â Â Â Â Â 69 Â and Â J. Â Douglas Â Bricker, Â 2002) Â (L. Â Ramirez Â et Â al., Â 2013). Â This Â prompted Â me Â to Â test Â the Â effect Â of Â GSH Â on Â growth Â defect Â of Â grx4 Â mutant Â in Â presence Â of Â iron Â after Â a Â period Â of Â iron Â starvation. Â Interestingly, Â the Â growth Â defect Â of Â the Â grx4 Â mutant Â was Â rescued Â by Â exogenous Â GSH Â as Â shown Â in Â Figure Â 2.11. Â This Â observation Â prompted Â us Â to Â investigate Â the Â potential Â physiological Â role Â of Â glutathione Â in Â iron Â homeostasis Â in Â C. Â neoformans, Â which Â has Â not Â been Â investigated Â in Â pathogenic Â fungi. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 70 Â Â Figure Â 2.10: Â Grx4 Â is Â required Â for Â growth Â in Â iron-­âdepleted Â media Â supplemented Â with Â FeCl3. Â The Â strains Â were Â pre-­âstarved Â for Â iron Â in Â LIM Â and Â grown Â in Â LIM Â supplemented Â with Â increasing Â concentrations Â of Â FeCl3: Â 25 Â μM Â (A), Â 50 Â μM Â (B), Â 100 Â μM Â (C). Â Growth Â was Â monitored Â by Â measuring Â the Â optical Â density Â at Â 600 Â nm. Â Â Data Â are Â presented Â as Â the Â average± Â S.D. Â Â As Â expected, Â none Â of Â the Â pre-­âstarved Â strains Â grew Â in Â the Â absence Â of Â iron Â (LIM). Â Â LIM0 24 48 72 96012345Time-hrsOD 600 nmWT-H99grx4!grx4!::GRX4Cir1!B Â A Â LIM+100µM Fecl30 24 48 72 96012345Time (H)OD 600 nmWT-H99grx4!::GRX4grx4!Cir1!LIM+ 25µM FeCl30 24 48 72 96012345Time-hrsOD 600 nmWT-H99grx4!grx4!::GRX4Cir1!C Â D Â Â Â Â Â Â Â Â Â Â Â Â Â Â 71 Â Â Â Figure Â 2.11: Â Addition Â of Â GSH Â restored Â growth Â of Â grx4 Â mutant Â under Â iron-­âreplete Â conditions. Â The Â strains Â were Â pre-­âstarved Â for Â iron Â in Â LIM Â and Â pre-­âgrown Â in Â LIM Â supplemented Â with Â 100 Â μM Â FeCl3 Â for Â 12 Â hours. Â Cells Â were Â washed Â and Â re-­âintroduced Â to Â LIM+ Â 100 Â μM Â FeCl3 Â (A) Â or Â LIM+ Â 100 Â μM Â FeCl3 Â + Â 5mM Â GSH Â dissolved Â in Â in Â 25 Â mM Â Hepes Â buffer Â pH Â 7.4 Â (B). Â Growth Â was Â then Â monitored Â by Â measuring Â the Â optical Â density Â at Â 600 Â nm Â for Â 72 Â hours. Â Â The Â experiments Â were Â performed Â three Â times Â and Â the Â data Â are Â presented Â as Â the Â average± Â S.D. Â Â Â 2.3.2.3 Â Grx4 Â is Â required Â for Â inhibition Â of Â iron Â uptake Â in Â the Â presence Â of Â iron Â Â Â The Â iron-­ârelated Â phenotypes Â of Â the Â grx4 Â mutant Â prompted Â me Â to Â investigate Â which Â C. Â neoformans Â genes Â involved Â in Â iron Â uptake Â might Â be Â influenced Â by Â loss Â of Â GRX4. Â I Â first Â compiled Â a Â list Â of Â genes Â that Â are Â known Â to Â influence Â iron Â uptake Â in Â C. Â neoformans Â including Â ferric Â reductases Â (FRE1-­â6), Â siderophore Â transporters Â (SIT1, Â STR3, Â MIRB), Â components Â of Â the Â high-­âaffinity Â uptake Â system Â (CFT1, Â CFO1), Â and Â candidate Â genes Â for Â a Â low-­âaffinity Â uptake Â system Â (CFT2, Â CFO2). Â Â I Â next Â compared Â the Â transcript Â levels Â of Â these Â genes Â in Â the Â WT Â strain Â Â Â Â Â Â Â Â Â Â Â Â Â Â 72 Â and Â the Â grx4 Â mutant Â under Â iron-­âdeplete Â or Â iron-­âreplete Â conditions Â by Â quantitative Â Real-­âTime Â PCR Â (q-­âRT-­âPCR) Â (See Â Materials Â and Â Methods, Â section Â 2.2.12). Â Under Â low-­âiron Â conditions, Â I Â did Â not Â detect Â significant Â differential Â transcript Â levels Â for Â the Â genes Â of Â interest Â in Â the Â grx4 Â mutant Â compared Â to Â the Â WT Â strain. Â Â However, Â under Â the Â high-­âiron Â condition, Â I Â found Â that Â the Â transcripts Â for Â the Â genes Â encoding Â the Â ferric Â reductases Â FRE2 Â and Â FRE6, Â the Â siderophore Â transporters, Â and Â the Â high-­âaffinity Â uptake Â system Â were Â highly Â upregulated Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â (Figure Â 2.11). Â Together, Â these Â results Â indicated Â that Â Grx4 Â is Â required Â for Â inhibition Â of Â the Â expression Â of Â iron Â acquisition Â functions Â in Â C. Â neoformans Â in Â high Â iron Â conditions. Â Â Also, Â loss Â of Â Grx4 Â leads Â to Â constitutive Â de-­ârepression Â of Â genes Â that Â are Â normally Â upregulated Â in Â the Â WT Â strain Â under Â low-­âiron Â conditions Â to Â provide Â a Â sufficient Â amount Â of Â iron Â for Â Fe-­âdependent Â pathways. Â Â This Â is Â in Â agreement Â with Â the Â iron-­ârelated Â phenotypes Â observed Â in Â the Â grx4 Â mutant, Â including Â sensitivity Â to Â elevated Â iron Â levels. Â It Â is Â likely Â that Â de-­ârepression Â of Â iron Â uptake/acquisition Â under Â high Â iron Â levels Â in Â the Â grx4 Â mutant Â leads Â to Â iron Â toxicity Â and Â increased Â oxidative Â stress; Â this Â in Â turn Â might Â account Â for Â the Â impaired Â growth Â of Â the Â grx4 Â mutant Â in Â the Â presence Â of Â iron. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 73 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Figure Â 2.12: Â Grx4 Â has Â a Â negative Â regulatory Â influence Â on Â the Â transcript Â levels Â for Â iron Â acquisition Â functions. Â Quantitative Â RT-­âPCR Â was Â used Â to Â measure Â the Â levels Â of Â transcripts Â of Â candidate Â genes Â in Â the Â grx Â mutant Â compared Â to Â the Â WT Â strain Â in Â iron-­âreplete Â conditions. Â The Â data Â were Â normalized Â using Â 18S Â rRNA Â as Â an Â internal Â control Â and Â are Â presented Â as Â relative Â expression Â in Â comparison Â to Â the Â WT Â value Â set Â at Â 1. Â The Â data Â from Â 3 Â separate Â experiments Â (biological Â replicates), Â each Â of Â which Â had Â 3 Â technical Â replicates Â was Â combined, Â and Â the Â bars Â represent Â standard Â deviations. Â Â 012345678910Relative expressionLIM+Fegrx4! vs. WTCNAG_07387 siderophore transporter 3 (STR3)CNAG_00815 siderophore-iron:H symporter (SIT1)CNAG_07751 siderophore iron transporter (MIRB)CNAG_02959 high-affinity iron transporter (CFT1)CNAG_02958 ferroxidase (CFO1)CNAG_06976, Ferric chelate reductase 6, (FRE6)CNAG_06821, Ferric chelate reductase 2, (FRE2) Â Â Â Â Â Â Â Â Â Â Â Â Â 74 Â 2.3.2.4 Â Â Grx4 Â influences Â the Â virulence Â of Â C. Â neoformans. Â 2.3.2.4.1 Â Â The Â grx4 Â mutant Â displays Â virulence-­ârelated Â phenotypes Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â The Â polysaccharide Â capsule Â is Â one Â of Â the Â major Â virulence Â traits Â of Â C. Â neoformans Â and, Â given Â that Â iron Â depletion Â results Â in Â an Â enlarged Â capsule, Â I Â evaluated Â the Â effect Â of Â GRX4 Â disruption Â on Â capsule Â formation. Â Â It Â had Â been Â shown Â previously Â that Â a Â cir1 Â mutant Â is Â acapsular Â in Â both Â serotype Â D Â and Â serotype Â A Â strains Â (W. Â H. Â Jung, Â 2006). Â Similar Â to Â the Â cir1 Â mutant, Â I Â did Â not Â detect Â a Â capsule Â on Â cells Â of Â the Â grx4 Â mutant; Â in Â contrast, Â both Â the Â WT Â and Â complemented Â strains Â displayed Â large Â capsules Â (Figure Â 2. Â 12 Â A). Â Â These Â results Â demonstrated Â that Â Grx4 Â is Â required Â for Â capsule Â formation, Â most Â likely Â through Â its Â influence Â on Â iron Â homeostasis. Â Â Â I Â next Â examined Â whether Â other Â important Â virulence Â traits Â such Â as Â the Â ability Â to Â grow Â at Â host Â temperature Â (37°C) Â and Â melanin Â formation Â were Â altered Â in Â the Â grx4 Â mutant. Â Â The Â growth Â of Â the Â grx4 Â and Â cir1 Â mutants Â resembled Â the Â WT Â and Â reconstituted Â grx4Î::GRX4 Â strains Â at Â 30°C, Â but Â both Â mutants Â grew Â poorly Â at Â 37°C Â (Figure Â 2. Â 13). Â Â It Â is Â known Â that Â Cir1 Â links Â iron Â regulation Â and Â expression Â of Â LAC1 Â C. Â neoformans Â (W. Â H. Â Jung, Â 2006). Â Here, Â I Â tested Â formation Â of Â melanin Â on Â DOPA Â medium Â at Â 0.1% Â glucose Â in Â the Â grx4 Â mutant Â and Â found Â that Â compared Â to Â WT, Â the Â grx4 Â mutant Â produced Â a Â slightly Â reduced Â amount Â of Â melanin Â represented Â by Â the Â light Â grey Â color Â pigment Â in Â grx4 Â mutant Â compared Â to Â the Â black Â one Â in Â WT Â (Figure Â 2.13 Â C). Â Overall Â these Â results Â show Â that Â Grx4 Â influences Â major Â virulence-­âassociated Â traits Â in Â C. Â neoformans. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 75 Â Â Â Â Â Â Â Â Â Â Figure Â 2.13: Â Â A Â grx4 Â mutant Â does Â not Â express Â virulence-­ârelated Â traits. Â Loss Â of Â the Â C-­âterminal Â Grx Â domain Â in Â a Â grx4 Â mutant Â leads Â to Â virulence-­ârelated Â phenotypes Â including Â loss Â of Â capsule Â formation Â (A), Â poor Â growth Â at Â 37°C Â (B) Â and Â altered Â melanin Â production Â (C). Â Â Â Â Â WT Â Â grx4Î Â Â grx4Î::GRX4 cir1Î B C Â 106 Â YPD-­â37°C Â L-­âDOPA Â Â Â Â Â Â Â Â Â Â Â Â Â Â 76 Â 2.3.2.4.2 Â Â Deletion Â of Â grx4 Â abrogates Â virulence Â in Â a Â mouse Â model Â of Â cryptococcosis Â Â Â My Â phenotypic Â analysis Â including Â the Â impact Â of Â the Â grx4 Â deletion Â on Â virulence-­ârelated Â phonotypes Â strongly Â suggested Â that Â the Â mutant Â would Â have Â a Â defect Â in Â its Â ability Â to Â cause Â disease Â in Â mice. Â This Â prediction Â was Â tested Â by Â inoculating Â 10 Â female Â BALB/c Â mice Â intranasally Â with Â cells Â of Â the Â WT Â strain, Â the Â grx4 Â mutant Â or Â the Â complemented Â strain. Â Â All Â mice Â infected Â with Â the Â WT Â and Â complemented Â cells Â succumbed Â to Â infection Â between Â days Â 18 Â and Â 21, Â while Â the Â mice Â infected Â with Â the Â grx4 Â mutant Â did Â not Â show Â disease Â symptoms Â and Â survived Â for Â the Â duration Â of Â the Â experiment Â (i.e., Â 60 Â days) Â (Figure Â 2.14). Â These Â results Â indicated Â that Â Grx4 Â is Â critical Â for Â the Â virulence Â of Â C. Â neoformans Â and Â I Â speculate Â that Â due Â to Â the Â significance Â of Â Grx4 Â in Â iron Â homeostasis Â and Â role Â of Â iron Â in Â regulation Â in Â virulence, Â Grx4 Â links Â iron Â homeostasis Â and Â virulence. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Figure Â 2.14: Â loss Â of Â GRX4 Â abolished Â virulence. Â Ten Â female Â BALB/c Â mice Â were Â infected Â intranasally Â with Â the Â WT Â strain Â (black Â triangles), Â the Â grx4 Â mutant Â (pink Â circles) Â or Â the Â complemented Â strain Â (filled Â brown Â triangles). Â Â The Â survival Â of Â the Â mice Â is Â shown Â versus Â *** Â Â Â Â Â Â Â Â Â Â Â Â Â 77 Â time Â in Â days. Â Â Statistical Â analysis Â was Â performed Â using Â ordinary Â one-­âway Â ANOVA. Â The Â (***) Â symbol Â represents Â significant Â differences Â with Â a Â P Â < Â 0.0006. Â Â This Â experiment Â was Â performed Â in Â collaboration Â with Â Dr. Â Guanggan Â Hu Â and Â Dr. Â Melissa Â Caza. Â Â Furthermore, Â the Â mice Â from Â each Â group Â were Â used Â at Â the Â time Â of Â sacrifice Â to Â analyze Â the Â distribution Â of Â fungal Â cells Â in Â different Â tissues Â including Â the Â lungs, Â kidney, Â liver, Â spleen, Â brain Â and Â blood. Â Â The Â numbers Â of Â fungal Â cells Â in Â the Â tissues Â of Â mice Â infected Â with Â WT Â or Â the Â complemented Â strain Â were Â comparable, Â but Â the Â grx4 Â mutant Â cells Â were Â 2-­â4 Â orders Â of Â magnitude Â lower Â than Â the Â WT Â level Â suggesting Â a Â defect Â in Â dissemination Â and/or Â colonization Â of Â organs Â by Â the Â mutant. Â (Figure Â 2.15) Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 78 Â Â Figure Â 2.15: Â Determination Â of Â fungal Â loads Â in Â mice Â tissue. Â The Â fungal Â burden Â in Â tissues Â Â from Â 3 Â mice Â from Â each Â group Â was Â determined Â by Â counting Â the Â CFU. Â The Â organs Â were Â LiverLog10 CFU/gWT grx4! grx4!::GRX40246LungsLog10 CFU/gWT grx4! grx4!::GRX40246810BrainLog10 CFU/gWT grx4! grx4!::GRX402468SpleenLog 10 CFU/gWT grx4! grx4!::GRX402468KidneyLog 10 CFU/gWT grx4! grx4!::GRX40246BloodLog10 CFU/mlWT grx4! grx4!::GRX401234 Â Â Â Â Â Â Â Â Â Â Â Â Â 79 Â collected Â at Â the Â endpoints Â on Â day Â 21 Â for Â WT, Â 26 Â for Â complemented Â strain, Â and Â 60 Â for Â grx4 Â mutant. Â 2.3.3 Â Â A Â pilot Â transcriptomic Â analysis Â using Â RNA-­âseq Â provided Â further Â insight Â into Â role Â of Â Grx4 Â and Â also Â revealed Â complexities Â in Â the Â transcript Â structure Â of Â GRX4. Â 2.3.3.1 Â Grx4 Â is Â required Â for Â broad Â transcriptional Â remodeling Â in Â response Â to Â iron Â in Â C. Â neoformans Â Â Â Â Fortuitously, Â I Â also Â had Â an Â opportunity Â to Â investigate Â the Â influence Â of Â Grx4 Â on Â transcriptional Â remodeling Â in Â response Â to Â iron Â levels Â in Â C. Â neoformans Â in Â a Â pilot Â RNA-­âSeq Â experiment Â offered Â by Â the Â NAPS Â Unit Â of Â the Â Michael Â Smith Â Laboratories. Â The Â NAPS Â unit Â needed Â RNA Â samples Â to Â test Â their Â methods Â for Â RNA-­âSeq Â library Â preparation Â and Â additionally Â offered Â complementary Â sequencing. Â Â I Â took Â advantage Â of Â this Â offer Â to Â add Â depth Â to Â the Â characterization Â of Â the Â grx4 Â mutant. Â Â Specifically, Â I Â wanted Â to Â further Â investigate Â the Â connection Â with Â iron Â homeostasis Â indicated Â by Â the Â aforementioned Â discovery Â of Â iron-­ârelated Â phenotypes Â (Figures Â 2.9-­â2.12) Â and Â the Â conserved Â role Â of Â Grxs Â in Â iron Â homeostasis Â in Â other Â fungi Â (N. Â Rouhier, Â et Â al., Â 2010). Â Â For Â this Â pilot Â experiment, Â the Â transcriptional Â profiles Â of Â the Â WT Â cells Â and Â the Â grx4 Â mutant Â were Â compared Â under Â iron-­âstarved Â and Â iron Â overload Â conditions Â using Â RNA-­âSeq. Â Â Briefly, Â iron-­âstarved Â cells Â were Â prepared Â for Â the Â WT Â strain Â and Â the Â grx4 Â mutant Â followed Â by Â growth Â for Â 6 Â h Â in Â LIM Â or Â LIM+Fe Â prior Â to Â RNA Â extraction Â (T. Â Lian, Â et Â al., Â 2005). Â Â It Â was Â previously Â shown Â that Â iron Â response Â becomes Â manifest Â within Â 6 Â hours Â See Â Materials Â and Â Methods, Â section Â 2.2.11, Â for Â the Â experimental Â set Â up). Â Â Due Â to Â the Â pilot Â nature Â of Â the Â experiment, Â RNA Â samples Â were Â prepared Â from Â only Â one Â biological Â replicate Â of Â the Â WT Â strain Â and Â the Â grx4 Â mutant Â to Â perform Â the Â transcriptome Â analyses Â Â (NAPS Â Unit, Â UBC). Â Â Â Â Â Â Â Â Â Â Â Â Â Â 80 Â Analysis Â of Â the Â RNA-­âSeq Â data Â was Â performed Â in Â collaboration Â with Â Dr. Â Daniel Â Croll Â using Â the Â R Â package Â cummeRbund Â ggplot2 Â version Â 0.9.3 Â (see Â Materials Â and Â Methods, Â 2.2.11). Â Â Â Although Â we Â did Â not Â have Â the Â biological Â and Â technical Â replicates Â to Â provide Â robust Â statistical Â confidence, Â we Â performed Â a Â preliminary Â analysis Â to Â identify Â differentially Â expressed Â genes Â (using Â a Â 3-­âfold Â cut Â off) Â in Â the Â grx4 Â mutant Â relative Â to Â the Â WT Â strain Â in Â LIM Â or Â LIM+Fe Â (Figure Â 2.16 Â and Â 2.17). Â Interestingly, Â differential Â transcript Â levels Â seen Â for Â genes Â such Â as Â the Â ferric Â reductase Â Fre Â 2, Â ferric-­âchelate Â reductase-­â Â Fre6 Â , Â siderophore-­âiron Â transporter Â Str3, Â and Â siderophore Â iron Â transporter Â mirB Â identified Â by Â Quantitative Â RT-­âPCR Â (Figure Â 2.11) Â also Â showed Â up Â in Â the Â RNA-­âSeq Â data. Â Â For Â the Â work Â in Â this Â thesis, Â we Â were Â able Â to Â analyze Â Gene Â Ontology Â (GO) Â terms Â in Â the Â RNA-­âseq Â data Â to Â generate Â an Â overview Â of Â the Â enriched Â functions Â among Â the Â differentially Â expressed Â genes Â in Â grx4 Â mutant Â compared Â to Â WT Â strain Â (GO Â terms Â available Â on Â locally Â installed Â Blast2GO Â were Â used Â as Â outlined Â in Â the Â Materials Â and Â Methods, Â section Â 2.2.11). Â Â As Â described Â below, Â the Â results Â from Â the Â GO Â term Â analysis Â revealed Â that Â the Â majority Â of Â differentially Â expressed Â genes Â in Â grx4 Â mutant Â were Â enriched Â for Â GO Â terms Â that Â particularly Â and Â notably Â were Â associated Â with Â iron Â sensing Â and Â the Â maintenance Â of Â iron Â homeostasis. Â Â 2.3.3.1.1 Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â grown Â under Â the Â iron-­âdeplete Â condition. Â Â We Â found Â that Â a Â total Â of Â 35 Â genes Â were Â down-­âregulated Â and Â 137 Â upregulated Â in Â the Â grx4 Â mutant Â in Â comparison Â with Â WT Â for Â cells Â grown Â under Â low-­âiron Â conditions. Â Â The Â GO Â term Â enrichment Â of Â these Â differentially-­âexpressed Â genes Â revealed Â that Â the Â main Â molecular Â Â Â Â Â Â Â Â Â Â Â Â Â Â 81 Â functions Â affected Â by Â the Â absence Â of Â Grx4 Â under Â the Â iron-­âdepleted Â condition Â included Â oxidoreductase Â activity Â (GO:0016491), Â Fe-­âS Â cluster Â binding Â (GO:0051536), Â and Â metal Â cluster Â binding Â (GO:0051540). Â The Â top Â 20 Â genes Â in Â these Â GO Â categories Â are Â listed Â in Â Appendix Â A: Â Table Â A2. Â Of Â note, Â the Â top-­âthree Â enriched Â GO Â terms Â in Â the Â grx4 Â mutant Â were Â iron-­âconsuming Â processes Â that Â were Â normally Â downregulated Â in Â the Â WT Â strain Â under Â iron-­âdeplete Â conditions. Â Â This Â result Â suggests Â that Â lack Â of Â Grx4 Â might Â lead Â to Â loss Â of Â inhibition Â of Â the Â expression Â of Â iron-­âconsuming Â functions Â during Â iron Â deficiency. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 82 Â Â Â Figure Â 2.16: Â Enrichment Â of Â genes Â differentially Â expressed Â in Â RNA-­âSeq Â analysis Â of Â the Â grx4 Â mutant Â and Â the Â WT Â strain Â under Â the Â iron-­âdeplete Â condition. Â Â GO Â term Â enrichment Â was Â based Â on Â molecular Â function Â and Â visualized Â using Â the Â R Â package Â ggplot2. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 83 Â 2.3.3.1.2 Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â grown Â under Â the Â iron-­âreplete Â condition. Â Â Â The Â analysis Â of Â the Â RNA-­âseq Â data Â from Â cells Â grown Â under Â the Â iron-­âreplete Â condition Â revealed Â 17 Â genes Â with Â reduced Â transcript Â levels Â and Â 228 Â with Â increased Â transcript Â levels Â in Â the Â grx4 Â mutant Â versus Â WT. Â Â The Â top Â three Â GO Â terms Â for Â these Â differentially Â expressed Â transcripts Â included Â oxidoreductase Â activity Â (GO:0016491), Â cation Â transporter Â activity Â (GO:00115101), Â and Â antioxidant Â activity Â (GO:0016209). Â The Â top Â 20 Â corresponding Â genes Â to Â the Â top Â three Â GO Â terms Â in Â the Â grx4 Â mutant Â in Â the Â iron-­âreplete Â condition Â are Â listed Â in Â Appendix Â A: Â Table Â A3. Â Â Interestingly, Â this Â list Â includes Â some Â of Â the Â iron Â acquisition Â genes Â such Â as Â the Â ferric Â reductases Â FRE2 Â and Â FRE6 Â and Â the Â siderophore Â transporters Â STR3 Â and Â MIRB Â that Â previously Â showed Â upregulation Â in Â the Â grx4 Â mutant Â compared Â to Â the Â WT Â in Â my Â qRT-­âPCR Â analysis Â under Â iron-­âreplete Â condition Â (Figure Â 2.12). Â Of Â note, Â these Â iron Â acquisition Â genes Â are Â normally Â upregulated Â in Â WT Â under Â iron Â starvation. Â Â Overall, Â the Â results Â from Â the Â pilot Â RNA-­âseq Â analysis Â further Â supported Â the Â hypothesis Â that Â Grx4 Â is Â required Â for Â inhibition Â of Â iron-­âacquiring Â functions Â in Â C. Â neoformans Â in Â high Â iron Â conditions, Â and Â also Â contributes Â to Â maintenance Â of Â iron Â homeostasis Â in Â C. Â neoformans. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 84 Â Â Â Â Figure Â 2.17: Â Enrichment Â of Â genes Â differentially Â expressed Â in Â the Â RNA-­âSeq Â analysis Â of Â the Â grx4 Â mutant Â and Â the Â WT Â strain Â in Â the Â iron-­âreplete Â condition. Â Â GO Â term Â enrichment Â was Â based Â on Â molecular Â function Â and Â visualized Â using Â the Â R Â package Â ggplot2. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 85 Â Â Furthermore, Â differentially Â regulated Â genes Â with Â similar Â and Â distinct Â patterns Â of Â expression Â in Â grx4 Â and Â cir1 Â mutants Â compared Â to Â the Â WT Â in Â iron-­âdeplete Â and Â iron Â replete Â conditions Â were Â listed Â for Â future Â reference Â (Appendix Â A, Â Table Â A4-­â Â A7) Â 2.3.3.2 Â Analysis Â of Â the Â data Â from Â the Â pilot Â RNA-­âseq Â analysis Â identified Â several Â putative Â transcripts Â associated Â with Â the Â GRX4 Â locus. Â Â Â During Â the Â analysis Â of Â the Â GRX4 Â deletion Â mutant, Â I Â realized Â that Â the Â annotation Â of Â the Â gene Â in Â the Â genome Â database Â (Broad) Â was Â modified Â with Â regard Â to Â intron-­âexon Â positions Â as Â part Â of Â the Â community Â effort Â to Â characterize Â and Â publish Â the Â genome Â of Â the Â WT Â strain Â H99 Â (G. Â Janbon Â et Â al., Â 2014). Â This Â change Â in Â the Â annotation Â prompted Â me Â to Â use Â the Â RNA-­âSeq Â data Â in Â hand Â to Â examine Â the Â experimental Â evidence Â for Â different Â isoforms Â from Â the Â GRX4 Â locus. Â Â For Â this Â analysis, Â the Â TopHat Â program Â (version Â 2.0.9) Â was Â used Â to Â align Â and Â to Â assemble Â the Â reads Â from Â the Â RNA-­âSeq Â analysis Â into Â transcripts, Â again Â in Â collaboration Â with Â Dr. Â D. Â Croll Â (Materials Â and Â Methods Â (section Â 2.2.11). Â Â The Â transcripts Â were Â then Â compared Â to Â the Â reference Â H99 Â C. Â neoformans Â serotype Â A Â genome Â annotation Â http://www.broadinstitute.org Â (Jan Â 2014) Â using Â Cufflinks Â 2.1.1, Â which Â was Â also Â used Â for Â further Â examination Â of Â transcriptomic Â data Â and Â de Â novo Â assembly Â of Â transcripts. Â Â Â This Â analysis Â led Â to Â identification Â of Â three Â putative Â unannotated Â transcripts Â associated Â with Â the Â GRX4 Â locus Â in Â addition Â to Â the Â main Â transcript Â that Â I Â have Â used Â as Â the Â gene Â model Â for Â the Â work Â described Â in Â this Â thesis Â (Figure Â 2.18). Â Â De Â novo Â assembly Â of Â sequences Â showed Â that Â the Â three Â additional Â putative Â transcripts Â isoforms Â contain Â the Â conserved Â C-­âterminal Â Grx Â domain Â and Â variation Â among Â these Â transcript Â versions Â arises Â from Â differential Â inclusion Â of Â exons Â from Â the Â 5â Â region Â of Â GRX4 Â locus. Â Â These Â results Â are Â Â Â Â Â Â Â Â Â Â Â Â Â Â 86 Â interesting Â because Â it Â is Â possible Â that Â the Â identified Â transcripts Â might Â encode Â variants Â of Â Grx4 Â with Â different Â functions, Â e.g., Â in Â binding Â proteins Â associated Â with Â Grx4. Â Of Â note, Â no Â isoform Â in Â our Â analysis Â was Â identical Â to Â the Â new Â GRX4 Â annotation Â available Â for Â the Â H99 Â genome Â provided Â by Â the Â Broad Â Institute Â (http://www.broadinstitute.org; Â January, Â 2014). Â Â This Â could Â possibly Â be Â due Â to Â differences Â in Â the Â conditions Â that Â were Â used Â in Â our Â RNA-­âseq Â analysis Â compared Â to Â those Â used Â in Â the Â studies Â that Â led Â to Â re-­âannotation Â of Â reference Â genome. Â Specifically, Â the Â conditions Â in Â our Â analysis Â were Â iron Â dependent Â whereas Â the Â conditions Â included Â in Â the Â published Â study Â included Â rich Â media Â (YPD), Â starvation Â medium Â (low Â glucose Â and Â low Â nitrogen Â medium), Â and Â pigeon Â guano Â broth. Â Â On Â the Â same Â note, Â a Â separate Â analysis Â performed Â by Â Dr. Â Croll Â on Â RNA-­âSeq Â data Â for Â the Â GRX4 Â transcripts Â obtained Â from Â available Â published Â RNA-­âseq Â libraries Â in Â different Â strain Â backgrounds Â and Â conditions Â also Â identified Â similar Â transcript Â variants Â (data Â not Â shown). Â Â The Â structures Â and Â expression Â patterns Â of Â the Â different Â transcript Â isoforms Â need Â to Â be Â independently Â validated Â in Â future Â experiments Â as Â a Â prelude Â to Â assessing Â their Â functional Â significance. Â Â Â Of Â note, Â the Â combined Â RNA-­âSeq Â analysis Â of Â the Â GRX4 Â region Â confirmed Â the Â existence Â of Â the Â transcript Â that Â was Â used Â for Â the Â studies Â reported Â here. Â Â Furthermore, Â this Â isoform Â encodes Â the Â polypeptide Â with Â the Â peptides Â that Â were Â experimentally Â identified Â in Â the Â initial Â mass Â spectrometry Â experiment Â outlined Â at Â the Â beginning Â of Â this Â chapter. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 87 Â Â Figure Â 2.18: Â Exonâintron Â organisation Â of Â C. Â neoformans Â GRX4 Â locus. Â The Â organization Â according Â to Â (A) Â H99 Â database Â Jan Â 2014 Â (B) Â H99 Â database Â 2009 Â (C-­âF). Â RNA-­âseq Â data Â in Â this Â study Â is Â shown. Â Â The Â boxes Â represent Â the Â exons, Â whilst Â the Â dashed Â line Â represents Â introns. Â One Â isoform Â in Â our Â RNA-­âseq Â data Â (F) Â matches Â the Â Broad Â annotation Â in Â 2009 Â (B). Â No Â isoform Â is Â identical Â to Â the Â new Â Broad Â annotation Â (2014). Â Â 2.3.3.3 Â Analysis Â of Â GRX4 Â essentiality Â 2.3.3.3.1 Â Deletion Â of Â the Â entire Â GRX4 Â including Â the Â N-­âterminal Â region Â in Â a Â stable Â diploid Â (strain Â AI187) Â background Â shows Â that Â the Â N-­âterminal Â region Â of Â GRX4 Â is Â not Â essential. Â Â The Â RNA-­âseq Â analysis Â of Â the Â C. Â neoformans Â transcriptome Â indicated Â the Â existence Â of Â different Â transcript Â isoforms Â from Â the Â GRX4 Â locus. Â These Â isoforms Â mainly Â showed Â variation Â in Â expression Â of Â the Â N-­âterminal Â region Â of Â Grx4 Â and Â this Â led Â us Â to Â speculate Â that Â N-­âterminal Â region Â contributes Â to Â the Â biological Â role Â of Â Grx4 Â in Â C. Â neoformans. Â Â Â Given Â that Â the Â deletion Â mutant Â characterized Â to Â date Â retained Â the Â coding Â sequence Â for Â the Â N-­âterminal Â Â Â Â Â Â Â Â Â Â Â Â Â Â 88 Â region, Â I Â wanted Â to Â investigate Â the Â outcome Â of Â deletion Â of Â entire Â GRX4 Â locus. Â Â A Â construct Â was Â generated Â using Â a Â (NEO) Â marker Â linked Â to Â 5â² Â and Â 3â² Â flanking Â sequences Â of Â the Â entire Â GRX4 Â by Â three-­âstep Â overlapping Â PCR Â (Figure Â 2.19 Â A), Â and Â the Â overlap Â PCR Â product Â was Â biolistically Â transformed Â into Â haploid Â H99 Â strain Â (See Â materials Â and Â methods Â 2.2.2.2). Â Surprisingly, Â numerous Â attempts Â to Â delete Â the Â entire Â locus Â in Â a Â haploid Â strain Â were Â unsuccessful. Â Â Â Â The Â complexity Â of Â the Â GRX4 Â locus Â structure Â shown Â in Â RNA-­âseq Â data, Â and Â the Â failure Â to Â delete Â the Â entire Â locus Â including Â the Â N-­âterminal Â region, Â led Â me Â to Â test Â whether Â the Â N-­âterminal Â region Â is Â essential. Â Â For Â this Â approach, Â I Â employed Â a Â commonly Â used Â technique Â to Â identify Â essential Â genes Â in Â C. Â neoformans Â and Â fungi Â in Â general, Â which Â is Â targeted Â gene Â replacement Â in Â a Â diploid Â strain Â with Â subsequent Â sporulation Â and Â phenotypic Â analysis Â of Â the Â resulting Â meiotic Â progeny Â (A. Â Idnurm, Â 2010). Â The Â deletion Â construct Â was Â biolistically Â transformed Â into Â the Â diploid Â strain Â AI187 Â (see Â section Â 2.2.2.2) Â and Â deletion Â of Â full-­âlength Â GRX4 Â in Â the Â diploid Â H99 Â background Â was Â confirmed Â by Â PCR. Â Â Â Â Positive Â transformants Â were Â subjected Â to Â sporulation Â (See Â Materials Â and Â Methods Â section Â 2.2.2.2.). Â Â Basidiospores Â were Â collected Â and Â incubated Â on Â YPD Â at Â 30â°C Â and Â then Â transferred Â individually Â to Â YPD Â medium Â containing Â NEO Â (Figure Â 2.19 Â B). Â Spores Â that Â grew Â in Â presence Â of Â NEO Â were Â selected Â for Â further Â analysis. Â Â Deletion Â of Â GRX4 Â was Â first Â confirmed Â by Â PCR Â in Â the Â colonies Â that Â formed Â on Â YPD+ Â NEO Â (Figure Â 2.19 Â B), Â while Â colonies Â formed Â on Â YPD Â but Â not Â YPD+ Â NEO Â were Â shown Â to Â carry Â the Â WT Â copy Â of Â GRX4. Â Â This Â was Â tested Â by Â PCR Â using Â primers Â that Â amplified Â an Â internal Â region Â within Â GRX4 Â to Â yield Â a Â fragment Â of Â 650 Â bp Â (Figure Â 2.19 Â A). Â As Â expected, Â colonies Â with Â deletion Â of Â GRX4, Â displayed Â increased Â cell Â surface Â reductase Â activity Â in Â the Â presence Â of Â TTC Â (Figure Â 2.19 Â B). Â Â Overall, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 89 Â these Â results Â show Â that Â sporulation Â of Â a Â stable Â diploid Â (strain Â AI187) Â generated Â a Â heterologous Â progeny Â population Â with Â respect Â to Â deletion Â of Â GRX4. Â Â Because Â spores Â with Â deletion Â of Â the Â entire Â GRX4 Â including Â the Â N-­âterminal Â region Â were Â viable, Â I Â concluded Â that Â the Â GRX4 Â N-­âterminal Â region Â is Â not Â essential Â in Â C. Â neoformans. Â The Â reason Â I Â was Â unsuccessful Â in Â deleting Â the Â entire Â locus Â including Â the Â N-­âterminal Â region Â in Â a Â haploid Â background Â remains Â to Â be Â investigated. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 90 Â Â Â Figure Â 2.19: Â Full Â deletion Â of Â GRX4 Â in Â a Â stable Â H99 Â diploid Â background. Â (A) Â GRX4 Â is Â shown Â in Â dark Â blue Â arrow Â and Â neomycin Â knockout Â cassette Â replacing Â the Â entire Â GRX4 Â locus Â is Â shown Â in Â red. Â (B) Â Spores Â were Â germinated Â on Â YPD Â and Â copied Â to Â YPD Â + Â 200μg/ml Â neomycin. Â Spores Â with Â deletion Â of Â GRX4 Â grew Â in Â presence Â of Â NEO Â and Â formed Â red Â colonies Â Â Â Â Â Â Â Â Â Â Â Â Â Â 91 Â in Â presence Â of Â TTC. Â Presence Â of Â the Â WT Â copy Â of Â GRX4 Â in Â Spores Â that Â only Â grew Â on Â YPD Â and Â not Â in Â presence Â of Â marker Â was Â confirmed Â by Â PCR. Â 2.3.3.3.2 Â Genetic Â segregation Â analysis Â of Â heterozygous Â auxotrophic Â markers Â in Â progeny Â isolated Â from Â a Â stable Â diploid Â with Â a Â confirmed Â deletion Â of Â GRX4 Â Â Â To Â confirm Â that Â meiosis Â had Â occurred Â during Â the Â generation Â of Â the Â basidiospores Â from Â the Â experiments Â described Â above Â in Â section Â 2.3.3.3.1, Â I Â undertook Â a Â more Â extensive Â genetic Â analysis Â of Â the Â collected Â spores. Â Â Â Specifically, Â I Â scored Â the Â progeny Â for Â the Â ade2 Â and Â ura5 Â genetic Â markers Â that Â were Â heterozygous Â in Â strain Â AI187 Â to Â confirm Â the Â expected Â single Â gene Â segregation Â of Â these Â markers Â in Â the Â spore Â population. Â Â The Â parent Â diploid Â strain Â AI187 Â was Â originally Â generated Â as Â a Â result Â of Â crosses Â between Â ade2 Â MATα Â x Â ura5 Â MATa Â strains Â (A. Â Idnurm, Â 2010). Â Basidiospores Â that Â were Â studied Â in Â section Â 2.3.3.3.1 Â above Â were Â transferred Â from Â YPD Â to Â YNB Â lacking Â adenine Â or Â uracil. Â Â Out Â of Â 52 Â colonies Â that Â were Â germinated Â on Â YPD, Â 29 Â (almost Â 55%) Â were Â ADE2 Â URA5 Â as Â shown Â by Â growth Â on Â both Â YNB-­âadenine Â and Â YNB-­âuracil, Â and Â 23 Â (44%) Â were Â ade2 Â ura5 Â as Â shown Â by Â no Â growth Â on Â either Â of Â the Â dropout Â plates Â (Figure Â 2.20). Â This Â analysis Â confirmed Â that Â meiotic Â reduction Â had Â indeed Â taken Â place. Â These Â results Â are Â consistent Â with Â previous Â results Â in Â the Â study Â done Â by Â Idnurm Â group Â where Â the Â sporulation Â event Â in Â diploid Â strain Â AI187 Â resulted Â in Â 77 Â total Â colonies Â of Â which Â 43 Â (56%) Â were Â ADE2 Â URA5, Â and Â 34 Â (47%) Â were Â ade2 Â ura5 Â (A. Â Idnurm, Â 2010). Â Overall, Â this Â extended Â genetic Â analysis Â confirmed Â that Â colonies Â studied Â in Â section Â 2.3.3.1 Â were Â the Â progeny Â produced Â by Â a Â meiotic Â event Â in Â strain Â AI187 Â upon Â sporulation. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 92 Â Â Â Figure Â 2.20: Â Analysis Â of Â progeny. Â A Â total Â of Â 52 Â progeny Â on Â YPD Â were Â replica Â plated Â to Â media Â to Â test Â for Â the Â ADE2 Â and Â URA5 Â alleles. Â 2.4 Â Discussion Â Â Understanding Â the Â mechanisms Â by Â which Â C. Â neoformans Â senses Â the Â mammalian Â host Â environment Â and Â regulates Â virulence Â factor Â expression Â is Â of Â great Â significance. Â Â Our Â group Â has Â previously Â shown Â that Â iron Â plays Â a Â central Â role Â in Â the Â pathogenesis Â of Â C. Â neoformans Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2011, Â W. Â H. Â Jung, Â et Â al., Â 2006, Â T. Â Lian, Â et Â al., Â 2005, Â S. Â Saikia Â et Â al., Â 2014). Â Â Â Â Specifically, Â the Â GATA-­âtype, Â zinc-­âfinger Â protein Â Cir1 Â was Â previously Â identified Â as Â the Â main Â transcription Â factor Â that Â controls Â the Â iron Â regulon Â and Â expression Â of Â the Â known Â major Â virulence Â factors Â in Â C. Â neoformans Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Thus Â understanding Â the Â mechanisms Â by Â which Â Cir1 Â senses Â iron Â availability Â and Â contributes Â to Â proliferation Â of Â the Â fungus Â in Â mammalian Â hosts Â and Â identification Â of Â the Â key Â iron Â sources Â used Â by Â the Â fungus Â during Â infection Â are Â important Â goals. Â The Â components Â and Â features Â that Â contribute Â to Â Cir1 Â functions Â are Â not Â completely Â defined. Â To Â address Â this Â issue, Â proteins Â that Â are Â candidate Â Â Â Â Â Â Â Â Â Â Â Â Â Â 93 Â interaction Â partners Â with Â Cir1 Â were Â identified Â using Â a Â binding Â assay Â and Â a Â proteomic Â approach; Â one Â such Â protein Â was Â a Â monothiol Â glutaredoxin Â (Grx) Â with Â a Â signature Â CGFS Â active Â site. Â Â Â Based Â on Â the Â conserved Â evolutionary Â functions Â of Â monothiol Â Grxs Â in Â binding Â iron-­âresponsive Â transcription Â factors Â and Â regulation Â of Â iron Â homeostasis Â in Â other Â fungi, Â the Â work Â in Â this Â chapter Â was Â conducted Â to Â understand Â the Â role Â of Â monothiol Â glutaredoxin Â Grx4 Â in Â C. Â neoformans. Â I Â focused Â on Â determining Â the Â role Â of Â Grx4 Â by Â characterizing Â the Â phenotypic Â and Â transcriptional Â changes Â resulting Â from Â deletion Â of Â GRX4. Â I Â found Â that Â Grx4 Â contributes Â to Â the Â maintenance Â of Â iron Â homeostasis Â and Â has Â a Â critical Â role Â in Â the Â virulence Â of Â the Â fungus. Â Importantly, Â this Â is Â the Â first Â functional Â characterization Â of Â a Â monothiol Â Grx Â in Â a Â pathogenic Â fungus. Â Â 2.4.1 Â Â The Â conserved Â C-­âterminal Â monothiol Â Grx Â domain Â of Â Grx4 Â binds Â to Â the Â N-­âterminal Â region Â of Â Cir1. Â Â In Â silico Â analysis Â indicated Â that Â CnGrx4 Â is Â a Â monothiol Â Grx Â with Â a Â highly Â conserved Â Grx Â domain. Â Studies Â in Â other Â fungi Â have Â revealed Â that Â monothiol Â Grxs Â inhibit Â iron-­âdependent Â transcription Â factors Â in Â response Â to Â both Â excess Â levels Â of Â iron Â and Â iron Â deficiency. Â Â These Â studies Â provide Â clues Â for Â the Â function Â of Â Grx4 Â in Â C. Â neoformans. Â Â Â For Â example, Â Aft1 Â in Â S. Â cerevisiae Â activates Â the Â expression Â of Â high-­âaffinity Â iron Â transport Â functions Â in Â response Â to Â iron Â deficiency, Â and Â is Â inactivated Â by Â iron Â repletion Â (J. Â Kaplan, Â et Â al., Â 2006). Â It Â has Â been Â shown Â that Â Grx3 Â and Â Grx4 Â (Grx3/4) Â interact Â directly Â with Â Aft1, Â and Â several Â other Â cellular Â components, Â including Â GSH, Â and Â the Â Fra1 Â and Â Fra2 Â (Fe Â repressors Â of Â activation) Â proteins, Â to Â communicate Â information Â about Â cellular Â iron Â levels Â to Â Aft1 Â and Â Â Â Â Â Â Â Â Â Â Â Â Â Â 94 Â inhibit Â its Â function Â upon Â iron Â repletion. Â Cells Â lacking Â Grx3/4 Â show Â constitutive Â expression Â of Â iron Â regulon Â genes Â (L. Â Ojeda, Â et Â al., Â 2006) Â Â Â Â In Â the Â fission Â yeast Â S. Â pombe, Â which Â may Â be Â a Â closer Â evolutionary Â relative Â to Â C. Â neoformans, Â the Â transcription Â factor Â Fep1 Â (a Â Cir1 Â orthologue) Â is Â activated Â by Â excess Â iron Â accumulation Â and Â represses Â the Â expression Â of Â the Â genes Â involved Â in Â the Â acquisition Â of Â iron. Â In Â contrast, Â when Â S. Â pombe Â undergoes Â a Â transition Â from Â conditions Â of Â iron Â sufficiency Â to Â iron Â deficiency, Â the Â repressor Â activity Â of Â Fep1 Â needs Â to Â be Â shut Â down Â to Â allow Â for Â expression Â of Â high-­âaffinity Â iron Â transporters Â (M. Â Jbel Â et Â al., Â 2009, Â B. Â Pelletier, Â et Â al., Â 2005). Â Of Â note, Â it Â has Â been Â shown Â that Â monothiol Â Grx4 Â is Â required Â for Â the Â iron-­âlimitation-­âdependent Â inhibition Â of Â Fep1 Â under Â conditions Â of Â low Â iron Â (M. Â Jbel, Â et Â al., Â 2011). Â Â Â In Â this Â study, Â I Â initially Â confirmed Â the Â binding Â between Â Grx4 Â and Â Cir1 Â in Â C. Â neoformans, Â which Â was Â originally Â identified Â by Â mass Â spectrometry Â analysis. Â I Â validated Â that Â monothiol Â Grx4 Â binds Â the Â N-­âterminal Â region Â of Â Cir1 Â as Â determined Â by Â ALPHAScreen Â analysis Â in Â vitro, Â and Â I Â was Â able Â to Â measure Â a Â dissociation Â constant Â of Â 2x10-­â6 Â M, Â which Â is Â indicative Â of Â a Â strong Â binding Â between Â Grx4 Â and Â Cir1. Â The Â recombinant Â full Â length Â Cir1 Â protein Â was Â however Â very Â susceptible Â to Â degradation, Â and Â I Â therefore Â purified Â a Â more Â stable, Â shorter Â MBP-­âfusion Â protein Â containing Â only Â the Â first Â 406 Â N-­âterminal Â amino Â acids Â (Figure Â 2.4). Â On Â this Â note, Â the Â full-­âlength Â Cir1 Â polypeptide Â is Â 952 Â amino Â acids Â in Â length Â with Â a Â cys-­ârich Â domain Â (amino Â acids Â 162-­â189) Â and Â a Â conserved Â zinc-­âfinger Â domain Â (amino Â acids Â 307-­â359) Â in Â the Â N-­âterminal Â region. Â Â It Â was Â previously Â shown Â that Â the Â N-­âterminal Â region Â (amino Â acids Â 1-­â406) Â of Â the Â protein Â was Â ironâresponsive Â and Â responsible Â for Â the Â iron-­âdependent Â stability Â of Â Cir1 Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2011). Â The Â contribution Â of Â the Â C-­âterminal Â portion Â of Â the Â Cir1 Â has Â not Â been Â previously Â shown Â and Â remains Â to Â be Â Â Â Â Â Â Â Â Â Â Â Â Â Â 95 Â characterized. Â Â Taken Â together, Â my Â study Â defines Â the Â first Â example Â of Â a Â multidomain Â monothiol Â Grx4 Â as Â a Â binding Â partner Â of Â a Â major Â iron Â regulation Â transcription Â factor Â in Â the Â pathogenic Â fungus Â C. Â neoformans. Â Â Defining Â the Â precise Â events Â underlining Â the Â binding Â between Â Cir1 Â and Â Grx4 Â needs Â further Â investigation, Â but Â what Â has Â been Â shown Â in Â S. Â cerevisiae Â and Â S. Â pombe Â can Â serve Â as Â a Â model Â of Â Cir1-­âGrx4 Â interaction Â in Â C. Â neoformans. Â Â The Â association Â of Â Grx3/Grx4 Â with Â Aft1 Â was Â shown Â to Â be Â independent Â of Â iron Â levels. Â Â In Â fact, Â Grx3/4 Â binding Â of Â Fe/S Â clusters Â serves Â as Â the Â iron Â sensor Â that Â communicates Â the Â cytosolic Â iron Â status Â to Â Aft1, Â and Â depletion Â of Â Grx3/4 Â leads Â to Â impairment Â of Â most Â iron-­âdependent Â processes, Â including Â formation Â of Â di-­âiron Â centers, Â and Â Fe-­âS Â cluster Â and Â heme Â biosynthesis. Â The Â Fe Â and/or Â FeâS Â cluster-­âsensing Â role Â of Â glutaredoxins Â is Â also Â conserved Â in Â humans Â and Â other Â fungi Â that Â utilize Â Fe-­âresponsive Â transcription Â factors Â (U. Â Muhlenhoff, Â et Â al., Â 2010). Â Â Â Â In Â S. Â pombe, Â iron-­âdependent Â modulation Â of Â Fep1 Â activity Â is Â exerted Â by Â its Â interaction Â with Â the Â N-­âterminal Â DNA-­âbinding Â of Â Grx4. Â The Â Fep1â Â Grx4 Â complex Â resides Â in Â the Â nucleus Â regardless Â of Â iron Â levels Â inside Â the Â cell. Â When Â iron Â is Â limited, Â Grx4 Â inhibits Â Fep1 Â function, Â which Â leads Â to Â dissociation Â of Â Fep1 Â from Â chromatin Â and Â derepression Â of Â the Â Fep1 Â regulon Â (M. Â Jbel, Â et Â al., Â 2011, Â K. Â D. Â Kim, Â et Â al., Â 2011, Â M. Â M. Â Molina-­âNavarro, Â et Â al., Â 2006, Â M. Â T. Â Rodriguez-­âManzaneque Â et Â al., Â 2002). Â The Â involvement Â of Â FeâS Â or Â Fe Â in Â this Â interaction Â was Â shown Â recently. Â Â Grx4 Â incorporates Â a Â glutathione-­âcontaining Â Fe-­âS Â cluster, Â and Â binds Â to Â iron-­âcontaining Â Fep1. Â Upon Â iron Â deprivation, Â the Â disassembly Â of Â the Â iron Â cluster Â promotes Â reverse Â metal Â transfer Â from Â Fep1 Â to Â Grx4, Â decreasing Â Fep1 Â interaction Â with Â promoters Â and Â de-­ârepression Â of Â iron-­âimport Â functions Â (J. Â Encinar Â del Â Dedo, Â et Â al., Â Â Â Â Â Â Â Â Â Â Â Â Â Â 96 Â 2015). Â As Â mentioned, Â Fep1 Â (S. Â pombe) Â and Â Cir1 Â (C. Â neoformans) Â are Â orthologs Â and Â share Â significant Â amino Â acid Â sequence Â identity. Â Therefore, Â it Â is Â possible Â that Â these Â two Â proteins Â use Â similar Â mechanisms Â or Â partners Â in Â their Â interactions Â with Â monothiol Â Grxs. Â Â Â It Â has Â also Â been Â shown Â that Â the Â monothiol Â Grxs Â are Â specific Â and Â efficient Â catalysts Â of Â deglutathionylation Â of Â their Â S-­âglutathionylated Â binding Â partners. Â Reversible Â glutathionylation Â is Â a Â prevalent Â form Â of Â cysteine Â modification Â and Â is Â recognized Â as Â a Â potential Â regulatory Â and Â a Â homeostatic Â protective Â mechanism Â that Â prevents Â cysteine Â oxidation Â to Â irreversible Â forms Â (C. Â L. Â Grek Â et Â al., Â 2013, Â M. Â D. Â Shelton Â and Â J. Â J. Â Mieyal, Â 2008). Â A Â large Â number Â of Â proteins Â have Â been Â identified Â as Â potentially Â regulated Â by Â reversible Â S-­âglutathionylation, Â including Â transcription Â factors Â such Â as Â p53 Â and Â NF-­âÎºB Â via Â conserved Â cysteines. Â This Â process Â affects Â their Â binding Â properties Â to Â DNA Â thereby Â regulating Â gene Â expression(J. Â J. Â Mieyal Â et Â al., Â 2008) Â (B. Â C. Â Liao Â et Â al., Â 2010) Â (C. Â S. Â Velu Â et Â al., Â 2007). Â It Â is Â therefore Â possible Â that Â Grx4 Â regulates Â the Â function Â of Â Cir1 Â through Â reversible Â S-­âglutathionylation, Â but Â this Â remains Â to Â be Â investigated. Â Â 2.4.2 Â Â The Â grx4 Â mutant Â displays Â iron-­ârelated Â and Â virulence-­ârelated Â phenotypes Â Â Â Â The Â original Â grx4 Â mutant Â (from Â our Â collaborators Â Dr. Â Missall Â and Â Dr. Â Lodge) Â has Â the Â conserved Â C-­âterminal Â Grx Â domain Â replaced Â by Â a Â NATr Â cassette. Â Â I Â was Â able Â to Â replace Â the Â deleted Â region Â with Â the Â wild-­âtype Â gene Â at Â the Â original Â locus Â to Â obtain Â a Â complemented Â strain Â (grx4Î::GRX4). Â Â This Â strain Â was Â valuable Â in Â my Â examination Â of Â the Â role Â of Â the Â Grx Â domain Â in Â GRX4 Â because Â it Â confirmed Â that Â the Â phenotypes Â of Â the Â deletion Â mutant Â were Â due Â to Â loss Â of Â Grx4. Â Â Â The Â phenotypic Â examination Â revealed Â that Â the Â association Â between Â Grx4 Â and Â Cir1 Â was Â reflected Â in Â the Â grx4 Â mutant Â sharing Â iron-­ârelated Â and Â virulence-­ârelated Â phenotypes Â with Â the Â cir1 Â mutant, Â including Â sensitivity Â to Â elevated Â levels Â of Â iron, Â increased Â Â Â Â Â Â Â Â Â Â Â Â Â Â 97 Â cell Â surface Â reductase Â activity Â and Â increased Â sensitivity Â to Â the Â pro-­âdrug Â phleomycin. Â Â These Â results Â strongly Â suggest Â that Â grx4 Â mutant Â has Â dysregulated Â iron Â homeostasis Â and Â indicated Â that Â the Â association Â between Â Grx4 Â and Â Cir1 Â has Â functional Â significance Â in Â C. Â neoformans. Â Â Another Â piece Â of Â evidence Â that Â indicates Â dysregulated Â iron Â homeostasis Â in Â the Â grx4 Â mutant Â was Â elevation Â in Â transcripts Â for Â iron Â acquisition Â functions Â including Â the Â high-­âaffinity Â iron Â uptake Â system Â (Cft1, Â Cfo1), Â siderophore Â transporters Â (Sit1, Â Str3, Â Mir1B) Â and Â ferric Â reductases Â (Fre2, Â Fre6) Â despite Â sufficient Â iron Â supply Â (Figure Â 2.11). Â The Â enhanced Â iron Â acquisition Â in Â the Â grx4 Â mutant Â in Â the Â presence Â of Â iron Â suggests Â that Â Grx4 Â has Â an Â inhibitory Â effect Â on Â iron Â acquisition Â in Â C. Â neoformans, Â which Â can Â potentially Â lead Â to Â increased Â intracellular Â iron Â content. Â This Â can Â also Â explain Â the Â increased Â sensitivity Â of Â grx4 Â mutant Â to Â the Â pro-­âdrug Â phleomycin Â compared Â to Â the Â WT. Â Phleomycin Â interacts Â with Â intracellular Â iron Â pools, Â causing Â the Â formation Â of Â ROS, Â which Â results Â in Â DNA Â damage Â and Â eventual Â cell Â death. Â Of Â note, Â sensitivity Â to Â phleomycin Â reflects Â the Â level Â of Â free Â intracellular Â iron Â (B. Â Pelletier, Â et Â al., Â 2002), Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Taken Â together Â these Â results Â support Â the Â role Â of Â Grx4 Â in Â maintenance Â of Â iron Â homeostasis Â in Â C. Â neoformans. Â This Â notion Â is Â further Â supported Â by Â my Â observation Â that Â the Â grx4 Â mutant Â exhibited Â a Â growth Â defect Â in Â the Â presence Â of Â iron, Â which Â was Â restored Â by Â addition Â of Â exogenous Â levels Â of Â GSH Â (5mM). Â Given Â the Â known Â toxicity Â of Â free Â iron, Â unrestricted Â iron Â uptake Â and Â enhanced Â free Â iron Â in Â the Â cell Â might Â be Â the Â cause Â of Â the Â poor Â growth Â of Â grx4 Â mutant Â cells Â in Â high Â iron Â media. Â Â Iron Â plays Â a Â determinant Â role Â through Â the Â generation Â of Â oxidative Â stress Â due Â to Â its Â ability Â to Â catalyze Â the Â generation Â of Â hydroxyl Â radicals Â (OH.) Â from Â H2O2 Â in Â the Â Fenton Â reaction Â (J. Â A. Â Imlay, Â 2008, Â J. Â A. Â Imlay, Â et Â al., Â 1988, Â J. Â A. Â Imlay Â and Â S. Â Linn, Â 1988). Â The Â results Â shown Â above Â suggest Â that Â lack Â of Â Grx4 Â ultimately Â leads Â to Â increased Â levels Â of Â intracellular Â iron, Â which Â Â Â Â Â Â Â Â Â Â Â Â Â Â 98 Â may Â accelerate Â the Â Fenton Â reaction. Â Â This Â is Â further Â supported Â by Â the Â observation Â that Â addition Â of Â GSH Â ameliorated Â the Â growth Â defect Â of Â grx4 Â mutant Â in Â the Â presence Â of Â iron. Â Â This Â can Â be Â explained Â by Â antioxidant Â properties Â of Â GSH, Â including Â Â its Â role Â in Â regulating Â cellular Â iron Â homeostasis Â and Â scavenging Â and Â detoxifying Â iron Â ions, Â leading Â to Â protection Â against Â oxidative Â stress Â caused Â by Â iron Â toxicity Â (L. Â M. Â Milchak Â and Â J. Â Douglas Â Bricker, Â 2002). Â On Â this Â note, Â GSH Â depletion Â in Â both Â S. Â cerevisiae Â and Â S. Â pombe Â leads Â to Â loss Â of Â iron-­âdependent Â inhibition Â of Â Aft1/Aft2 Â and Â Fep1, Â presumably Â stemming Â from Â the Â requirement Â for Â GSH Â as Â a Â [2Fe-­â2S] Â cluster Â ligand Â in Â monothiol Â Grxs. Â Â Â Alternatively, Â as Â described Â in Â section Â 1.5.1.1, Â GSH Â is Â a Â major Â component Â of Â the Â labile Â iron Â pool Â (LIP) Â of Â available Â iron Â in Â cells, Â and Â this Â adds Â significance Â to Â the Â role Â of Â GSH Â in Â iron Â homeostasis Â (R. Â C. Â Hider Â and Â X. Â L. Â Kong, Â 2011). Â Â In Â this Â regard, Â GSH Â acts Â as Â a Â major Â cytosolic Â ligand Â for Â Fe Â (II). Â Â Although Â the Â properties Â and Â features Â of Â this Â cytosolic Â pool Â are Â not Â completely Â understood, Â LIP Â is Â defined Â as Â the Â freely Â exchangeable Â iron Â that Â is Â loosely Â coordinated Â by Â water, Â small Â molecules, Â and Â GSH. Â Therefore, Â one Â can Â speculate Â that Â addition Â of Â GSH Â to Â the Â culture Â leads Â to Â restored Â growth Â of Â the Â grx4 Â mutant Â by Â chelating Â free Â excess Â iron Â and Â repression Â of Â iron Â acquisition Â components. Â Nevertheless, Â further Â studies Â are Â necessary Â to Â unravel Â the Â role Â of Â GSH Â in Â C. Â neoformans Â both Â in Â association Â with Â Grx4 Â or Â independently. Â 2.4.3 Â Â Grx4 Â has Â a Â central Â role Â in Â maintenance Â of Â iron Â homeostasis Â in Â C. Â neoformans Â Â My Â preliminary Â RNA-­âseq Â analysis Â of Â the Â impact Â of Â the Â grx4 Â mutation Â further Â consolidated Â my Â findings Â on Â the Â role Â of Â Grx4 Â in Â maintenance Â of Â iron Â homeostasis. Â Â Recent Â genetic Â and Â biochemical Â analysis Â of Â monothiol Â Grxs Â have Â shown Â that Â their Â central Â role Â in Â the Â control Â of Â iron Â homeostasis Â (at Â the Â transcriptional Â and Â post-­âtranscriptional Â levels) Â is Â Â Â Â Â Â Â Â Â Â Â Â Â Â 99 Â an Â important Â component Â of Â the Â cellular Â response Â to Â iron Â deprivation. Â I Â hypothesized Â that Â the Â positive Â and Â negative Â influence Â of Â monothiol Â Grxs Â on Â iron Â homeostasis Â may Â result Â from Â downstream Â regulators Â that Â transcriptionally Â or Â post-­âtranscriptionally Â control Â the Â genes Â for Â iron Â homeostasis. Â To Â address Â this Â question, Â I Â performed Â RNA-­âseq Â analysis Â on Â the Â grx4 Â mutant Â and Â found Â that Â Grx4 Â is Â an Â important Â component Â of Â intracellular Â iron Â homeostasis. Â Â In Â C. Â neoformans, Â iron Â starvation Â causes Â extensive Â transcriptional Â remodeling Â that Â is Â mediated Â by Â the Â transcription Â factors Â such Â as Â Cir1 Â and Â HapX Â to Â activate Â iron-­âand Â siderophore Â uptake Â pathways Â and Â repress Â iron-­âdependent Â and Â iron-­âconsuming Â metabolic Â pathways Â such Â as Â heme Â biosynthesis, Â respiration Â and Â ribosome Â biogenesis Â during Â iron Â starvation Â (to Â spare Â iron). Â Â This Â system Â is Â reversed Â during Â iron Â sufficiency Â in Â order Â to Â avoid Â toxic Â effects. Â Â Â In Â our Â RNA-­âseq Â data, Â I Â found Â that Â under Â the Â iron Â sufficient Â conditions, Â the Â transcripts Â for Â genes Â encoding Â the Â ferric Â reductases Â FRE2 Â and Â FRE6 Â and Â the Â siderophore Â transporters Â STR3 Â and Â MIRB Â (also Â designated Â SIT6) Â were Â highly Â upregulated Â compared Â to Â the Â WT. Â Â These Â genes Â previously Â showed Â upregulation Â in Â the Â grx4 Â mutant Â compared Â to Â the Â WT Â in Â my Â qRT-­âPCR Â analysis Â under Â iron-­âreplete Â condition. Â Of Â note, Â these Â iron Â acquisition Â functions Â are Â normally Â upregulated Â in Â WT Â in Â response Â to Â iron Â starvation. Â Â My Â data Â from Â the Â RNA-­âseq Â analysis Â are Â also Â in Â agreement Â with Â my Â findings Â on Â the Â sensitivity Â of Â grx4 Â to Â elevated Â iron Â levels, Â as Â observed Â in Â my Â phenotypic Â assays Â and Â growth Â curves. Â Â On Â the Â same Â note, Â I Â found Â that Â DNA Â repair Â helicases Â and Â repair Â enzymes Â were Â highly Â upregulated Â in Â grx4 Â mutant Â particularly Â in Â the Â iron-­âreplete Â condition Â and Â this Â is Â another Â clue Â leading Â to Â the Â conclusion Â that Â loss Â of Â Grx4 Â results Â in Â over Â accumulation Â of Â iron Â and Â subsequent Â DNA Â Â Â Â Â Â Â Â Â Â Â Â Â Â 100 Â damage. Â Â Â In Â general, Â my Â RNA-­âSeq Â data Â support Â the Â hypothesis Â that Â Grx4 Â may Â constitute Â part Â of Â an Â âiron Â sensing Â complexâ Â (possibly Â through Â binding Â iron Â or Â Fe-­âS Â clusters) Â that Â responds Â to Â alterations Â in Â iron Â levels Â and Â transmits Â signals Â for Â transcriptional Â activation Â though Â interaction Â with Â Cir1 Â or Â other Â binding Â partners. Â Â Furthermore, Â owing Â to Â the Â central Â metabolic Â role Â of Â iron, Â iron Â regulation Â and Â maintenance Â of Â iron Â homeostasis Â are Â interconnected Â with Â various Â other Â regulatory Â circuits Â in Â C. Â neoformans Â including Â pH Â regulation, Â gluconeogenesis, Â zinc Â metabolism, Â and Â oxidative Â stress Â response Â and Â many Â various Â metabolic Â pathways Â that Â are Â linked Â to Â iron Â homeostasis. Â Therefore, Â it Â is Â suggested Â that Â Grx4 Â affects Â both Â maintenance Â of Â iron Â homeostasis Â and Â linked Â pathways. Â 2.4.4 Â Â Â Grx4 Â is Â required Â for Â virulence Â in Â C. Â neoformans Â Â Â As Â an Â important Â part Â of Â my Â current Â study, Â I Â could Â show Â Grx4 Â is Â also Â important Â for Â virulence Â in Â C. Â neoformans. Â Â Several Â studies Â have Â shown Â that Â pathogenic Â microbes Â link Â the Â availability Â of Â iron Â with Â virulence Â attributes Â (see Â 2.2.1) Â and Â my Â work Â shows Â a Â similar Â link Â between Â the Â iron Â homeostasis Â system Â and Â virulence Â via Â Grx4 Â and Â Cir1. Â Â Remarkably, Â I Â found Â that Â the Â grx4 Â mutant Â lost Â the Â ability Â to Â elaborate Â capsule, Â produce Â melanin Â and Â grow Â at Â 37°C Â which Â is Â representative Â of Â human Â body Â condition. Â Importantly, Â deletion Â of Â GRX4 Â leads Â to Â complete Â loss Â of Â virulence Â in Â inhalation Â model Â of Â infection Â in Â mice. Â Â Â I Â speculate Â that Â the Â lack Â of Â virulence Â of Â the Â grx4 Â mutant Â most Â likely Â results Â from Â the Â combined Â defects Â in Â iron Â homeostasis Â in Â this Â mutant, Â since Â iron Â is Â critical Â for Â fundamental Â cellular Â processes Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2008, Â W. Â H. Â Jung, Â et Â al., Â 2006). Â The Â connections Â between Â Â Â Â Â Â Â Â Â Â Â Â Â Â 101 Â Grx4 Â functions Â and Â iron Â homeostasis Â can Â also Â occur Â at Â multiple Â levels, Â including Â the Â association Â with Â Cir1 Â and Â regulation Â of Â expression Â of Â the Â iron Â regulon, Â capsule Â formation, Â laccase, Â phospholipase, Â cell Â wall Â and Â membrane Â biosynthesis, Â in Â addition Â to Â signaling Â pathways Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Cir1 Â also Â regulates Â the Â expression Â of Â genes Â associated Â with Â iron Â uptake Â from Â heme Â (CIG1), Â siderophores Â (SIT1), Â and Â the Â production Â of Â melanin, Â in Â addition Â to Â other Â iron-­ârelated Â genes Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â I Â found Â that Â the Â grx4 Â mutant Â also Â shares Â virulence-­ârelated Â phenotypes Â associated Â with Â the Â cir1 Â mutant. Â It Â has Â previously Â been Â shown Â that Â cir1 Â mutant Â has Â a Â defect Â in Â capsule Â and Â melanin Â production, Â as Â well Â as Â reduced Â growth Â at Â 37°C, Â and Â is Â also Â avirulent Â in Â a Â mouse Â model Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Â Overall, Â my Â study Â revealed Â that Â Grx4 Â binds Â Cir1 Â and Â subsequently Â influences Â iron Â homeostasis, Â transcriptional Â control, Â and Â the Â production Â of Â virulence-­ârelated Â factors Â in Â C. Â neoformans. Â 2.4.5 Â Â The Â N-­âterminal Â of Â Grx4 Â is Â not Â essential Â and Â exists Â in Â some Â but Â not Â transcript Â isoforms Â from Â the Â GRX4 Â locus Â Â Re-­âanalysis Â of Â the Â RNA-­âseq Â data Â led Â to Â the Â identification Â of Â three Â putative Â unannotated Â transcripts Â associated Â with Â the Â GRX4 Â locus, Â in Â addition Â to Â the Â main Â transcript Â that Â I Â have Â used Â as Â the Â gene Â model Â for Â the Â work Â described Â in Â this Â thesis Â (Figure Â 2.18). Â Â De Â novo Â assembly Â of Â sequences Â showed Â that Â these Â additional Â putative Â transcripts Â contain Â the Â conserved Â C-­âterminal Â Grx Â domain Â and Â variation Â among Â these Â transcript Â versions Â arises Â from Â differential Â inclusion Â of Â exons Â from Â the Â 5â Â region Â of Â the Â GRX4 Â locus. Â Â Additionally Â the Â 2014 Â annotation Â of Â the Â GRX4 Â gene Â in Â the Â genome Â database Â (Broad) Â was Â modified Â with Â regard Â to Â intron-­âexon Â positions Â in Â the Â N-­âterminal Â of Â GRX4 Â as Â part Â of Â the Â community Â effort Â Â Â Â Â Â Â Â Â Â Â Â Â Â 102 Â to Â characterize Â and Â publish Â the Â genome Â of Â the Â WT Â strain Â H99 Â (G. Â Janbon, Â et Â al., Â 2014). Â Â On Â this Â note, Â a Â notable Â characteristic Â of Â C. Â neoformans Â is Â the Â heterogeneity Â of Â genome Â sequences Â from Â different Â clinical Â and Â environmental Â isolates, Â and Â in Â many Â cases, Â this Â genetic Â variation Â results Â in Â altered Â expression Â patterns Â of Â genes Â (G. Â Hu, Â et Â al., Â 2011) Â . Â Also, Â alternative Â splicing Â is Â common Â in Â C. Â neoformans Â and Â intron-­âdependent Â regulation Â of Â gene Â expression Â could Â play Â a Â major Â role Â in Â the Â biology Â of Â the Â fungus Â (R. Â Tarrio Â et Â al., Â 2008). Â Â C. Â neoformans Â genes Â are Â intron-­âdense Â with Â an Â average Â of Â 5.7 Â per Â gene Â and Â the Â introns Â are Â generally Â small Â in Â size Â (median Â size: Â 56 Â nucleotides) Â (G. Â Janbon, Â et Â al., Â 2014). Â Also Â of Â significance Â is Â the Â fact Â that Â alternative Â splicing Â is Â a Â common Â feature Â for Â genes Â located Â on Â chromosome Â 3 Â (G. Â Janbon, Â et Â al., Â 2014) Â where Â GRX4 Â is Â also Â located. Â Another Â point Â that Â complicates Â the Â expression Â pattern Â of Â the Â GRX4 Â locus Â is Â its Â antisense Â direction. Â The Â patterns Â of Â transcription Â on Â genes Â in Â antisense Â direction Â are Â regulated Â by Â growth Â conditions, Â suggesting Â a Â complex Â mechanism Â of Â gene Â expression Â regulation Â in Â these Â genes Â including Â GRX4. Â This Â prompted Â me Â to Â test Â the Â phenotypic Â outcome Â of Â deletion Â of Â the Â N-­âterminal Â region Â of Â the Â GRX4 Â locus Â and Â investigate Â the Â role Â of Â this Â region Â in Â C. Â neoformans. Â Â However, Â despite Â numerous Â efforts, Â I Â was Â not Â able Â to Â acquire Â a Â deletion Â mutant Â when Â targeting Â this Â region Â and Â deleting Â the Â complete Â GRX4 Â locus Â in Â the Â haploid Â strain Â H99. Â Â This Â led Â me Â to Â hypothesize Â that Â this Â region Â might Â be Â essential Â in Â C. Â neoformans, Â especially Â because Â it Â has Â previously Â been Â shown Â that Â the Â N-­âterminal Â region Â of Â GRX4 Â is Â essential Â for Â in Â vivo Â function Â in Â S. Â cerevisiae Â (B. Â Hoffmann Â et Â al., Â 2011, Â U. Â Muhlenhoff, Â et Â al., Â 2010). Â I Â tested Â this Â hypothesis Â by Â deleting Â the Â complete Â locus Â including Â the Â N-­âterminal Â region Â of Â GRX4 Â in Â a Â diploid Â strain Â of Â C. Â neoformans Â by Â biolistic Â transformation. Â The Â results Â of Â this Â experiment Â showed Â that Â the Â meiotic Â progeny Â with Â deletion Â of Â GRX4 Â were Â viable Â under Â normal Â growth Â Â Â Â Â Â Â Â Â Â Â Â Â Â 103 Â conditions, Â indicating Â that, Â unexpectedly, Â the Â N-­âterminal Â region Â of Â GRX4 Â is Â not Â essential Â in Â a Â haploid Â background. Â At Â this Â point Â it Â is Â not Â clear Â why Â I Â was Â not Â able Â to Â delete Â the Â N-­âterminal Â region Â in Â the Â haploid Â strain Â H99. Â One Â possible Â explanation Â is Â that Â this Â region Â might Â be Â required Â for Â chromosome Â stability Â of Â the Â locus presumably Â because Â of Â an Â essential Â gene(s) Â in Â the Â locus Â (K. Â L. Â Ormerod Â and Â J. Â A. Â Fraser, Â 2013). Â On Â this Â note, Â N-­âterminal Â of Â GRX4 Â locus Â is Â in Â close Â proximity Â to Â CNAG_02948, Â which Â encodes Â a Â peptide Â chain Â release Â factor Â 1. Â Â This Â is Â an Â essential Â gene Â and Â it Â shares Â a Â promoter Â with Â GRX4 Â promoter, Â which Â might Â explain Â why Â I Â was Â not Â able Â to Â interfere Â with Â the Â N-­âterminal Â region Â in Â a Â haploid Â background. Â Combined, Â these Â data Â indicate Â a Â unique Â unusual Â arrangement Â of Â the Â GRX4 Â locus Â in Â C Â neoformans. Â Â In Â summary Â of Â this Â study, Â I Â investigated Â the Â role Â of Â the Â monothiol Â Grx4 Â and Â obtained Â evidence Â that Â it Â binds Â a Â major Â iron Â transcription Â factor Â Cir1 Â and Â grx4 Â and Â cir1 Â mutants Â share Â similar Â iron-­ârelated Â and Â virulence Â related Â phenotypes. Â Investigating Â the Â mechanisms Â for Â iron Â homeostasis Â in Â C. Â neoformans Â will Â allow Â a Â better Â understanding Â of Â how Â this Â pathogen Â survives Â the Â environments Â encountered Â during Â infection Â and Â should Â provide Â additional Â potential Â targets Â for Â therapeutic Â intervention. Â My Â study Â on Â Grx4 Â underscores Â the Â significance Â of Â iron Â sensing Â in Â the Â expression Â of Â virulence Â factors Â leading Â to Â cryptococcosis. Â The Â knowledge Â of Â the Â role Â of Â iron Â in Â fungal Â infections Â has Â advanced Â immensely Â in Â recent Â years, Â and Â its Â potential Â application Â in Â treatment Â and Â diagnosis Â of Â fungal Â infections Â requires Â deeper Â and Â further Â insights. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 104 Â Chapter Â 3: Â Analysis Â of Â the Â roles Â of Â glutathione Â synthetase Â in Â the Â AIDS-­âassociated Â pathogen Â Cryptococcus Â neoformans Â 3.1 Â Introduction Â Â The Â work Â presented Â in Â chapter Â 2 Â identified Â and Â characterized Â the Â role Â of Â Grx4 Â in Â iron Â homeostasis Â in Â C. Â neoformans. Â As Â described Â in Â section Â 2.3.2.2 Â I Â found Â that Â after Â 48 Â hours Â of Â iron Â starvation, Â the Â grx4 Â mutant Â had Â a Â growth Â defect Â in Â the Â presence Â of Â iron Â compared Â to Â WT Â (Figure Â 2.10). Â I Â speculated Â that Â grx4 Â mutant Â might Â be Â susceptible Â to Â oxidative Â stress Â upon Â iron Â starvation Â and/or Â iron Â repletion Â leading Â to Â impaired Â growth. Â Â On Â this Â note, Â I Â also Â found Â that Â the Â growth Â defect Â of Â the Â grx4 Â mutant Â was Â rescued Â by Â addition Â of Â exogenous Â GSH Â at Â concentration Â of Â 5 Â mM Â (Figure Â 2.11). Â This Â observation Â prompted Â me Â to Â further Â investigate Â the Â role Â of Â glutathione Â in Â C. Â neoformans. Â The Â experiments Â presented Â in Â this Â chapter Â of Â my Â thesis Â focused Â on Â examining Â the Â roles Â of Â GSH Â in Â iron Â homeostasis, Â cell Â wall Â integrity Â and Â expression Â of Â virulence Â factors Â in Â C. Â neoformans. Â Â Â Â The Â tripeptide Â glutathione Â (GSH) Â or Â Î³-­âglutamyl-­âcysteinyl-­âglycine Â (Figure Â 3.1) Â is Â the Â most Â abundant Â low-­âmolecular-­âweight Â thiol Â in Â eukaryotic Â organisms Â and Â gram-­ânegative Â bacteria Â (R. Â C. Â Fahey Â et Â al., Â 1978, Â H. Â Sies, Â 1999). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 105 Â Â Â Figure Â 3.1: Â Structure Â of Â GSH: Â The Â tripeptide Â GSH Â consists Â of Â the Â amino Â acids Â glutamate, Â cysteine Â and Â glycine. Â A Â glutamyl Â amide Â linkage Â ligates Â glutamate Â and Â cysteine. Â Â Â GSH Â serves Â in Â many Â biological Â processes Â including Â the Â synthesis Â of Â proteins Â and Â DNA, Â transport, Â enzyme Â activity, Â detoxification Â of Â heavy Â metals, Â xenobiotics Â and Â drugs, Â and Â defense Â against Â reactive Â oxygen Â species Â (ROS) Â (Figure Â 3.2) Â (A. Â Meister Â and Â M. Â E. Â Anderson, Â 1983). Â The Â âclassicâ Â role Â of Â GSH Â as Â thiol Â redox Â buffer Â and Â cofactor Â of Â antioxidant Â enzymes Â is Â particularly Â important Â because Â living Â organisms Â need Â to Â maintain Â an Â intracellular Â reducing Â environment Â (C. Â Mytilineou Â et Â al., Â 2002). Â GSH Â guarantees Â maintenance Â of Â the Â intracellular Â reducing Â environment Â and Â also Â acts Â as Â a Â cofactor Â for Â enzymes Â such Â as Â glutathione Â peroxidases Â and Â glutathione-­âS-­âtransferases Â (GST) Â (K. Â Becker Â et Â al., Â 2003, Â A. Â Meister Â and Â M. Â E. Â Anderson, Â 1983). Â These Â enzymes Â are Â involved Â in Â protecting Â cells Â against Â reactive Â oxygen Â species Â (ROS), Â toxic Â metabolic Â intermediates Â and Â xenobiotics. Â Â GSH Â is Â also Â known Â as Â a Â non-­âenzymatic Â antioxidant Â either Â alone Â or Â in Â conjunction Â Â Â Â Â Â Â Â Â Â Â Â Â Â 106 Â with Â vitamin Â C Â and Â vitamin Â E Â (C. Â L. Â Linster Â and Â E. Â Van Â Schaftingen, Â 2007). Â For Â example, Â it Â has Â been Â demonstrated Â that Â GSH Â and Â vitamin Â E Â protect Â against Â iron Â toxicity Â and Â inhibit Â iron-­âinduced Â cell Â death Â in Â rat Â hepatocytes Â (L. Â M. Â Milchak Â and Â J. Â Douglas Â Bricker, Â 2002). Â It Â has Â also Â been Â shown Â that Â GSH Â and Â Vitamin Â C Â supplementation Â protects Â Arabidopsis Â seedlings Â from Â iron Â deficiency, Â preserves Â cell Â redox Â homeostasis Â and Â improves Â internal Â iron Â availability Â (L. Â Ramirez, Â et Â al., Â 2013). Â Intracellular Â glutathione Â also Â hinders Â the Â progression Â of Â heavy Â metal-­âinitiated Â cell Â injuries Â by Â chelating Â and Â sequestering Â the Â metal Â ions Â in Â ectomycorrhizal Â fungi Â (M. Â Bellion Â et Â al., Â 2006) Â Â Research Â on Â GSH Â has Â expanded Â rapidly Â in Â the Â last Â 10 Â years, Â and Â recent Â findings Â are Â also Â challenging Â the Â traditional Â view Â of Â GSH Â as Â the Â main Â player Â in Â redox Â control. Â Â In Â particular, Â recent Â studies Â present Â glutathione Â as Â a Â critical Â signaling Â molecule. Â For Â instance, Â it Â was Â recently Â shown Â that Â GSH Â activates Â the Â virulence Â of Â the Â intracellular Â pathogen Â Listeria Â monocytogenes Â (M. Â L. Â Reniere Â et Â al., Â 2015). Â Another Â fast-­âdeveloping Â topic Â in Â GSH Â research Â is Â âprotein Â glutathionylationâ Â which Â has Â been Â suggested Â to Â have Â a Â major Â role Â in Â regulating Â the Â activity Â of Â metabolic Â and Â structural Â enzymes Â (P. Â Ghezzi Â et Â al., Â 2002) Â Â (D. Â A. Â Davis Â et Â al., Â 2003). Â Another Â critical Â role Â of Â GSH Â is Â in Â regulation Â of Â iron Â homeostasis Â (C. Â Kumar, Â et Â al., Â 2011, Â C. Â Berndt Â et Â al., Â 2014, Â C. Â H. Â Lillig Â and Â C. Â Berndt, Â 2013, Â C. Â E. Â Outten Â and Â A. Â N. Â Albetel, Â 2013, Â F. Â W. Â Outten, Â 2007, Â K. Â Sipos, Â et Â al., Â 2002). Â It Â has Â been Â shown Â that Â GSH Â is Â involved Â in Â iron Â metabolism Â by Â partnering Â with Â monothiol Â Grxs Â to Â assemble Â FeâS Â clusters Â as Â described Â in Â section Â 1.5. Â Â GSH Â also Â serves Â as Â a Â major Â component Â of Â the Â labile Â iron Â pool Â (LIP) Â of Â available Â iron Â in Â cells, Â (R. Â C. Â Hider Â and Â X. Â L. Â Kong, Â 2011). Â Â C. Â neoformans Â is Â exposed Â to Â numerous Â stresses Â during Â its Â life Â cycle Â in Â the Â hostile Â conditions Â of Â the Â human Â host, Â including Â nutrient Â deprivation, Â oxidative Â and Â nitrosative Â Â Â Â Â Â Â Â Â Â Â Â Â Â 107 Â stress, Â and Â high Â temperature. Â Â The Â defense Â antioxidant Â mechanisms Â that Â enable Â C. Â neoformans Â to Â survive Â these Â stress Â conditions Â include Â thioredoxins, Â catalases, Â peroxidases Â and Â glutathione Â peroxidases Â (Gpx), Â as Â well Â as Â the Â mitogen-­âactivated Â protein Â kinase Â (HOG1) Â signal Â transduction Â and Â calcineurin/calmodulin Â pathways. Â Â Â Â Â Figure Â 3.2: Â Biological Â functions Â associated Â with Â GSH. Â A Â partial Â list Â of Â GSH-­âdependent Â cellular Â pathways Â and Â proteins Â are Â illustrated. Â Â Â In Â all Â GSH-­âcontaining Â species, Â GSH Â is Â synthesized Â de-­ânovo Â by Â the Â sequential Â action Â of Â the Â Î³-­âglutamylcysteine Â synthetase Â (Î³-­âGCS) Â and Â GSH Â synthetase Â enzymes Â (Figure Â 3.3) Â These Â two Â enzymes Â are Â encoded Â by Â separate Â genes, Â GSH1 Â and Â GSH2, Â in Â eukaryotes Â (gshA Â and Â gshB, Â respectively, Â in Â prokaryotes Â (S. Â C. Â Lu, Â 2009). Â Â The Â enzyme Â Î³-­âGCS Â conjugates Â glutamic Â acid Â with Â cysteine Â and Â this Â is Â the Â rate-­âlimiting Â step Â of Â GSH Â synthesis Â that Â is Â also Â Â Â Â Â Â Â Â Â Â Â Â Â Â 108 Â subject Â to Â feedback Â inhibition Â by Â GSH Â (A. Â Meister Â and Â M. Â E. Â Anderson, Â 1983). Â The Â gene Â encoding Â Î³-­âGCS Â (GSH1) Â is Â essential Â in Â S. Â cerevisiae, Â S. Â pombe Â and Â C. Â albicans. Â Â Deletion Â of Â the Â genes Â encoding Â Î³-­âGCS Â in Â these Â microorganisms Â leads Â to Â GSH Â autotrophy Â under Â normal Â growth Â conditions, Â indicating Â an Â essential Â role Â of Â (GSH1) Â in Â these Â fungi Â (B. Â Chaudhuri, Â et Â al., Â 1997) Â (Y. Â U. Â Baek, Â et Â al., Â 2004, Â C. Â M. Â Grant, Â et Â al., Â 1996, Â D. Â Wu, Â et Â al., Â 1994). Â Of Â note, Â it Â has Â been Â shown Â that Â GSH Â depletion Â causes Â an Â arrest Â of Â cell Â cycle Â at Â the Â G1 Â phase Â and Â cell Â death Â via Â apoptosis Â in Â S. Â cerevisiae Â and Â C. Â albicans Â (Y. Â U. Â Baek, Â et Â al., Â 2004, Â T. Â Russo Â et Â al., Â 1995, Â D. Â Spector Â et Â al., Â 2001). Â GSH1 Â is Â also Â essential Â in Â the Â model Â plant Â Arabidopsis Â thaliana, Â as Â lack Â of Â Î³-­âglutamylcysteine Â synthetase Â abolishes Â root Â growth Â and Â cell Â division Â (T. Â Vernoux Â et Â al., Â 2000). Â In Â mammalian Â cells, Â mutation Â of Â GSH1 Â results Â in Â embryonic Â lethality, Â and Â blastocysts Â isolated Â from Â gsh1 Â mutant Â strain Â cannot Â grow Â without Â GSH. Â Â The Â exact Â reason Â why Â GSH Â is Â important Â for Â development Â and Â growth Â is Â still Â unknown Â (Z. Â Z. Â Shi Â et Â al., Â 2000). Â Â Â Â In Â the Â second Â step, Â glutathione Â synthetase Â catalyzes Â the Â ATP-­âdependent Â synthesis Â of Â GSH Â by Â ligation Â of Â Î³-­âglutamylcysteine Â (Î³-­âGlu-­âCys) Â with Â glycine. Â Genes Â encoding Â GSH Â synthetase Â have Â been Â identified Â in Â several Â bacterial Â and Â eukaryotic Â species Â (H. Â Gushima Â et Â al., Â 1983, Â N. Â Mutoh Â et Â al., Â 1991). Â It Â was Â shown Â that Â gsh2 Â deletion Â mutant Â in Â S. Â cerevisiae Â was Â viable Â but Â dependent Â on Â exogenous Â GSH Â for Â wild-­âtype Â growth Â rates. Â Â It Â was Â also Â shown Â that Â the Â gsh2 Â mutant Â lacked Â GSH Â but Â accumulated Â the Â dipeptide Â Î³-­âGlu-­âCys, Â an Â intermediate Â in Â GSH Â biosynthesis. Â Of Â note, Â it Â has Â been Â shown Â that Â the Â Î³-­âGlu-­âCys Â intermediate Â was Â at Â least Â as Â good Â as Â GSH Â in Â protecting Â yeast Â cells Â against Â an Â oxidant Â challenge Â and Â served Â as Â an Â antioxidant Â and Â substitute Â for Â GSH Â in Â the Â cell; Â however, Â Î³-­âGlu-­âCys Â did Â not Â substitute Â for Â the Â essential Â function Â of Â GSH Â such Â as Â detoxification Â of Â harmful Â intermediates Â that Â are Â generated Â during Â normal Â cellular Â metabolism Â (C. Â M. Â Grant, Â et Â al., Â 1996) Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 109 Â Â Figure Â 3.3: Â Enzymatic Â pathway Â for Â GSH Â synthesis Â in Â eukaryotes. Â GSH Â is Â synthesized Â de-­ânovo Â by Â the Â sequential Â action Â of Â Î³-­âglutamylcysteine Â synthetase Â (Î³-­âGCS) Â and Â GSH Â synthetase. Â Â In Â spite Â of Â critical Â roles Â of Â glutathione Â in Â a Â plethora Â of Â biological Â processes, Â the Â roles Â of Â GSH Â and Â GSH Â biosynthesis Â in Â C. Â neofomans Â have Â not Â been Â explored. Â Â In Â the Â present Â study Â I Â generated Â mutants Â deficient Â in Â GSH Â by Â a Â targeted Â deletion Â of Â GSH2 Â (CNAG_04647) Â Â Â Â Â Â Â Â Â Â Â Â Â Â 110 Â encoding Â glutathione Â synthetase. Â I Â then Â examined Â the Â role Â of Â GSH Â by Â detailed Â analysis Â of Â phenotypic Â outcomes Â manifested Â upon Â deletion Â of Â the Â GSH2 Â gene Â in Â C. Â neoformans. Â Â 3.2 Â Materials Â and Â methods Â Â 3.2.1 Â Â In Â silico Â protein Â analysis Â Â Â The Â amino Â acid Â sequence Â of Â GSH Â synthetase Â from Â the Â C. Â neoformans Â serotype Â A Â strain Â H99 Â (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans, Â 2014) Â was Â used Â in Â a Â blastp Â search(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) Â to Â identify Â orthologs Â in Â Â other Â fungal Â species Â and Â eukaryotes. Â Â Multiple Â sequence Â alignments Â were Â performed Â using Â Clustal Â Omega Â (http://www.ebi.ac.uk/clustalO/). Â Â 3.2.2. Â Yeast Â strains, Â plasmids Â and Â growth Â media Â Â The Â serotype Â A Â strain Â H99 Â of Â C.âneoformans Â var. Â grubii Â was Â used Â as Â the Â WT Â strain Â along Â with Â three Â individual Â gsh2 Â mutants, Â and Â the Â complemented Â strain Â gsh2Î::GSH2 Â (Table Â 3.1). Â Â The Â strains Â were Â maintained Â on Â YPD Â medium Â (1% Â yeast Â extract, Â 2% Â peptone, Â 2% Â dextrose Â and Â 2% Â agar). Â Â Selectable Â markers Â for Â transformation Â were Â from Â plasmids Â pJAF15 Â (hygromycin Â resistance) Â for Â mutants, Â and Â pJAF1 Â (neomycin Â resistance) Â for Â the Â complemented Â strain. Â Â Hygromycin Â was Â used Â for Â selection Â at Â 100Î¼gâmlâ1 Â and Â neomycin Â was Â used Â at Â 200Î¼gâmlâ1. Â Â Â Low Â iron Â medium Â (LIM) Â was Â prepared Â as Â described Â previously Â (S. Â E. Â Vartivarian, Â et Â al., Â 1993) Â Briefly Â LIM Â was Â composed Â of Â 6.7g Â YNB Â plus Â 2% Â glucose Â in Â 1 Â L Â of Â iron-­âchelated Â H2O Â using Â BIORAD Â chelex-­â100 Â and Â supplemented Â with Â the Â membrane Â impermeable Â iron Â chelator Â BPS. Â The Â pH Â was Â adjusted Â to Â 7.2 Â and Â the Â medium Â was Â filter Â sterilized. Â To Â prepare Â iron-­âreplete Â media, Â hemin Â was Â added Â at Â 100 Â μM, Â or Â FeCl3 Â was Â added Â at Â 10 Â μM Â or Â 100 Â μM Â final Â concentrations Â to Â low-­âiron Â medium. Â Cells Â were Â pre-­âcultured Â in Â YPD Â overnight Â at Â 30°C Â Â Â Â Â Â Â Â Â Â Â Â Â Â 111 Â and Â starved Â in Â LIM Â for Â 48 Â hours Â to Â make Â sure Â intracellular Â iron Â was Â depleted Â before Â harvesting Â and Â transferring Â cells Â to Â LIM Â or Â iron-­âreplete Â media. Â Â Where Â indicated, Â 5 Â mM Â GSH Â dissolved Â in Â chelexed Â 25 Â mM Â Hepes Â buffer Â pH Â 7.4 Â was Â added Â to Â LIM Â cultures Â at Â 12 Â hours Â prior Â to Â harvesting Â the Â cells. Â Â Â For Â spot Â assays Â on Â solid Â media, Â ten-­âfold Â serial Â dilutions Â of Â cells Â were Â spotted Â onto Â the Â agar Â media Â indicated Â in Â the Â text. Â Plates Â were Â incubated Â at Â 30°C Â for Â 2-­â3 Â days Â before Â being Â photographed. Â Table Â 3.1: Â Strains Â used Â in Â chapter Â 3 Â Strain Â Â Description Â Â Provided Â by/ Â Prepared Â by Â Â H99 Â Â C. Â neoformans Â wild-­âtype Â strain Â Â Dr. Â Â Joseph Â Heitman Â Â gsh2Î1 Â and Â gsh2Î2 Â gsh2 Â deletion Â mutants Â in Â H99 Â background Â Dr. Â Guanggan Â Hu Â Â gshÎ3 Â gsh2 Â deletion Â mutant Â in Â H99 Â background Â Rodgoun Â Attarian Â Â gsh2Î::GSH2 Â GSH2 Â complemented Â strain Â in Â the Â H99 Â strain Â background Â Rodgoun Â Attarian Â Â 3.2.3 Â Â Generation Â of Â gsh2 Â mutants Â and Â gsh2Î::GSH2 Â complemented Â strain Â Â Primers Â used Â for Â construction Â of Â all Â strains Â are Â listed Â in Â Table Â 3.2. Â Table Â 3.2: Â Primer Â sequences Â for Â strain Â construction Â Allele Â constructed Â Â Primer Â identification Â Â Primer Â sequence Â Â Â Â gsh2:HYG Â Â Â Â Â Â Â Â Â Â Gls1-­â1 Â Â Gls1-­â2 Â Â Gls1-­â3 Â Â Gls1-­â4 Â Â Â GGGTGCAGCTAATGTCACAA Â Â CATACATCCTCTTCTTCCCCACCGCTGCGAGGATGTGAG Â Â AGCTCACATCCTCGCAGCGGTGGGGAAGAAGAGGATGT Â Â TAGTTTCTACATCTCTTCTACGCGAACTGCTTCTTCTGT Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 112 Â Â Three Â individual Â gsh2 Â deletion Â mutants Â were Â constructed Â by Â homologous Â recombination Â using Â a Â hygromycin Â (HYGr Â ) Â marker Â linked Â to Â 5â² Â and Â 3â² Â flanking Â sequences Â of Â GSH2 Â by Â three-­âstep Â overlapping Â PCR Â (primer Â list Â 3.2). Â Â The Â overlap Â PCR Â product Â was Â biolistically Â transformed Â into Â the Â WT Â strain Â H99, Â and Â deletion Â was Â confirmed Â by Â PCR Â (Appendix, Â Figure Â B.2.). Â To Â reconstitute Â the Â deleted Â GSH2 Â in Â the Â mutant Â at Â the Â original Â locus, Â a Â genomic Â DNA Â fragment Â containing Â 1.1 Â Kb Â of Â upstream Â promoter Â region Â and Â a Â 1 Â Kb Â Allele Â constructed Â Primer Â identification Â Â Gls1-­â5 Â Â Â Gls1-­â6 Â Â Gls1-­â7NE Â Â Gls1-­â8NE Â Â Gls1-­â9PO Â Â Gls1-­â10PO Â Â Primer Â sequence Â Â Â Â GACACAGAAGAAGCAGTTCGCGTAGAAGAGATGTAGAAACTA Â Â GCCAAGCAACAAGCAATGTATGA Â Â GGATCAGTTGGTGCAGAGGAAGCA Â Â TCGGTGGGGTAGTCGGTAGG Â Â GCTCTTTCTGGATCCGATCTAGT Â Â CTGAACATCACTGTAAGGTGCGTC Â gsh2Î::GSH2 Â P9-­âPO Â Â P3C Â Â Â P2C Â Â Â P5C Â Â Â P4C Â Â Â P6C Â Â GCTCTTTCTGGATCCGATCTAGT Â Â CACTGGCGGCCGTTACTAGTTCTCTTGAGAAGCGCGTCCGACGT Â Â ACGTCGGACGCGCTTCTCAAGAGAACTAGTAACGGCCGCCAGTG Â Â GACGCGATTTCACGCACCTTTAAGCTGCGAGGATGTGAGCT Â Â AGCTCACATCCTCGCAGCTTAAAGGTGCGTGAAATCGCGTC Â Â CACTAGGAGTACTCACTGAAGCGTACATC Â Â Â Â Â Â Â Â Â Â Â Â Â Â 113 Â downstream Â terminator Â region Â carrying Â the Â GSH2 Â gene Â was Â amplified Â by Â PCR. Â Â This Â PCR Â fragment Â was Â fused Â with Â the Â neomycin Â (NEOr) Â selectable Â marker Â (1.9 Â Kb) Â at Â its Â C Â terminus Â in Â an Â overlap Â PCR Â reaction. Â Â The Â overlap Â PCR Â product Â was Â introduced Â into Â the Â gsh2 Â mutant Â by Â biolistic Â transformation. Â Targeted Â integration Â was Â confirmed Â by Â PCR Â (Appendix, Â Figure Â B.2) Â and Â phenotypic Â assays. Â 3.2.4. Â Â Phenotypic Â and Â biochemical Â analysis Â 3.2.4.1. Â Growth Â Assay Â Â The Â growth Â phenotypes Â of Â gsh2 Â mutants Â compared Â to Â the Â WT Â strain Â and Â the Â complemented Â strain Â gsh2Î::GSH2 Â were Â tested Â in Â rich Â YPD Â and Â minimal Â YNB Â media. Â Briefly, Â strains Â were Â grown Â overnight Â in Â YPD Â or Â YNB, Â cells Â were Â harvested Â and Â serially Â diluted Â with Â sterile Â ddH2O. Â Serial Â dilution Â of Â cells Â starting Â at Â 106 Â were Â spotted Â on Â YPD Â or Â YNB Â agar Â medium Â and Â incubated Â at Â 30°C Â for Â 2 Â days. Â Â Growth Â phenotypes Â of Â the Â strains Â was Â also Â determined Â in Â a Â parallel Â experiment Â where Â strains Â initially Â underwent Â iron-­âstarvation Â for Â a Â period Â of Â 48 Â hours Â and Â were Â then Â plated Â on Â YPD, Â YNB, Â iron-­âdeplete Â and Â iron-­âreplete Â agar Â media Â as Â described Â in Â section Â 3.2.2. Â Â This Â experiment Â was Â also Â performed Â with Â introduction Â of Â glutathione Â to Â the Â culture Â Â (described Â in Â 3.2.2). Â Plates Â were Â incubated Â at Â 30° Â C Â for Â 2-­â3 Â days Â before Â being Â photographed. Â Â 3.2.4.2 Â Quantification Â of Â the Â intracellular Â content Â of Â total Â (reduced Â and Â oxidized Â or Â GSHt) Â glutathione Â Â Â To Â determine Â the Â endogenous Â levels Â of Â glutathione Â in Â WT, Â gsh2 Â mutants Â and Â the Â complemented Â strain Â gsh2Î::GSH2 Â cells Â were Â grown Â in Â YPD Â overnight Â and Â and Â the Â next Â day Â 2x105 Â cells Â were Â harvested Â and Â were Â re-­âintroduced Â to Â fresh Â 50 Â ml Â YPD Â or Â 50 Â ml Â YNB, Â cells Â Â Â Â Â Â Â Â Â Â Â Â Â Â 114 Â were Â then Â harvested Â at Â 24 Â h Â and Â 48 Â h Â for Â the Â assay. Â Â Â In Â parallel, Â the Â intracellular Â glutathione Â content Â of Â WT, Â gsh2 Â mutants Â and Â the Â complemented Â strain Â gsh2Î::GSH2 Â was Â measured Â in Â an Â experimental Â set Â up Â where Â strains Â initially Â underwent Â 48 Â hours Â of Â iron Â starvation Â before Â the Â assay Â was Â performed. Â Then Â iron-­âstarved Â cells Â (described Â in Â 3.2.2.) Â were Â re-­âintroduced Â to Â fresh Â YNB Â with Â or Â without Â 5 Â mM Â GSH, Â and Â were Â harvested Â at Â 24 Â h Â and Â 48 Â h. Â Â The Â intracellular Â total Â glutathione Â levels Â (GSHt) Â were Â then Â quantified. Â Whole Â cells Â from Â each Â experimental Â set Â up Â were Â assayed Â for Â glutathione Â content Â using Â the Â BIOXYTECH® Â GSH/GSSG-­â412 Â colorimetric Â assay Â (OxisResearch; Â Portland, Â OR). Â Â This Â assay Â follows Â the Â principles Â of Â Tietze Â methods, Â which Â is Â an Â enzymatic Â method Â for Â quantitative Â determination Â of Â amounts Â of Â total Â (reduced Â and Â oxidized Â or Â GSHt) Â glutathione Â (M. Â S. Â Anderson Â et Â al., Â 1996, Â O. Â W. Â Griffith, Â 1980, Â H. Â Guntherberg Â and Â I. Â Rademacher, Â 1966, Â H. Â Guntherberg Â and Â J. Â Rost, Â 1966, Â F. Â Tietze, Â 1969) Â This Â method Â also Â employs Â Ellmanâs Â reagent Â (chromogen) Â 5,5'-­â Â dithiobis-­â2-­ânitrobenzoic Â acid Â or Â DTNB), Â which Â reacts Â with Â GSH Â to Â form Â a Â spectrophotometrically Â detectable Â product Â at Â 412 Â nm. Â Â In Â this Â assay, Â GSSG Â is Â determined Â by Â the Â reduction Â of Â GSSG Â to Â GSH, Â which Â is Â then Â determined Â by Â the Â reaction Â with Â Ellmanâs Â reagent. Â Â Â Here, Â to Â quantify Â reduced Â GSH, Â samples Â were Â obtained Â using Â a Â 5% Â cold Â metaphosphoric Â acid Â (MPA) Â extraction. Â The Â oxidized Â glutathione Â sample Â was Â obtained Â using Â the Â same Â type Â of Â extraction Â after Â scavenging Â the Â free Â thiols Â with Â scavenger Â 1-­âmethyl-­â2-­âvinylpyridinium Â trifluoromethanesulphonate Â (M2VP). Â To Â quantify Â GSH, Â 108 Â cells Â (in Â 1ml Â of Â culture) Â were Â harvested Â and Â were Â frozen Â atâ70°C. Â To Â quantify Â GSSH, Â cells Â were Â first Â mixed Â gently Â with Â 200 Â μl Â M2VP Â in Â a Â microcentrifuge Â tube Â and Â were Â frozen Â at Â â70°C. Â Â After Â thawing, Â cells Â were Â resuspended Â in Â cold Â 5% Â MPA Â on Â ice Â (at Â 1/4 Â dilution Â of Â the Â volume Â of Â Â Â Â Â Â Â Â Â Â Â Â Â Â 115 Â original Â sample, Â GSSG Â samples Â were Â mixed Â with Â 300 Â μl Â 5% Â MPA, Â GSH Â samples Â were Â mixed Â with Â 250 Â μl Â 5% Â MPA) Â and Â were Â mixed Â with Â glass Â beads. Â Subsequently, Â the Â cells Â were Â homogenized Â by Â bead Â beating Â (3x Â 90sec, Â with Â 1min Â interval Â cooling Â on Â ice). Â After Â centrifugation Â at Â 4,500 Â rpm Â for Â 10 Â min Â at Â 4°C, Â 50 Â μl Â of Â the Â MPA Â extract Â was Â mixed Â with Â 700 Â μL Â of Â the Â assay Â buffer Â (Na·PO4) Â followed Â by Â addition Â of Â glutathione Â reductase, Â Î²âNicotinamide Â adenine Â dinucleotide Â phosphate Â (NADPH) Â and Â the Â DTNB Â to Â each Â sample. Â The Â reaction Â mix Â was Â transferred Â to Â glass Â cuvettes Â and Â the Â change Â in Â color Â development Â of Â the Â chromogen Â DTNB Â during Â the Â reaction Â was Â measured Â by Â absorbance Â at Â 412 Â nm Â by Â spectrophotometer. Â The Â reaction Â was Â monitored Â for Â 5 Â minutes. Â In Â the Â blank Â reaction Â 50 Â μl Â of Â MPA Â was Â mixed Â with Â 700 Â μl Â of Â assay Â buffer Â only. Â Â The Â reduced Â and Â oxidized Â glutathione Â were Â calculated Â by Â measuring Â the Â change Â in Â absorbance Â at Â 412 Â nm Â as Â a Â linear Â function Â of Â the Â glutathione Â concentration Â in Â the Â sample. Â The Â concentration Â of Â GSH Â and Â GSSH Â in Â the Â sample Â was Â calculated Â by Â comparing Â with Â the Â rates Â of Â the Â standards Â (0.1, Â 0.25, Â 0.5, Â 1.5, Â 3 Â μM). Â Concentration Â of Â GSH Â and Â GSSG Â were Â calculated Â after Â subtracting Â the Â values Â generated Â by Â the Â blank Â reactions. Â The Â sum Â of Â quantified Â GSH Â and Â GSSG Â in Â each Â sample Â was Â calculated Â as Â total Â (reduced Â and Â oxidized Â or Â GSHt) Â glutathione Â content. Â Statistical Â significance Â was Â determined Â using Â a Â single Â factor Â ANOVA Â variance Â test. Â Â 3.2.4.3 Â Capsule Â formation, Â melanin Â production, Â and Â growth Â at Â 37°C Â Â Capsule Â formation Â was Â examined Â by Â differential Â interference Â microscopy Â (DIC). Â Briefly, Â cells Â were Â pre-­âgrown Â in Â YPD, Â washed Â twice Â with Â low Â iron Â water Â (Li-­âH2O), Â diluted Â to Â 106 Â cells/ml Â in Â Capsule Â Inducing Â Media Â (CIM), Â and Â grown Â for Â 24 Â hours Â at Â 30°C. Â Â CIM Â was Â prepared Â by Â dissolving Â 5g Â glucose, Â 5g Â asparagine, Â 4.78g Â HEPES, Â 0.4g Â K2HPO4, Â 0.25g Â CaCl2.2H2O, Â 0.08g Â MgSO4.7H2O, Â 1.85g Â NaHCO3, Â add Â 1ml Â of Â 1000X Â salt Â stock Â solution Â in Â 1L Â Â Â Â Â Â Â Â Â Â Â Â Â Â 116 Â of Â Li-­âH2O, Â pH Â adjusted Â to Â 7.2 Â using Â 1M Â MOPS Â made Â with Â low Â iron Â water, Â sterilized Â and Â 100 Â μl Â sterile Â thiamine Â (4 Â mg/ml) Â added. Â Â The Â cells Â were Â then Â resuspended Â in Â 1/10th Â volume Â of Â CIM, Â mixed Â 1:1 Â in Â India Â ink Â and Â visualized Â by Â negative Â staining Â on Â a Â Zeiss Â Axioplan Â 2 Â Imaging Â microscope Â by Â differential Â interference Â microscopy Â (DIC). Â Â Â Â Melanin Â production Â was Â examined Â on Â l-­â3,4-­âdihydroxyphenylalanine Â (l-­âDOPA) Â agar Â containing Â 0.1% Â glucose. Â Â Cells Â were Â pre Â grown Â in Â YPD, Â washed Â once, Â counted, Â and Â adjusted Â to Â 2 Â Ã Â 106 Â cells Â mlâ1 Â in Â YPD. Â Â Serial Â dilutions Â were Â performed, Â and Â 5 Â μl Â of Â each Â dilution Â was Â spotted Â onto Â agar Â medium. Â Â Plates Â were Â incubated Â at Â 30°C. Â Â Â Â Â The Â ablity Â of Â the Â gsh2 Â mutants Â to Â grow Â at Â host Â temperature Â was Â tested Â by Â plating Â on Â solid Â media Â with Â subsequent Â incubation Â at Â 37°C. Â 3.2.4.4 Â Stress Â and Â drug Â response Â assays Â Â Â Overnight Â pre-­âcultured Â cells Â of Â the Â wild-­âtype, Â gsh2 Â mutants, Â and Â reconstituted Â gsh2Î::GSH2 Â strains Â were Â washed, Â resuspended Â in Â H2O, Â and Â adjusted Â to Â 2 Â Ã Â 105 Â cells/μl. Â The Â cell Â suspensions Â were Â serially Â diluted Â 10-­âfold, Â and Â 5 Â μl Â of Â each Â dilution Â was Â spotted Â onto Â YPD Â plates Â with Â or Â without Â 1.5 Â M Â KCl, Â 1.5 Â M Â NaCl, Â 0.5 Â mg/ml Â Congo Â Red, Â 1 Â mM Â diamide, Â 10 Â μg/ml Â fluconazole, Â 0.1 Â μg/ml Â miconazole, Â or Â 1 Â μg/ml Â amphotericin Â B. Â Plates Â were Â incubated Â for Â 3 Â to Â 5 Â days Â at Â 30°C Â and Â photographed. Â Â 3.2.4.5 Â Â Sensitivity Â to Â inhibitors Â of Â mitochondria Â electron Â transport Â chain Â Â Â Â Overnight Â pre-­âcultured Â cells Â of Â the Â WT, Â gsh2 Â mutants, Â and Â reconstituted Â gsh2Î::GSH2 Â strains Â were Â washed, Â resuspended Â in Â H2O, Â and Â adjusted Â to Â 2 Â Ã Â 105 Â cells/μl. Â The Â cell Â suspensions Â were Â serially Â diluted Â 10-­âfold, Â and Â 5 Â μl Â of Â each Â dilution Â was Â spotted Â onto Â YPD Â plates Â supplemented Â with Â or Â without Â 75μM Â carbonyl Â cyanide Â m-­âchlorophenyl Â hydrazone Â (CCCP), Â 75 Â μg/ml Â Rotenone), Â 10mM Â potassium Â cyanide Â (KCN), Â 3 Â μg/ml Â Â Â Â Â Â Â Â Â Â Â Â Â Â 117 Â antimycin Â or Â 5 Â mM Â salicylhydroxamic Â acid Â (SHAM). Â Â Plates Â were Â incubated Â up Â to Â 5 Â days Â at Â 30°C Â and Â photographed. Â 3.3 Â Results Â Â 3.3.1 Â Â GSH Â synthetase Â encoded Â by Â GSH2 Â in Â C. Â neoformans Â belongs Â to Â the Â eukaryotic Â glutathione Â synthetase Â (eu-­âGS) Â family Â in Â the Â ATP-­âgrasp Â superfamily. Â Â The Â predicted Â glutathione Â synthetase Â encoded Â by Â GSH2 Â (CNAG_04647) Â in Â C Â neoformans Â had Â a Â length Â of Â 516 Â amino Â acids. Â BLAST Â analysis Â with Â this Â polypeptide Â sequence Â showed Â that Â GSH Â synthetase Â belongs Â to Â the Â Eukaryotic Â Glutathione Â Synthetase Â (eu-­âGS) Â family Â in Â the Â ATP-­âgrasp Â superfamily. Â Â The Â members Â of Â this Â group Â of Â ATP-­âbinding Â proteins Â cleave Â ATP Â to Â ADP Â and Â Pi, Â via Â the Â formation Â of Â an Â active Â intermediate Â y-­âL-­âglutamyl-­âL-­âcysteinyl Â phosphate Â (A. Â Meister Â and Â M. Â E. Â Anderson, Â 1983). Â Â An Â analysis Â of Â the Â amino Â acid Â sequence Â similarity Â of Â GSH Â synthetase Â in Â C. Â neoformans Â with Â other Â members Â of Â ATP-­âbinding Â eu-­âGS Â family Â of Â proteins Â revealed Â a Â conserved Â GSH-­âbinding Â domain, Â and Â ATP-­âbinding Â domain, Â and Â a Â magnesium Â binding Â domain, Â in Â addition Â to Â a Â C-­â Â terminal Â glycine-­ârich Â loop Â and Â an Â alanine-­ârich Â segment. Â The Â C-­âterminal Â glycine-­ârich Â loop Â and Â an Â alanine-­ârich Â segment Â have Â with Â high Â degree Â of Â conservation Â and Â are Â proposed Â to Â be Â critical Â for Â the Â catalytic Â function Â of Â GSH Â synthetases Â (C. Â Fan Â et Â al., Â 1995)(Figure Â 3.4 Â A). Â Â Â Â A Â Clustal Â Omega Â alignment Â of Â the Â amino Â acid Â sequence Â of Â the Â GSH Â synthetase Â of Â C. Â neoformans Â with Â four Â selected Â GSH Â synthetase Â sequences Â from Â other Â organisms Â revealed Â that Â the Â GSH Â synthetase Â protein Â sequence Â shares Â 37-­â39% Â identity Â with Â the Â four Â other Â eu-­âGS Â family Â gene Â products Â (39% Â amino Â acid Â sequence Â identity Â with Â GSH Â synthetase Â from Â S. Â pombe, Â 38% Â with Â GSH Â synthetase Â from Â S. Â cerevisiae, Â 39% Â with Â GSH Â synthetase Â from Â C. Â albicans, Â and Â 37 Â % Â identity Â with Â GSH Â synthetase Â in Â Arabidopsis Â thaliana Â Â (Figure Â 3.4 Â B). Â Â Â Â Â Â Â Â Â Â Â Â Â Â 118 Â A Â Â B Â Â Figure Â 3.4: Â Domain Â structure Â and Â Blastp Â analysis Â of Â Gsh2 Â in Â C. Â neoformans Â Â (A) Â The Â identified Â domains Â underlined Â in Â GSH Â synthetase Â (C. Â neoformans Â var. Â grubii Â H99) Â are Â as Â follows: Â amino Â acids Â 16-­â51, Â dimerization Â site; Â 129-­â Â 503, Â GSH Â binding Â pocket; Â 133-­â493, Â ATP-­âbinding Â pocket; Â 147-­â401 Â magnesium Â binding Â site; Â 399-­â409, Â glycine-­ârich Â loop; Â 495-­â507, Â alanine-­ârich Â loop. Â (B) Â Percentage Â of Â identity Â in Â amino Â acid Â sequences Â in Â GSH Â synthetases Â across Â selected Â species Â as Â determined Â by Â Blastp Â analysis. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 119 Â 3.3.2 Â Â GSH Â synthetase Â (Gsh2) Â is Â dispensable Â for Â growth Â under Â normal Â conditions Â in Â C. Â neoformans. Â Â Â Â The Â growth Â of Â three Â individual Â gsh2 Â mutants Â each Â with Â a Â total Â gene Â deletion Â was Â compared Â to Â growth Â of Â the Â WT Â and Â the Â complemented Â strain Â gsh2Î::GSH2 Â on Â YPD Â medium. Â Â The Â gsh2 Â mutants Â were Â viable Â on Â rich Â YPD Â and Â minimal Â YNB Â media, Â indicating Â that Â GSH2 Â is Â not Â an Â essential Â gene Â for Â growth Â under Â normal Â conditions Â for Â C. Â neoformans. Â Â This Â is Â in Â consistence Â with Â findings Â for Â Gsh2 Â in Â S. Â cerevisiae, Â which Â has Â also Â been Â shown Â to Â be Â dispensable Â for Â growth Â under Â normal Â conditions Â (C. Â M. Â Grant Â et Â al., Â 1997) Â Â Â Â Â Â Â Figure Â 3.5: Â Gsh2 Â is Â dispensable Â for Â growth Â under Â normal Â conditions Â in Â C. Â neoformans. Â Spot Â assays Â starting Â with Â 106 Â cells Â in Â the Â first Â spot Â and Â 10-­âfold Â dilutions Â in Â the Â spots Â to Â the Â right Â were Â plated Â on Â YPD Â or Â YNB Â agar. Â Â 3.3.3 Â Â gsh2 Â mutants Â are Â sensitive Â to Â iron Â starvation Â and Â require Â exogenous Â GSH Â for Â growth Â on Â minimal Â or Â iron-­âreplete Â media Â after Â iron Â starvation Â Â Â Â Â The Â growth Â phenotype Â of Â gsh2 Â mutants Â post Â iron-­âstarvation Â was Â tested Â in Â comparison Â with Â the Â WT Â strain Â and Â the Â complemented Â gsh2Î::GSH2 Â strain. Â Each Â strain Â was Â starved Â for Â iron Â in Â LIM Â for Â 48 Â hours Â and Â plated Â on Â LIM Â with Â 10μM Â FeCl3, Â 100 Â μM Â FeCl3 Â or Â 100 Â μM Â hemin Â (See Â materials Â and Â methods Â 3.2.4.1). Â All Â five Â strains Â including Â the Â gsh2 Â WT gsh2Î1 gsh2Î2 gsh2Î3 gsh2Î::GSH2 Â Â Â Â Â Â Â Â Â Â Â Â Â 120 Â mutants Â displayed Â a Â robust Â growth Â phenotype Â on Â YPD Â plate Â after Â iron Â starvation. Â Â However, Â when Â plated Â on Â minimal Â YNB Â agar, Â gsh2 Â mutants Â displayed Â a Â prominent Â growth Â defect Â compared Â to Â the Â WT Â and Â gsh2Î::GSH2 Â strains, Â indicating Â that Â the Â nutrient-­âlimited Â condition Â of Â YNB Â did Â not Â support Â the Â growth Â of Â the Â gsh2 Â mutants Â after Â iron Â starvation. Â I Â speculated Â that Â this Â is Â likely Â due Â to Â lack Â of Â GSH Â levels Â in Â the Â gsh2 Â mutants Â that Â underwent Â iron-­âstarvation. Â It Â is Â possible Â that Â the Â detrimental Â side Â effects Â of Â iron Â starvation Â are Â more Â pronounced Â in Â the Â presence Â of Â low Â levels Â of Â GSH Â in Â gsh2 Â mutants. Â Of Â note, Â it Â has Â been Â shown Â that Â GSH Â accumulates Â in Â the Â vacuole, Â and Â serves Â as Â a Â nitrogen Â and Â amino Â acid Â source Â during Â nitrogen Â starvation Â of Â S. Â cerevisiae Â (K. Â Mehdi Â and Â M. Â J. Â Penninckx, Â 1997). Â Â Furthermore, Â as Â expected Â none Â of Â the Â strains Â grew Â on Â LIM Â agar Â plates Â (YNB+ Â 150 Â μM Â BPS). Â Â Testing Â the Â growth Â phenotype Â of Â the Â strains Â on Â LIM Â agar Â supplemented Â with Â iron Â showed Â that Â gsh2 Â mutants Â had Â a Â growth Â defect Â in Â the Â presence Â of Â 10 Â μM Â FeCl3, Â 100 Â μM Â FeCl3 Â or Â 100 Â μM Â hemin Â ccompared Â to Â the Â WT Â and Â gsh2Î::GSH2 Â strains. Â These Â results Â led Â to Â speculation Â that Â a Â potential Â lack Â of Â GSH Â in Â gsh2 Â mutants Â might Â be Â underlying Â these Â observations Â as Â well. Â Â Therefore, Â I Â next Â sought Â to Â test Â the Â effect Â of Â exogenous Â levels Â of Â GSH Â on Â the Â observed Â phenotypes Â in Â gsh2 Â mutants. Â Â To Â test Â this, Â I Â pre-­âtreated Â the Â gsh2 Â mutants, Â and Â the Â WT Â and Â gsh2Î::GSH2 Â strains Â with Â exogenous Â GSH Â (5 Â mM). Â Â GSH Â was Â added Â to Â the Â cultures Â during Â the Â period Â that Â they Â were Â undergoing Â iron-­âstarvation Â at Â 12 Â hours Â before Â harvest. Â Â Harvested Â cells Â were Â then Â plated Â on Â LIM Â plates Â + Â 10 Â μM Â FeCl3, Â 100 Â μM Â FeCl3 Â or Â 100 Â μM Â hemin. Â Â The Â results Â showed Â that Â addition Â of Â GSH Â recued Â the Â growth Â defect Â of Â the Â gsh2 Â mutants, Â as Â the Â mutants Â had Â a Â solid Â growth Â phenotype Â compared Â to Â the Â WT Â and Â gsh2Î::GSH2 Â Â strains Â on Â YNB Â and Â iron-­âreplete Â media Â (Figure Â 3.6) Â . Â Â Â These Â results Â reflect Â the Â importance Â of Â Gsh2 Â and Â ultimately Â GSH Â for Â growth Â under Â Â Â Â Â Â Â Â Â Â Â Â Â Â 121 Â conditions Â that Â are Â comparable Â to Â the Â conditions Â that Â C. Â neoformans Â may Â encounter Â in Â the Â human Â host. Â Â It Â is Â possible Â that Â lack Â of Â GSH Â in Â gsh2 Â mutants Â makes Â them Â more Â susceptible Â to Â the Â effects Â of Â oxidative Â stress Â brought Â upon Â by Â iron Â starvation, Â and Â therefore Â addition Â of Â GSH Â restores Â the Â growth Â phenotype Â of Â gsh2 Â mutants Â as Â a Â result Â of Â protective Â role Â of Â GSH Â against Â oxidative Â stress, Â or Â the Â auxotrophy Â of Â gsh2 Â mutants Â for Â GSH Â itself. Â Â Also, Â given Â the Â proven Â role Â of Â GSH Â in Â sequestering Â iron Â and Â detoxification Â of Â metal Â ions Â it Â is Â likely Â that Â introduction Â of Â iron Â to Â gsh2 Â mutants Â with Â significantly Â low Â levels Â of Â GSH Â leads Â to Â presence Â of Â unbound Â iron Â in Â the Â cell Â (M. Â Jozefczak Â et Â al., Â 2012). Â Free Â iron Â is Â known Â to Â have Â toxic Â effects Â on Â cellular Â metabolism Â (J. Â W. Â Eaton Â and Â M. Â Qian, Â 2002). Â Therefore Â addition Â of Â GSH Â to Â gsh2 Â mutants Â potentially Â leads Â to Â chelating Â iron Â and Â reducing Â the Â amount Â of Â free Â metal Â inside Â cells, Â which Â ultimately Â results Â in Â undisturbed Â metabolism Â and Â solid Â growth Â in Â gsh2 Â mutants Â in Â presence Â of Â iron. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 122 Â Â Figure Â 3.6: Â Growth Â phenotypes Â of Â gsh2 Â mutants Â in Â YNB Â and Â iron-­âreplete Â media Â after Â Â iron Â starvation. Â (A Â & Â B) Â Growth Â of Â strains Â on Â YPD, Â YNB, Â and Â LIM Â agar Â supplemented Â with Â iron Â sources. Â (C Â & Â D) Â Strains Â were Â pre-­âtreated Â with Â 5 Â mM Â GSH Â and Â growth Â was Â evaluated Â on Â YPD, Â YNB, Â and Â LIM Â supplemented Â with Â different Â iron Â sources Â as Â shown. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 123 Â 3.3.4 Â Â The Â gsh2 Â mutants Â have Â reduced Â intracellular Â total Â glutathione Â (GSHt) Â compared Â to Â the Â WT Â strain Â Â Â The Â results Â of Â the Â growth Â assays Â in Â 3.3.3 Â suggested Â that Â the Â gsh2 Â mutants Â had Â a Â decreased Â level Â of Â intracellular Â glutathione. Â I Â sought Â to Â examine Â this Â possibility Â by Â measuring Â intracellular Â levels Â of Â GSH Â in Â gsh2 Â mutants Â grown Â under Â normal Â conditions Â without Â prior Â iron-­âstarvation Â (Figure Â 3.7 Â A) Â or Â with Â prior Â iron-­âstarvation Â (Figure Â 3.7 Â B) Â (See Â materials Â and Â method Â for Â details, Â section Â 3.2.4.2). Â Â This Â analysis Â revealed Â an Â average Â of Â 55 Â nmol Â of Â GSHt/mg Â of Â protein Â in Â the Â lysate Â from Â the Â WT Â cells, Â 45 Â nmol Â of Â GSHt/mg Â of Â protein Â in Â the Â lysate Â from Â the Â complemented Â strain Â cells, Â and Â an Â average Â of Â 5 Â nmol Â of Â GSHt/mg Â of Â protein Â in Â the Â lysate Â from Â the Â gsh2 Â mutants Â cells Â grown Â in Â YPD. Â The Â GSHt Â content Â was Â slightly Â lower Â in Â strains Â grown Â in Â YNB Â compared Â to Â strains Â grown Â in Â YPD. Â This Â slight Â difference Â between Â GSHt Â levels Â calculated Â in Â YNB Â compared Â to Â YPD Â was Â not Â statistically Â significant. Â There Â was Â also Â a Â small Â decrease Â in Â GSHt Â levels Â in Â all Â strains Â at Â 48 Â h Â compared Â to Â 24 Â h, Â but Â this Â difference Â was Â not Â statistically Â significant. Â Â The Â 10X Â fold Â decrease Â in Â GSHt Â levels Â in Â gsh2 Â mutants Â compared Â to Â the Â WT Â at Â both Â time Â points Â were Â statistically Â significant Â (p Â value<0.05). Â These Â results Â confirmed Â that Â gsh2 Â mutants Â have Â lower Â GSH Â content Â compared Â to Â the Â WT Â strain Â and Â the Â complemented Â strain Â under Â normal Â growth Â conditions. Â Despite Â significantly Â low Â levels Â of Â GSH Â quantified Â in Â gsh2 Â mutants Â in Â C. Â neoformans, Â these Â mutants Â are Â viable Â due Â to Â the Â potential Â accumulation Â of Â y-­âGlu-­âCys Â intermediate, Â which Â remains Â to Â be Â further Â confirmed. Â On Â this Â note, Â it Â was Â previously Â shown Â that Â the Â gsh2 Â mutant Â lacked Â GSH Â but Â accumulated Â the Â dipeptide Â Î³-­âGlu-­âCys Â intermediate Â in Â GSH Â biosynthesis. Â Importantly, Â it Â has Â also Â been Â shown Â that Â the Â Î³-­âGlu-­âCys Â intermediate Â was Â at Â least Â as Â good Â as Â GSH Â in Â protecting Â yeast Â cells Â against Â an Â oxidant Â Â Â Â Â Â Â Â Â Â Â Â Â Â 124 Â challenge Â and Â served Â as Â an Â antioxidant Â and Â substitute Â for Â GSH Â in Â the Â cell Â (C. Â M. Â Grant, Â et Â al., Â 1997). Â Â According Â to Â results Â outlined Â in Â section Â 3.3.3 Â and Â shown Â in Â Figure Â 3.6 Â of Â this Â chapter, Â I Â found Â that Â gsh2 Â mutants Â did Â not Â grow Â in Â YNB Â after Â 48 Â hours Â of Â starvation, Â but Â pre-­âtreatment Â of Â the Â cells Â with Â 5 Â mM Â GSH Â restored Â growth Â of Â gsh2 Â mutants Â on Â YNB Â plates. Â Therefore, Â I Â sought Â to Â further Â validate Â that Â the Â addition Â of Â GSH Â resulted Â in Â restoration Â of Â growth Â in Â gsh2 Â mutants. Â To Â do Â this, Â strains Â were Â initially Â starved Â for Â 48 Â hours Â and Â were Â then Â grown Â in Â YNB Â or Â YNB Â supplemented Â with Â 5 Â mM Â GSH Â for Â 24 Â h Â and Â 48 Â h Â for Â GSHt Â assay. Â The Â results Â from Â the Â GSHt Â assay Â showed Â an Â average Â level Â of Â 60 Â nmol Â of Â GSHt/mg Â of Â protein Â in Â the Â lysate Â from Â the Â WT Â cells Â pre-­âstarved Â in Â iron Â and Â grown Â in Â YNB Â for Â 24 Â hours Â and Â 80 Â nmol Â of Â GSHt/mg Â of Â protein Â in Â the Â lysate Â from Â WT Â cells Â pre-­âstarved Â in Â iron, Â grown Â in Â YNB Â +5 Â mM Â GSH Â for Â 24 Â hours Â (1.3 Â fold Â higher) Â which Â was Â statistically Â significant. Â The Â results Â from Â this Â assay Â showed Â that Â the Â levels Â of Â GSHt Â increased Â in Â gsh2 Â mutant Â after Â growth Â in Â YNB+ Â 5 Â mM Â GSH Â (compared Â to Â YNB) Â from Â 2.5 Â nmol Â of Â of Â GSHt/mg Â of Â protein Â in Â the Â lysate Â to Â 20 Â nmol Â of Â GSHt/mg Â of Â lysate Â respectively Â (almost Â a Â 10-­âfold Â increase). Â Â Â Overall, Â these Â findings Â lead Â to Â the Â conclusion Â that Â gsh2 Â mutants Â have Â a Â significantly Â lower Â amount Â of Â cellular Â glutathione, Â and Â addition Â of Â exogenous Â levels Â of Â GSH Â restores Â GSH Â content Â in Â these Â mutants. Â Lack Â of Â GSH Â levels Â in Â gsh2 Â mutants Â is Â most Â likely Â the Â underlying Â reason Â for Â the Â growth Â defect Â phenotype Â of Â gsh2 Â mutants Â after Â iron-­âstarvation Â and Â introduction Â to Â YNB Â or Â ion-­âreplete Â conditions. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 125 Â Â Â Figure Â 3.7: Â Total Â glutathione Â (GSHt) Â levels Â in Â the Â gsh2 Â mutants. Â (A) Â GSHt Â levels Â in Â gsh2 Â mutants Â were Â measured Â and Â compared Â to Â the Â WT Â and Â the Â complemented Â strains Â grown Â in Â YPD Â (brown Â bars) Â or Â YNB Â (yellow Â bars). Â Samples Â were Â taken Â at Â 24 Â h Â and Â 48 Â h. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 126 Â (Statistical Â analysis Â Â *p< Â 0.05). Â (B) Â Total Â glutathione Â (GSHt) Â levels Â in Â the Â gsh2 Â mutants Â were Â measured Â and Â compared Â to Â WT-­âH99 Â and Â the Â complemented Â strains Â that Â were Â iron Â starved Â and Â transferred Â to Â YNB Â (Black Â bar) Â or Â YNB+ Â 5 Â mM Â GSH Â (purple Â bars). Â Samples Â were Â taken Â at Â 24 Â h Â and Â 48 Â h). Â 3 Â biological Â replicates Â for Â each Â strain Â were Â prepared Â for Â each Â growth Â condition. Â (Statistical Â analysis Â was Â performed Â using Â using Â a Â single Â factor Â ANOVA Â variance Â test Â *p< Â 0.05 Â and Â *p< Â 0.0001 Â 3.3.5 Â Â gsh2 Â is Â involved Â in Â multi-­âstress Â resistance Â Â The Â role Â of Â GSH Â as Â the Â major Â cellular Â redox Â buffer Â is Â well Â established Â (K. Â Aquilano Â et Â al., Â 2014). Â Given Â the Â significance Â of Â GSH Â role Â in Â anti-­âoxidant Â defense, Â I Â sought Â to Â further Â examine Â the Â outcome Â of Â deletion Â of Â GSH2 Â and Â reduced Â GSH Â in Â C. Â neoformans, Â by Â phenotypic Â analysis Â of Â gsh2 Â mutants Â in Â presence Â of Â stressors. Â I Â found Â that Â gsh2 Â mutants Â were Â dispensable Â for Â C. Â neoformans Â growth Â in Â the Â presence Â of Â common Â ROS-­âgenerating Â stressors Â such Â as Â H2O2 Â (data Â not Â shown), Â however Â gsh2 Â mutants Â were Â in Â particular Â sensitive Â to Â diamide. Â Â Â The Â growth Â defect Â of Â gsh2 Â mutants Â in Â presence Â of Â diamide Â are Â also Â consistent Â with Â my Â finding Â of Â low Â levels Â of Â GSHt Â in Â gsh2 Â mutants Â compared Â to Â the Â WT. Â Diamide Â is Â a Â thiol-­âoxidizing Â agent Â that Â mediates Â the Â direct Â oxidation Â of Â reduced Â glutathione Â (GSH) Â to Â GSSG Â (N. Â S. Â Kosower Â et Â al., Â 1969). Â Insufficient Â levels Â of Â GSH Â in Â gsh2 Â mutants Â can Â lead Â to Â loss Â of Â protection Â against Â toxicity Â of Â diamide. Â This Â potentially Â results Â in Â slower Â growth Â of Â gsh2 Â mutants Â compared Â to Â the Â WT Â and Â the Â complemented Â strain Â in Â presence Â of Â diamide Â (Figure Â 3.8). Â Â The Â C. Â neoformans Â cell Â wall Â provides Â protection Â from Â the Â environment Â and Â determines Â the Â cell Â morphology Â during Â development Â of Â C. Â neoformans Â (S. Â R. Â Waterman Â et Â Â Â Â Â Â Â Â Â Â Â Â Â Â 127 Â al., Â 2007). Â To Â assess Â the Â role Â of Â Gsh2 Â in Â cell Â integrity, Â the Â WT Â strain, Â gsh2 Â mutants Â and Â the Â gsh2Î::GSH2 Â strain Â were Â incubated Â on Â media Â containing Â Congo Â Red Â which Â is Â known Â to Â impair Â cell Â wall Â assembly, Â and Â to Â interfere Â with Â cell Â morphology. Â Â Additionally, Â resistance Â to Â salt Â stress Â has Â been Â correlated Â with Â C. Â neoformans Â survival Â in Â vivo, Â and Â genes Â involved Â and/or Â regulated Â by Â the Â HOG Â pathway Â are Â expressed Â during Â osmotic Â stress Â (Y. Â J. Â Ko Â et Â al., Â 2009). Â To Â test Â these Â stress Â phenotypes, Â the Â WT Â strain, Â the Â gsh2 Â mutants Â and Â the Â gsh2Î::GSH2 Â strain Â were Â incubated Â on Â media Â containing Â high Â salt Â concentrations. Â Â The Â gsh2 Â mutants Â displayed Â a Â marked Â sensitivity Â to Â both Â Congo Â Red Â and Â high Â salt Â concentrations Â (Figure Â 3.8). Â This Â information Â suggests Â that Â Gsh2 Â is Â required Â for Â assembly Â of Â cell Â wall Â or Â expression Â of Â genes Â for Â cell Â wall Â synthesis. Â Furthermore, Â the Â confirmed Â lack Â of Â GSHt Â in Â gsh2 Â mutants Â may Â also Â account Â for Â an Â overall Â higher Â oxidative Â state Â of Â the Â cells. Â Â Â Figure Â 3.8: Â The Â gsh2 Â mutants Â have Â increased Â susceptibility Â to Â salt Â stress Â and Â thiol Â oxidizing Â agent Â diamide. Â Tenfold Â serial Â dilution Â of Â cells Â of Â the Â indicated Â strains Â were Â spotted Â onto Â solid Â YPD Â without Â or Â with Â 1.5 Â M Â NaCl, Â 1.5 Â M Â KCl, Â or Â 1mM Â diamide. Â The Â plates Â were Â incubated Â at Â 30°C Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 128 Â 3.3.6 Â Â Â Lack Â of Â Gsh2 Â affects Â virulence Â factors Â in Â C. Â neoformans Â Â Growth Â at Â human Â body Â temperature, Â production Â of Â melanin, Â and Â capsule Â are Â classical Â virulence Â factors Â for Â C. Â neoformans. Â For Â example, Â it Â has Â been Â shown Â that Â melanized Â cells Â are Â more Â resistant Â to Â ROS Â and Â reactive Â nitrogen Â intermediates Â (RNI) Â than Â non-­âmelanized Â cells Â (Y. Â Wang Â and Â A. Â Casadevall, Â 1994). Â Strains Â that Â do Â not Â produce Â melanin Â are Â reported Â to Â have Â attenuated Â virulence Â in Â animal Â models Â (K. Â J. Â Kwon-­âChung Â and Â J. Â C. Â Rhodes, Â 1986). Â I Â tested Â the Â expression Â of Â these Â major Â virulence Â traits Â in Â gsh2 Â mutants Â and Â compared Â the Â resulting Â phenotypes Â to Â those Â in Â the Â WT Â and Â reconstituted Â strains. Â Â I Â found Â that Â that Â gsh2 Â mutants Â did Â not Â produce Â capsule Â or Â melanin, Â and Â lost Â the Â ability Â to Â grow Â at Â 37°C. Â Â These Â findings Â indicate Â that Â an Â intact Â GSH Â biosynthesis Â pathway Â and Â sufficient Â levels Â of Â GSH Â are Â required Â for Â the Â elaboration Â of Â virulence Â traits Â in Â C. Â neoformans. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 129 Â Â Â Figure Â 3.9: Â The Â gsh2 Â mutants Â are Â impaired Â in Â virulence-­ârelated Â traits. Â Loss Â of Â GSH2 Â leads Â to Â virulence-­ârelated Â phenotypes Â including Â loss Â of Â capsule Â formation, Â an Â altered Â melanin Â production, Â and Â poor Â growth Â at Â 37°C. Â 3.3.7 Â Â gsh2 Â mutants Â display Â increased Â susceptibility Â to Â antifungal Â drugs Â Â The Â roles Â of Â GSH Â in Â the Â detoxification Â of Â drug Â compounds Â and Â resistance Â against Â antibiotics Â have Â been Â described Â in Â Cyanobacteria, Â E. Â coli, Â and Â C. Â albicans Â (J. Â C. Â Cameron Â and Â H. Â B. Â Pakrasi, Â 2011, Â M. Â Kominkova Â et Â al., Â 2015, Â B. Â Maras Â et Â al., Â 2014). Â For Â instance, Â it Â has Â been Â shown Â that Â GSH Â metabolism Â plays Â an Â important Â role Â in Â the Â global Â organization Â of Â C. Â albicans Â cells Â for Â resistance Â to Â fluconazole Â (B. Â Maras, Â et Â al., Â 2014). Â Â Â This Â led Â me Â to Â hypothesize Â that Â gsh2 Â mutants Â in Â C. Â neoformans Â will Â have Â an Â Â Â Â Â Â Â Â Â Â Â Â Â Â 130 Â impaired Â protection Â against Â antifungal Â challenge, Â given Â their Â significantly Â low Â levels Â of Â GSH. Â I Â tested Â this Â hypothesis Â by Â phenotypic Â analysis Â of Â gsh2 Â mutants Â in Â presence Â of Â the Â antifungal Â drugs Â that Â are Â currently Â used Â to Â treat Â cryptococcosis. Â Compared Â to Â the Â WT Â and Â the Â complemented Â strain, Â the Â gsh2 Â mutants Â displayed Â increased Â sensitivity Â to Â the Â azole Â drugs Â including Â fluconazole Â and Â miconazole, Â which Â are Â known Â to Â target Â and Â inhibit Â ergosterol Â biosynthesis. Â The Â gsh2 Â mutants Â displayed Â sensitivity Â to Â amphotericin, Â which Â binds Â irreversibly Â to Â ergosterol, Â resulting Â in Â disruption Â of Â membrane Â integrity. Â Taken Â together Â results Â support Â the Â idea Â that Â Gsh2 Â and Â cellular Â GSH Â alter Â susceptibility Â of Â C Â neoformans Â to Â antifungal Â drugs. Â Â Â Â Figure Â 3.10: Â Deletion Â of Â GSH2 Â Increases Â susceptibility Â to Â antifungal Â agents.The Â growth Â of Â strains Â in Â media Â containing Â the Â antifungal Â drugs Â fluconazole, Â miconazole Â and Â amphotericin Â B Â is Â shown. Â Ten-­âfold Â serial Â dilutions Â of Â cells Â (starting Â at Â 106 Â cells) Â were Â spotted Â onto Â YPD Â plates Â with Â and Â without Â the Â antifungal Â drug Â indicated. Â Plates Â were Â incubated Â at Â 30°C Â for Â 3 Â days. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 131 Â 3.3.8 Â Â Â gsh2 Â mutants Â display Â subtle Â susceptibility Â to Â mitochondria Â inhibitors Â Â Â It Â is Â known Â that Â GSH Â is Â synthesized Â in Â the Â cytosol Â and Â transported Â into Â the Â mitochondria Â via Â an Â energy-­âdependent Â transporter Â (A. Â Meister, Â 1988a, Â b). Â GSH Â is Â important Â for Â the Â maintenance Â of Â optimum Â mitochondrial Â function. Â Distinguished Â from Â cytosolic Â GSH, Â mitochondria Â also Â contain Â GSH Â (mGSh) Â and Â it Â has Â also Â been Â suggested Â that Â decreases Â in Â cellular Â GSH Â availability Â in Â the Â mammalian Â cells Â promote Â mitochondrial Â damage Â via Â increased Â ROS Â (A. Â Jain Â et Â al., Â 1991). Â Â Â Â This Â led Â to Â the Â hypothesis Â that Â loss Â of Â GSH2 Â in Â C. Â neoformans Â and Â lack Â of Â synthesis Â of Â GSH Â in Â these Â mutants Â will Â affect Â mitochondrial Â functions. Â I Â tested Â this Â hypothesis Â by Â phenotypic Â analysis Â of Â gsh2 Â mutants Â along Â with Â the Â WT Â and Â the Â complemented Â strain Â in Â presence Â of Â inhibitors Â of Â mitochondrial Â functions, Â including Â 75μM Â CCCP, Â 75 Â μg/ml Â rotenone, Â 10 Â mM Â (KCN), Â 3 Â μg/ml Â antimycin Â or Â 5 Â mM Â SHAM. Â Results Â show Â that Â gsh2 Â mutants Â have Â a Â subtle Â sensitivity Â compare Â to Â the Â WT Â and Â the Â complemented Â strain Â to Â respiratory Â chain Â enzymes Â inhibitors. Â These Â results Â suggest Â that Â GSH2 Â and Â cellular Â levels Â of Â GSH Â influence Â mitochondrial Â functions. Â Â The Â role Â of Â mGSH Â in Â C. Â neoformans Â remains Â to Â be Â investigated. Â Â Figure Â 3.11: Â Â gsh2 Â mutants Â display Â a Â subtle Â susceptibility Â to Â ETC Â inhibitors. Â The Â growth Â of Â strains Â in Â media Â containing Â the Â inhibitors Â of Â mitochondrial Â function Â is Â shown. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 132 Â Ten-­âfold Â serial Â dilutions Â of Â cells Â (starting Â at Â 106 Â cells) Â were Â spotted Â onto Â YPD Â plates Â with Â and Â without Â the Â compounds Â indicated. Â Plates Â were Â incubated Â at Â 30°C Â for Â 5 Â days. Â 3.4 Â Discussion Â Â Â GSH Â is Â essential Â in Â eukaryotes Â and Â plays Â critical Â roles Â in Â many Â cellular Â processes. Â For Â example, Â GSH Â participates Â in Â antioxidant Â defense Â systems, Â detoxification Â of Â xenobiotics/heavy Â metals, Â and Â regulation Â of Â gene Â expression. Â Â In Â eukaryotes, Â GSH Â is Â essential Â for Â assembling Â cytoplasmic Â FeâS Â clusters Â and Â maintenance Â of Â mitochondrial Â stability Â and Â functions Â (K. Â Sipos, Â et Â al., Â 2002, Â C. Â M. Â Grant, Â et Â al., Â 1996, Â A. Â Jain, Â et Â al., Â 1991, Â C. Â Kumar, Â et Â al., Â 2011). Â GSH Â is Â also Â required Â for Â storage Â and Â transport Â of Â sulfur Â which Â is Â an Â essential Â element Â for Â growth Â of Â intracellular Â pathogens Â (N. Â Rouhier Â et Â al., Â 2008, Â K. Â Hoffmann Â et Â al., Â 2013). Â Â Â Â The Â biosynthesis Â of Â GSH Â has Â been Â studied Â in Â a Â variety Â of Â organisms Â such Â as Â E. Â coli, Â S. Â cerevisiae, Â C. Â albicans Â and Â Trypanosoma Â brucei Â (S. Â Bouter Â et Â al., Â 1988, Â J. Â C. Â Lee Â et Â al., Â 2001, Â Y. Â U. Â Baek, Â et Â al., Â 2004, Â T. Â T. Â Huynh Â et Â al., Â 2003). Â It Â was Â shown Â that Â GSH Â biosynthesis Â is Â not Â essential Â in Â these Â microorganisms Â as Â long Â as Â they Â are Â supplemented Â with Â an Â exogenous Â high Â concentration Â of Â GSH Â in Â the Â extracellular Â environment. Â In Â contrast, Â glutathione Â synthetase Â deletion Â mutants Â in Â S. Â cerevisiae Â do Â not Â require Â exogenous Â GSH Â for Â growth, Â as Â they Â accumulate Â and Â utilize Â the Â GSH Â precursor Â Î³-­âglutamylcysteine Â (Î³-­âGlu-­âCys), Â as Â a Â substitute Â under Â normal Â growth Â conditions Â (C. Â M. Â Grant, Â et Â al., Â 1997). Â Â Â In Â C. Â neoformans, Â glutathione Â metabolism Â genes Â were Â induced Â during Â heat Â shock, Â nitrosative Â stress Â or Â during Â phagocytosis Â by Â macrophages Â (T. Â A. Â Missall Â et Â al., Â 2006). Â Â Interestingly, Â glutathione Â reductase Â (Glr1), Â which Â reduces Â the Â disulphide Â form Â of Â glutathione Â (GSSG) Â to Â GSH, Â has Â been Â shown Â to Â be Â important Â for Â virulence Â in Â C. Â neoformans, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 133 Â as Â a Â glr1 Â deletion Â mutant Â was Â found Â to Â be Â avirulent, Â and Â was Â more Â sensitive Â to Â nitrosative Â stress Â and Â macrophage Â killing Â (T. Â A. Â Missall, Â et Â al., Â 2006). Â This Â further Â highlights Â the Â importance Â of Â glutathione Â biosynthesis Â and Â metabolism Â in Â C. Â neoformans. Â Â However, Â the Â role Â of Â glutathione Â in Â C. Â neoformans Â has Â not Â been Â explored Â so Â far. Â Â In Â this Â chapter, Â I Â characterized Â the Â role Â of Â GSH2 Â gene, Â which Â encodes Â GSH Â synthetase Â in Â the Â glutathione Â biosynthesis Â pathway, Â and Â I Â uncovered Â a Â pleiotropic Â consequence Â of Â lack Â of Â GSH Â due Â to Â deletion Â of Â GSH2 Â in Â C. Â neoformans. Â Â 3.4.1 Â GSH Â synthetase Â is Â dispensable Â for Â growth Â under Â normal Â growth Â conditions Â Â Â A Â total Â gene Â deletion Â of Â GSH2 Â in Â C. Â neoformans Â was Â generated Â by Â replacement Â of Â the Â entire Â GSH2 Â open Â reading Â frame Â with Â HYGr Â gene. Â Despite Â significantly Â low Â amount Â of Â GSHt Â measured Â in Â gsh2 Â mutants, Â they Â were Â viable Â on Â rich Â YPD Â and Â minimal Â YNB Â without Â exogenous Â glutathione. Â This Â indicated Â that Â GSH2 Â is Â not Â an Â essential Â gene Â for Â normal Â growth Â under Â non-­âstressed Â conditions. Â My Â findings Â were Â in Â agreement Â with Â findings Â on Â Gsh2 Â in Â S. Â cerevisiae. Â It Â was Â shown Â that Â Gsh2 Â was Â dispensable Â for Â growth Â under Â normal Â conditions Â in Â S. Â cerevisiae Â due Â to Â accumulation Â of Â the Â dipeptide Â Î³-­âGlu-­âCys Â intermediate. Â This Â intermediate Â is Â the Â product Â of Â GSH1 Â and Â was Â shown Â to Â protect Â yeast Â cells Â against Â an Â oxidant Â challenge Â and Â served Â as Â an Â antioxidant Â and Â substitute Â for Â GSH Â in Â the Â cell Â (C. Â M. Â Grant, Â et Â al., Â 1997). Â Similarly, Â I Â predict Â that Â accumulation Â of Â the Â Î³-­âGlu-­âCys Â intermediate Â in Â gsh2 Â mutants Â in Â C.neoformans Â is Â sufficient Â for Â growth Â in Â YPD Â and Â YNB. Â This Â prediction Â will Â require Â further Â studies Â for Â confirmation. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 134 Â 3.4.2 Â Â Phenotypic Â analysis Â of Â gsh2 Â mutants Â suggest Â an Â interplay Â between Â GSH Â homeostasis Â and Â iron Â homeostasis Â in Â C. Â neoformans Â Â Â The Â growth Â phenotypes Â of Â gsh2 Â mutants Â subjected Â to Â iron Â starvation Â revealed Â a Â further Â understanding Â of Â the Â role Â of Â Gsh2 Â in Â C. Â neoformans. Â Â I Â found Â that Â iron-­âstarved Â gsh2 Â mutants Â had Â a Â normal Â growth Â phenotype Â similar Â to Â the Â WT Â and Â the Â complemented Â strains Â when Â plated Â on Â rich Â YPD Â agar, Â but Â showed Â a Â pronounced Â growth Â defect Â when Â plated Â on Â minimal Â YNB Â agar. Â One Â potential Â explanation Â for Â the Â observed Â difference Â is Â that Â compared Â to Â YNB, Â YPD Â has Â a Â higher Â content Â of Â amino Â acids Â including Â methionine, Â cysteine Â and Â their Â constituent Â sulfur. Â Sulfur Â also Â serves Â as Â a Â constituent Â of Â organic Â molecules Â like Â coenzyme-­âA, Â GSH Â and Â Fe-­âS Â clusters. Â The Â importance Â of Â sulfur Â in Â fungal Â species Â such Â as Â C. Â albicans, Â C. Â glabrata Â and Â Aspergillus Â fumigatus Â has Â been Â demonstrated Â (Marzluf Â et Â al., Â 1997). Â I Â speculate Â that Â iron Â depletion Â in Â gsh2 Â mutants Â in Â the Â absence Â of Â GSH Â may Â lead Â to Â a Â higher Â nutritional Â demand Â for Â growth Â in Â gsh2 Â mutants, Â which Â is Â satisfied Â in Â the Â nutrient-­ârich Â condition Â of Â YPD Â compared Â to Â YNB. Â Importantly, Â addition Â of Â iron Â to Â YNB Â did Â not Â restore Â growth Â in Â gsh2 Â mutants, Â but Â pre-­âtreatment Â of Â gsh2 Â mutants Â with Â GSH Â led Â to Â restoration Â of Â growth Â on Â YNB. Â Overall, Â these Â results Â clearly Â indicated Â that Â depletion Â of Â iron Â in Â gsh2 Â mutants Â led Â to Â auxotrophy Â for Â GSH. Â I Â also Â measured Â GSHt Â content Â in Â cells Â grown Â in Â YNB Â after Â undergoing Â iron Â starvation. Â I Â first Â found Â that Â iron-­âstarved Â gsh2 Â mutants Â had Â slightly Â a Â lower Â amount Â of Â GSHt Â compared Â to Â the Â non-­âstarved Â cells Â and, Â importantly, Â pre-­âtreatment Â of Â gsh2 Â mutants Â with Â GSH Â led Â to Â a Â significant Â increase Â in Â GSHt Â levels. Â Â Â Â GSH Â requirement Â for Â viability Â of Â gsh2 Â mutants Â in Â C. Â neoformans Â in Â iron-­âstarvation Â conditions Â can Â theoretically Â be Â linked Â to Â antioxidant Â properties Â of Â GSH Â in Â this Â situation. Â It Â has Â previously Â been Â shown Â that Â GSH Â treatment Â in Â A. Â thaliana Â prevented Â the Â accumulation Â Â Â Â Â Â Â Â Â Â Â Â Â Â 135 Â of Â ROS Â induced Â by Â iron Â deficiency Â (L. Â Ramirez, Â et Â al., Â 2013). Â It Â is Â also Â known Â that Â iron Â is Â a Â constituent Â of Â several Â components Â of Â the Â electron Â transport Â chain Â in Â mitochondria Â and Â thus Â iron Â deficiency Â disrupts Â normal Â electron Â transfer Â resulting Â in Â the Â overproduction Â of Â ROS Â (M. Â Graziano Â and Â L. Â Lamattina, Â 2005, Â W. Â H. Â Tong Â and Â T. Â A. Â Rouault, Â 2006) Â (L. Â Ramirez, Â et Â al., Â 2013). Â Under Â these Â conditions, Â the Â high Â levels Â of Â ROS Â generated Â exceed Â the Â control Â capacity Â of Â the Â antioxidant Â system, Â causing Â cell Â oxidative Â damage. Â Â It Â is Â known Â that Â the Â decrease Â in Â GSHt Â content Â during Â iron Â deficiency Â could Â negatively Â affect Â the Â cell Â redox Â homeostasis, Â and Â addition Â of Â the Â exogenous Â of Â GSH Â controls Â the Â redox Â imbalance Â generated Â from Â an Â excess Â of Â ROS Â production Â under Â iron-­âdeficient Â conditions Â ((L. Â Ramirez, Â et Â al., Â 2013). Â Alternatively, Â GRXs Â can Â modulate Â protein Â activity Â by Â glutathionylation, Â a Â reversible Â post-­âtranslational Â modification Â involving Â disulfide Â formation Â between Â a Â protein Â thiol Â and Â GSH. Â This Â modification Â occurs Â more Â frequently Â in Â response Â to Â increased Â ROS, Â and Â protects Â the Â proteins Â from Â irreversible Â oxidation Â (I. Â Dalle-­âDonne Â et Â al., Â 2008). Â So Â it Â is Â possible Â that Â very Â low Â levels Â of Â GSH Â in Â gsh2 Â mutants Â renders Â GSH-­âdependent Â proteins Â such Â as Â glutaredoxin Â (Grx4), Â glutathione-­âS-­âtransferases Â (GSTs), Â and Â glutathione Â peroxides Â (GPXs) Â in Â C. Â neoformans, Â unable Â to Â make Â use Â of Â GSH Â as Â a Â cofactor. Â Consequently, Â this Â would Â result Â in Â an Â imbalance Â of Â the Â intracellular Â redox Â homeostasis Â and Â impairment Â of Â cellular Â functions Â dependent Â on Â these Â enzymes. Â Nevertheless, Â further Â studies Â are Â required Â to Â determine Â whether Â specific Â molecular Â mechanisms Â triggered Â by Â GSH Â could Â be Â occurring Â to Â preserve Â cellular Â homeostasis Â during Â iron Â deficiency Â in Â C. Â neoformans. Â Â Â The Â GSH Â requirement Â for Â viability Â of Â gsh2 Â mutants Â after Â iron Â starvation Â could Â also Â be Â theoretically Â linked Â to Â iron-­âmetabolism Â and Â iron-­âsensing Â functions Â of Â GSH Â (H. Â R. Â Lopez-­âMirabal Â et Â al., Â 2007) Â (C. Â Kumar, Â et Â al., Â 2011) Â (H. Â R. Â Lopez-­âMirabal, Â et Â al., Â 2007). Â The Â role Â of Â Â Â Â Â Â Â Â Â Â Â Â Â Â 136 Â GSH Â for Â sensing Â iron Â is Â conserved Â between Â S. Â cerevisiae Â and Â S. Â pombe. Â It Â has Â been Â shown Â that Â regulating Â gene Â expression Â as Â a Â function Â of Â iron Â availability Â and Â sensing Â the Â iron Â inhibitory Â signals Â in Â C. Â neoformans Â share Â common Â components Â and Â iron-­âresponsive Â transduction Â pathways Â with Â S. Â cerevisiae Â and Â S. Â pombe Â (D. Â J. Â Kosman, Â 2003). Â Therefore Â it Â is Â possible Â that Â iron Â starved Â gsh2 Â mutants Â are Â auxotrophic Â for Â GSH Â because Â GSH Â is Â required Â for Â iron Â sensing Â (H. Â Li Â and Â C. Â E. Â Outten, Â 2012), Â which Â further Â explains Â why Â addition Â of Â iron Â to Â YNB, Â did Â not Â restore Â growth Â of Â gsh2 Â mutants. Â It Â is Â also Â possible Â that Â GSH Â is Â required Â for Â iron-­âdependent Â regulation Â of Â Cir1, Â or Â other Â transcription Â factors Â in Â iron Â network Â of Â C. Â neoformans, Â including Â HapX. Â These Â predictions Â need Â to Â be Â further Â investigated. Â Another Â possible Â aspect Â is Â that Â GSH Â depletion Â is Â shown Â to Â cause Â induction Â of Â an Â iron Â starvation-­âlike Â response Â that Â impairs Â cytoplasmic Â FeâS Â protein Â maturation Â in Â yeast Â species Â due Â to Â the Â role Â of Â GSH Â and Â Grx Â partners Â in Â stabilizing Â [2Fe-­â2S] Â groups Â (H. Â Li Â and Â C. Â E. Â Outten, Â 2012, Â J. Â C. Â Rutherford, Â et Â al., Â 2005) Â (K. Â Sipos, Â et Â al., Â 2002) Â , Â (S. Â Bandyopadhyay, Â et Â al., Â 2008a). Â Â Â Â GSH Â also Â plays Â a Â critical Â role Â in Â stabilizing Â the Â labile Â iron Â pool Â that Â supplies Â iron Â for Â the Â synthesis Â of Â heme Â or Â [Fe-­âS] Â clusters Â (R. Â C. Â Hider Â and Â X. Â L. Â Kong, Â 2011). Â It Â is Â possible Â that Â lack Â of Â GSH Â in Â gsh2 Â mutants Â may Â lead Â to Â the Â presence Â of Â unbound Â iron Â in Â gsh2 Â mutants, Â which Â is Â not Â bioavailable Â for Â growth. Â Therefore Â addition Â of Â GSH Â to Â gsh2 Â mutants Â results Â in Â binding Â and Â stabilizing Â iron, Â which Â becomes Â bioavailable Â to Â support Â growth Â of Â gsh2 Â mutants. Â Â Â Overall, Â these Â findings Â suggest Â that Â an Â inadequate Â supply Â of Â GSH Â in Â gsh2 Â mutants Â in Â the Â absence Â of Â endogenous Â biosynthesis Â likely Â affects Â the Â ability Â of Â the Â C. Â neoformans Â to Â survive Â the Â harsh Â iron-­â Â and Â nutrient-­ârestricted Â conditions Â of Â the Â host. Â I Â speculate Â that Â this Â is Â possibly Â due Â to Â the Â role Â of Â GSH Â in Â iron Â homeostasis Â in Â C. Â neoformans. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 137 Â Â Â Â Taken Â together, Â my Â results Â suggest Â that Â GSH Â plays Â a Â central Â role Â under Â iron Â deficiency-­âtriggered Â stress Â conditions, Â protects Â C. Â neoformans Â from Â iron Â deficiency, Â preserves Â cell Â redox Â homeostasis Â and Â is Â required Â for Â internal Â iron Â availability. Â This Â suggests Â that Â specific Â depletion Â of Â the Â glutathione Â in Â C. Â neoformans Â would Â impinge Â on Â its Â survival Â in Â the Â host, Â also Â raising Â the Â possibility Â of Â targeting Â GSH Â synthesis Â pathway Â to Â treat Â cryptococcal Â infections. Â Interestingly, Â an Â antifungal Â compound Â from Â garlic, Â diallyl Â sulphide, Â and Â the Â protein Â kinase Â inhibitor Â staurosporine, Â have Â both Â been Â shown Â to Â cause Â their Â antifungal Â activity Â by Â depletion Â of Â glutathione Â in Â C. Â albicans Â (S. Â Yousuf Â et Â al., Â 2011) Â (K. Â M. Â Lemar Â et Â al., Â 2007). Â 3.4.3 Â Â Phenotypic Â characterization Â of Â gsh2 Â mutants Â revealed Â that Â Gsh2 Â is Â required Â for Â a Â plethora Â of Â functions Â in Â C. Â neoformans Â Â Â As Â a Â key Â discovery Â of Â the Â phenotypic Â analyses Â of Â gsh2 Â mutants, Â I Â found Â that Â Gsh2 Â has Â pleiotropic Â functions Â in Â C. Â neoformans Â including Â the Â response Â to Â diverse Â antifungal Â drugs Â including Â azoles Â and Â polyene Â drugs, Â cell Â wall Â integrity, Â salt Â stress Â responses, Â and Â virulence Â factor Â production. Â Of Â note, Â the Â pleotropic Â role Â of Â GSH Â in Â eukaryotes Â is Â known, Â and Â given Â the Â very Â low Â levels Â of Â GSHt Â in Â in Â gsh2 Â mutants, Â elaboration Â of Â several Â phenotypes Â is Â expected. Â Â Â Â In Â this Â study, Â I Â found Â that Â gsh2 Â mutants Â exhibited Â sensitivity Â to Â external Â stressors Â such Â as Â high Â temperature, Â high Â salts, Â and Â antifungal Â agents. Â The Â ability Â to Â sense Â and Â respond Â to Â a Â hostile Â host Â environment Â is Â a Â crucial Â element Â for Â virulence Â of Â C. Â neoformans. Â Various Â extracellular Â or Â intracellular Â stresses Â such Â as Â high Â temperature, Â high Â salt, Â and Â antifungal Â drugs Â lead Â to Â accumulation Â of Â misfolded Â or Â damaged Â proteins Â inside Â the Â cell, Â and Â cause Â cell Â damage Â that Â is Â prevented Â by Â their Â removal Â through Â several Â response Â Â Â Â Â Â Â Â Â Â Â Â Â Â 138 Â mechanisms Â in Â C. Â neoformans. Â These Â responses Â are Â evoked Â and Â coordinated Â by Â diverse Â signaling Â cascades, Â including Â the Â stress-­âactivated Â HOG Â pathway, Â PKC1/MAPK, Â and Â Ca2+/ Â calcineurin Â pathways. Â These Â signaling Â pathways Â are Â further Â regulated Â by Â the Â ubiquitin-­âproteasome Â system(C. Â S. Â Hemenway Â and Â J. Â Heitman, Â 1999) Â (Y. Â S. Â Bahn Â et Â al., Â 2005, Â K. Â W. Â Jung Â et Â al., Â 2012, Â K. Â T. Â Lee Â et Â al., Â 2014) Â (Y. Â J. Â Ko, Â et Â al., Â 2009) Â (K. Â W. Â Jung Â and Â Y. Â S. Â Bahn, Â 2009) Â (T. Â B. Â Liu Â et Â al., Â 2011) Â (Y. Â S. Â Bahn Â and Â K. Â W. Â Jung, Â 2013). Â My Â findings Â suggest Â the Â presence Â of Â a Â crosstalk Â between Â GSH Â homeostasis Â and Â these Â response Â pathways Â in Â C. Â neoformans. Â Â Â Â In Â C. Â neoformans, Â gsh2 Â mutants Â were Â sensitized Â to Â salt Â stress Â (1.5M Â NaCl Â and Â KCl). Â Similar Â effects Â have Â been Â reported Â in Â plants Â (A. Â thaliana, Â Brassica Â napus, Â and Â Solanum Â lycopersicum) Â where Â GSH Â protects Â against Â exogenous Â osmotic Â stress Â (J. Â H. Â Chen Â et Â al., Â 2012, Â V. Â Mittova Â et Â al., Â 2003) Â (J. Â M. Â Ruiz Â and Â E. Â Blumwald, Â 2002). Â The Â salinity Â stress Â in Â plants Â leads Â to Â excessive Â generation Â of Â ROS, Â which Â results Â from Â impaired Â electron Â transport Â processes Â in Â chloroplast Â and Â mitochondria Â as Â well Â as Â photorespiration. Â Resistance Â to Â salt Â stress Â is Â usually Â correlated Â with Â GSH Â acting Â as Â an Â antioxidant. Â My Â results Â suggest Â that Â GSH Â might Â have Â a Â role Â in Â mitigating Â salt-­âinduced Â oxidative Â stress Â in Â C. Â neoformans Â Â Â I Â also Â found Â that Â gsh2 Â mutants Â displayed Â increased Â susceptibility Â to Â major Â antifungal Â drugs Â including Â amphotericin Â B, Â fluconazole Â and Â miconazole Â compared Â to Â the Â WT Â and Â complemented Â strains. Â These Â drugs Â are Â commonly Â used Â to Â treat Â cryptococcal Â meningitis. Â Previously, Â the Â role Â of Â GSH Â in Â resistance Â to Â antifungal Â drugs Â has Â been Â demonstrated Â in Â C. Â albicans, Â and Â it Â was Â shown Â that Â increased Â levels Â of Â GSH Â lead Â to Â a Â more Â efficient Â elimination Â of Â fluconazole Â from Â the Â cells Â (B. Â Maras, Â et Â al., Â 2014). Â This Â could Â be Â due Â to Â the Â role Â of Â GSH Â in Â detoxification Â of Â drugs Â and Â xenobiotics Â or Â in Â ergosterol Â biosynthesis Â Â Â Â Â Â Â Â Â Â Â Â Â Â 139 Â pathway. Â In Â this Â context, Â it Â has Â been Â shown Â that Â depletion Â of Â GSH Â leads Â to Â an Â iron Â starvationâlike Â response Â and Â repression Â of Â ergosterol Â biosynthetic Â enzymes Â in Â yeast Â (C. Â Kumar, Â et Â al., Â 2011). Â Furthermore, Â gsh2 Â mutants Â display Â a Â subtle Â susceptibility Â to Â 75 Â μM Â CCCP, Â 75 Â μg/ml Â rotenone, Â 10mM Â KCN, Â 3 Â μg/ml Â antimycin Â or Â 5 Â mM Â SHAM, Â compared Â to Â the Â WT Â and Â complemented Â strains. Â This Â finding Â suggests Â that Â the Â role Â of Â Gsh2 Â in Â GSH Â production Â may Â have Â an Â impact Â on Â mitochondrial Â functions Â in Â C. Â neoformans. Â It Â has Â been Â shown Â that Â depletion Â of Â GSH Â leads Â to Â repression Â of Â genes Â encoding Â FeâS Â and Â heme Â proteins Â of Â mitochondrial Â respiration, Â and Â the Â tricarboxylic Â acid Â cycle Â in Â yeast Â (C. Â Kumar, Â et Â al., Â 2011). Â Of Â note, Â despite Â its Â exclusive Â synthesis Â in Â the Â cytosol, Â GSH Â is Â distributed Â in Â intracellular Â organelles Â including Â the Â endoplasmic Â reticulum Â (ER), Â nucleus, Â and Â mitochondria. Â In Â mitochondria, Â however, Â GSH Â represents Â a Â minor Â fraction Â of Â the Â total Â GSH Â pool Â (10â15%) Â (M. Â Mari Â et Â al., Â 2009). Â Given Â the Â volume Â of Â the Â mitochondrial Â matrix, Â the Â concentration Â of Â mitochondrial Â GSH Â (mGSH) Â is Â similar Â to Â that Â of Â cytosol Â (10â14âmM) Â (M. Â Mari, Â et Â al., Â 2009) Â (C. Â Garcia-­âRuiz Â et Â al., Â 1992) Â (O. Â W. Â Griffith Â and Â A. Â Meister, Â 1985). Â Therefore, Â it Â is Â possible Â to Â speculate Â that Â lack Â of Â intracellular Â GSH Â in Â gsh2 Â mutants Â may Â affect Â the Â mGSH Â levels Â and Â impair Â mGSH Â functions. Â mGSh Â protects Â mitochondrial Â functions Â in Â oxidative Â phosphorylation Â and Â ATP Â production Â and Â maintains Â mitochondria Â genome Â stability(M. Â Mari, Â et Â al., Â 2009). Â The Â importance Â of Â mitochondrial Â functions Â has Â been Â demonstrated Â in Â other Â cryptococcal Â studies, Â in Â which Â deletion Â of Â a Â mitochondrial Â gene Â encoding Â the Â alternative Â oxidase Â of Â mitochondria Â resulted Â in Â defects Â in Â tolerance Â to Â the Â stress Â environment Â in Â macrophages Â and Â attenuation Â in Â cryptococcal Â virulence Â (S. Â Akhter Â et Â al., Â 2003). Â Importantly, Â gsh2 Â mutants Â also Â displayed Â enhanced Â sensitivity Â to Â cell Â wall Â damaging Â agent Â Congo Â red. Â This Â suggests Â that Â GSH Â might Â serve Â as Â a Â signalling Â component Â Â Â Â Â Â Â Â Â Â Â Â Â Â 140 Â in Â regulation Â of Â actin Â cytoskeletal Â architecture Â and Â cell Â wall Â integrity Â in Â C. Â neoformans. Â In Â this Â context, Â cell Â wall Â biogenesis, Â composition Â and Â the Â maintenance Â of Â cell Â integrity Â in Â C. Â neoformans Â is Â regulated Â by Â the Â protein Â kinase Â C Â (PKC1) Â signal Â transduction Â pathway Â (K. Â J. Â Gerik Â et Â al., Â 2008). Â It Â has Â been Â shown Â that Â GSH Â can Â antagonize Â PKC Â isozymes Â in Â mammalian Â cells. Â There Â is Â evidence Â that Â glutathione Â inhibits Â PKC Â isozymes Â by Â a Â yet Â unknown Â non-­âredox Â mechanism. Â Also, Â depletion Â of Â the Â intracellular Â GSH Â pool Â is Â proposed Â to Â lead Â to Â loss Â of Â a Â negative Â regulatory Â mechanism Â over Â PKC Â (N. Â E. Â Ward Â et Â al., Â 1998). Â Whether Â or Â how Â PKC1 Â is Â regulated Â by Â glutathione Â in Â C. Â neoformans Â is Â not Â known Â and Â remains Â to Â be Â investigated. Â Â Â Â I Â also Â found Â that Â gsh2 Â mutants Â did Â not Â express Â classical Â virulence Â traits Â in Â C. Â neoformans Â including Â capsule Â production, Â melanin Â formation Â and Â growth Â at Â 37°C. Â Â Capsule Â production Â is Â a Â complex Â and Â dynamic Â process Â that Â includes Â intracellular Â polysaccharide Â synthesis, Â secretion Â of Â capsular Â polysaccharide Â via Â vesicles, Â and Â extracellular Â assembly Â (A. Â Yoneda Â and Â T. Â L. Â Doering, Â 2006). Â Cell Â wall Â components Â such Â as Â Î±-­â1,3-­âglucan Â and Â chitin-­âderived Â molecules Â are Â also Â required Â for Â GXM Â (major Â capsule Â component) Â anchoring Â to Â the Â cell Â surface Â and Â cell Â wall Â integrity Â contributes Â to Â formation Â of Â capsule Â (T. Â L. Â Doering, Â 2009). Â Elaboration Â of Â virulence-­ârelated Â phenotypes Â in Â gsh2 Â mutants Â suggest Â that Â Gsh2 Â is Â potentially Â required Â for Â the Â establishment Â of Â the Â proper Â capsule Â or Â cell Â wall Â structure Â required Â for Â anchoring Â of Â capsule Â polysaccharides Â to Â Î±-­â1, Â 3-­âglucan Â at Â the Â cell Â surface. Â It Â is Â also Â possible Â that Â Gsh2 Â via Â GSH Â is Â required Â for Â proper Â trafficking Â of Â materials Â for Â capsule Â production. Â Similarly, Â loss Â of Â Gsh2 Â may Â interrupt Â the Â processing Â and/or Â transport Â of Â laccase, Â resulting Â in Â loss Â of Â melanization. Â Â One Â of Â the Â key Â factors Â in Â the Â virulence Â of Â C. Â neoformans Â is Â the Â ability Â to Â grow Â at Â human Â body Â temperature Â (P. Â R. Â Kraus, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 141 Â et Â al., Â 2004). Â In Â the Â present Â investigation, Â I Â speculate Â that Â defective Â growth Â of Â gsh2 Â mutants Â at Â 37°C Â is Â due Â to Â lack Â of Â GSH Â in Â these Â mutants. Â On Â this Â note, Â it Â has Â been Â shown Â that Â intracellular Â GSH Â levels Â are Â elevated Â during Â heat Â shock Â in Â mammalian Â cells Â and Â depletion Â of Â GSH Â leads Â to Â thermal Â sensitivity Â in Â mammalian Â cells Â and Â maize Â roots Â (J. Â B. Â Mitchell Â et Â al., Â 1983) Â (J. Â Nieto-­âSotelo Â and Â T. Â H. Â Ho, Â 1986)). Â Emphasizing Â on Â the Â role Â of Â GSH Â in Â virulence, Â it Â is Â interesting Â to Â add Â that Â in Â a Â recent Â study Â reported Â by Â Reniere, Â et Â al., Â it Â was Â shown Â that Â glutathione Â activates Â virulence Â gene Â expression Â of Â intracellular Â bacterial Â pathogen Â Listeria Â monocytogenes Â (M. Â L. Â Reniere, Â et Â al., Â 2015). Â The Â intracellular Â lifecycle Â of Â L. Â monocytogenes Â is Â entirely Â dependent Â on Â the Â thermoregulator Â PrfA. Â PrfA Â is Â a Â member Â of Â the Â cAMP Â -­âFnr Â family Â of Â transcription Â factors Â and Â is Â referred Â to Â as Â the Â master Â regulator Â of Â virulence Â as Â it Â directly Â regulates Â the Â transcription Â of Â nine Â virulence Â factors Â in Â L. Â monocytogenes Â (N. Â E. Â Freitag Â et Â al., Â 2009). Â In Â the Â report Â by Â Reniere Â et Â al., Â it Â was Â shown Â that Â PrfA Â senses Â the Â glutathione Â concentration Â to Â regulate Â its Â biphasic Â lifestyle Â and Â the Â switch Â from Â saprophyte Â to Â pathogen, Â and Â lack Â of Â GSH Â leads Â to Â complete Â loss Â of Â virulence Â in Â L. Â monocytogenes Â Â (M. Â L. Â Reniere, Â et Â al., Â 2015). Â Overall, Â it Â is Â possible Â that Â GSH Â has Â conserved Â and Â similar Â activating Â roles Â in Â virulence Â gene Â expression Â in Â other Â intracellular Â pathogens Â such Â as Â C. Â neoformans. Â Given Â the Â many Â faces Â and Â roles Â of Â GSH, Â it Â is Â predicted Â that Â lack Â of Â sufficient Â GSH Â in Â gsh2 Â mutants Â would Â impair Â virulence Â in Â C. Â neoformans Â as Â a Â result Â of Â combined Â defects Â of Â gsh2 Â mutants Â outlined Â in Â this Â thesis, Â including Â loss Â of Â capsule Â production, Â impaired Â melanization Â and Â loss Â of Â ability Â to Â grow Â at Â 37°C. Â Â Â Experimental Â infections Â in Â mice Â could Â be Â used Â to Â investigate Â this Â prediction. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 142 Â Chapter Â 4: Â Discussion Â Â 4.1 Â Â Iron Â Homeostasis Â in Â C. Â neoformans Â Â Â The Â research Â presented Â in Â this Â thesis Â provides Â further Â understanding Â of Â mechanisms Â of Â iron Â homeostasis Â in Â C. Â neoformans. Â Iron Â homeostasis Â is Â directly Â related Â to Â virulence Â of Â AIDS-­âassociated Â fungal Â pathogen Â C. Â neoformans, Â as Â iron Â regulates Â the Â elaboration Â of Â major Â virulence Â factors Â in Â C. Â neoformans Â including Â the Â pigment Â melanin Â and Â the Â polysaccharide Â capsule Â (E. Â S. Â Jacobson Â and Â J. Â D. Â Hong, Â 1997) Â (K. Â L. Â Tangen, Â et Â al., Â 2007) Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2008). Â Therefore, Â identifying Â the Â genes Â and Â gene Â products Â that Â contribute Â to Â the Â iron Â regulatory Â network Â is Â essential Â to Â understanding Â the Â pathogenesis Â of Â C. Â neoformans. Â Â Several Â transcription Â factors Â in Â C. Â neoformans Â regulate Â iron Â homeostasis Â and Â other Â aspects Â of Â adaptation Â to Â the Â host Â environment Â (J. Â Kronstad Â et Â al., Â 2012). Â Our Â group Â has Â previously Â shown Â that Â Cir1 Â is Â the Â master Â iron Â regulator Â that Â influences Â the Â expression Â of Â iron Â and Â virulence-­ârelated Â genes Â including Â genes Â associated Â with Â iron Â uptake Â from Â heme Â (CIG1), Â a Â siderophore Â transporter Â (SIT1), Â and Â the Â production Â of Â melanin Â (LAC1). Â Cir1 Â functions Â also Â as Â an Â activator Â for Â growth Â at Â 37°C Â (host Â temperature) Â which Â is Â an Â important Â virulence Â trait Â in Â C. Â neoformans Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â The Â mechanisms Â by Â which Â C. Â neoformans Â senses Â the Â mammalian Â host Â environment Â and Â regulates Â virulence Â factor Â expression Â are Â promising Â areas Â for Â therapeutic Â intervention. Â Â These Â areas Â include Â the Â identification Â of Â targets Â for Â new Â antifungal Â drugs Â and Â candidates Â for Â vaccine Â development Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 143 Â 4.2 Â Â Monothiol Â Grx4 Â is Â required Â for Â iron Â homeostasis Â and Â virulence Â in Â C. Â neoformans Â Â Â A Â preliminary Â proteomic Â analysis Â of Â potential Â binding Â partners Â for Â Cir1 Â was Â performed Â in Â 2009 Â in Â our Â lab, Â and Â identification Â of Â a Â monothiol Â Grx4 Â (gene Â ID: Â CNAG_02950) Â as Â a Â potential Â Cir1-­âassociated Â protein Â set Â the Â foundation Â of Â the Â work Â outlined Â in Â my Â thesis. Â Monothiol Â Grxs Â are Â reported Â in Â other Â eukaryotic Â organisms Â and Â fungal Â species Â to Â regulate Â iron Â homeostasis, Â iron Â trafficking Â and Â iron Â signaling Â events Â (N. Â Rouhier, Â et Â al., Â 2010) Â (H. Â Li Â and Â C. Â E. Â Outten, Â 2012). Â In Â the Â first Â chapter Â of Â my Â thesis, Â several Â lines Â of Â evidence Â indicate Â that Â Grx4 Â in Â C. Â neoformans Â has Â a Â major Â role Â in Â this Â process Â in Â C. Â neoformans. Â Importantly, Â I Â also Â demonstrated Â that Â in Â addition Â to Â iron Â homeostasis, Â Grx4 Â participates Â in Â virulence Â in Â C. Â neoformans. Â Â Â Â Initially, Â I Â demonstrated Â that Â Grx4 Â binds Â N-­âterminal Â region Â of Â Cir1 Â containing Â the Â DNA Â binding Â domain Â by Â ALPHAScreen Â protein-­âprotein Â interaction Â assay Â and Â by Â Y2H Â analysis. Â Â The Â physical Â interaction Â between Â an Â iron Â transcription Â factor Â and Â a Â monothiol Â Grx Â has Â previously Â been Â shown Â in Â other Â fungal Â species. Â For Â instance, Â interaction Â between Â Grx4 Â and Â Fep1 Â in Â S. Â pombe Â as Â analyzed Â by Â yeast Â two Â hybrid Â assays Â has Â been Â shown, Â which Â Â is Â consistent Â with Â what Â I Â observed Â in Â my Â study Â (M. Â Jbel, Â et Â al., Â 2009). Â Â They Â also Â showed Â that Â the Â conserved Â C-­âterminal Â Grx Â domain Â containing Â the Â CGFS Â motif Â in Â Grx4 Â governs Â iron-­âdependent Â modulation Â of Â Fep1 Â activity Â through Â close Â association Â with Â the Â DNA-­âbinding Â domain. Â Additionally, Â in Â a Â more Â recent Â study Â by Â Encinar Â del Â Dedo Â et Â al., Â it Â was Â ascertained Â that Â Grx4 Â incorporates Â a Â glutathione-­âcontaining Â Fe-­âS Â cluster Â and Â participates Â in Â iron Â starvation Â signaling Â by Â binding Â and Â controlling Â the Â functions Â of Â Fep1 Â repressor Â through Â metal Â transfer Â (J. Â Encinar Â del Â Dedo, Â et Â al., Â 2015). Â Â Â Â Â Â Â Â Â Â Â Â Â Â 144 Â Â Â Â It Â is Â important Â to Â investigate Â the Â cellular Â compartment Â for Â the Â interaction Â between Â Grx4 Â and Â Cir1 Â that Â was Â identified Â in Â this Â study. Â According Â to Â observations Â made Â by Â Dr. Â Guanggan Â Hu Â in Â our Â laboratory, Â localization Â of Â Cir1 Â is Â unaffected Â by Â iron Â status Â when Â expressed Â in Â C. Â neoformans Â under Â both Â iron-­âlimiting Â and Â iron-­âreplete Â conditions Â (Dr. Â Guanggan Â Hu Â personal Â communications). Â Given Â the Â nuclear Â localization Â of Â Cir1 Â and Â the Â high Â amino Â acid Â sequence Â identity Â between Â Cir1 Â and Â Fep1, Â I Â hypothesize Â that Â Grx4 Â interaction Â with Â Cir1 Â most Â likely Â takes Â place Â in Â the Â nucleus. Â In Â this Â theoretical Â scenario, Â Grx4 Â has Â an Â inhibitory Â influence Â on Â Cir1 Â function Â by Â binding Â Cir1 Â in Â presence Â of Â iron Â and Â dissociating Â from Â Cir1 Â under Â iron-­âstarvation Â conditions. Â Â Â Â Furthermore, Â the Â Fe-­âS Â binding Â capacity Â of Â monothiol Â Grxs Â via Â CGFS Â motif Â plays Â a Â critical Â role Â for Â their Â functions Â in Â iron Â homeostasis. Â A Â report Â by Â Muhlenhoff Â et Â al. Â revealed Â the Â function Â of Â Grx4 Â homologs Â Grx3/Grx4 Â in Â S. Â cerevisiae Â in Â iron Â trafficking Â and Â iron Â sensing Â (U. Â Muhlenhoff, Â et Â al., Â 2010). Â It Â was Â also Â shown Â that Â Grx3/Grx4 Â form Â an Â iron-­âsensor Â complex Â via Â their Â bound Â GSH Â and Â Fe-­âS Â clusters Â (Muhlenhoff, Â 2010) Â (B. Â Hoffmann, Â et Â al., Â 2011). Â It Â has Â also Â been Â shown Â that Â Grx4 Â is Â required Â for Â iron Â limitation-­âdependent Â inhibition Â of Â Fep1 Â via Â Fe-­âS Â binding Â capacity Â of Â Grx4 Â in Â S. Â pombe Â (M. Â Jbel, Â et Â al., Â 2009) Â (J. Â Encinar Â del Â Dedo, Â et Â al., Â 2015). Â Therefore, Â it Â is Â also Â possible Â that Â Grx4 Â in Â C. Â neoformans Â serves Â as Â an Â iron Â sensor Â relaying Â the Â levels Â of Â iron Â to Â Cir1 Â in Â the Â nucleus Â by Â shuttling Â between Â the Â cytosol Â and Â nucleus. Â Whether Â and Â how Â FeâS Â or Â Fe Â is Â involved Â in Â the Â Grx4-­âCir1 Â interaction Â is Â a Â challenging Â question Â to Â be Â tackled Â in Â the Â future. Â Â Â Next, Â I Â sought Â to Â determine Â the Â role Â of Â conserved Â Grx Â domain Â of Â Grx4 Â in Â C. Â neoformans. Â I Â used Â a Â grx4 Â disruption Â strain Â in Â which Â the Â C-­âterminal Â conserved Â Grx Â domain Â was Â deleted Â and Â I Â generated Â the Â complemented Â grx4Î::GRX4 Â strain Â with Â the Â deleted Â Grx Â Â Â Â Â Â Â Â Â Â Â Â Â Â 145 Â domain Â integrated Â back Â in Â locus. Â Transcriptome Â and Â phenotypic Â analysis Â of Â the Â grx4 Â mutant Â consolidated Â the Â role Â of Â Grx4 Â in Â iron Â homeostasis Â in Â C. Â neoformans. Â Remarkably, Â I Â found Â that Â loss Â of Â conserved Â C-­âterminal Â Grx4 Â domain Â in Â GRX4 Â leads Â to Â elaboration Â of Â similar Â iron-­ârelated Â phenotypes Â seen Â in Â cir1 Â mutants, Â as Â described Â by Â Jung Â et Â al. Â 2006 Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Â Â Regulation Â of Â iron Â homeostasis Â largely Â depends Â on Â the Â regulation Â of Â iron Â uptake, Â iron Â storage Â and Â consumption Â in Â response Â to Â iron Â (J. Â W. Â Kronstad, Â 2013) Â (J. Â W. Â Kronstad, Â et Â al., Â 2013) Â (W. Â H. Â Jung Â and Â J. Â W. Â Kronstad, Â 2008). Â In Â fungi, Â iron Â starvation Â increases Â expression Â of Â genes Â required Â for Â iron Â acquisition Â and Â siderophore-­âmediated Â iron Â uptake, Â Meanwhile, Â iron-­âconsuming Â pathways Â are Â downregulated Â to Â optimize Â the Â use Â of Â the Â limited Â iron Â resources Â (B. Â Mei Â et Â al., Â 1993) Â (H. Â Haas, Â 2003) Â (D. Â L. Â Greenshields Â et Â al., Â 2007); Â (M. Â Schrettl Â et Â al., Â 2007). Â One Â key Â finding Â from Â my Â analysis Â was Â based Â on Â the Â gene Â expression Â data Â I Â obtained Â for Â the Â grx4 Â mutant Â compared Â to Â WT Â in Â presence Â of Â iron Â (Figure Â 2.11). Â I Â found Â that Â Grx4 Â had Â an Â inhibitory Â effect Â on Â the Â expression Â of Â iron Â acquisition Â gene Â because Â these Â genes Â were Â highly Â upregulated Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â in Â the Â presence Â of Â iron. Â Â The Â regulated Â functions Â included Â iron Â reductases, Â siderophores Â transporters, Â and Â components Â of Â the Â high Â affinity Â uptake Â system. Â Given Â the Â aforementioned Â conserved Â role Â of Â monothiol Â Grxs Â in Â iron Â homeostasis, Â it Â is Â possible Â that Â similar Â to Â the Â scenario Â in Â S. Â pombe, Â Grx4 Â in Â C. Â neoformans Â serves Â as Â an Â inhibitory Â partner Â for Â Cir1 Â in Â response Â to Â iron Â by Â physically Â binding Â Cir1 Â and/or Â Fe-­âS Â clusters. Â This Â can Â also Â result Â from Â an Â influence Â of Â Grx4 Â on Â downstream Â regulators Â that Â transcriptionally Â or Â post-­âtranscriptionally Â control Â the Â genes Â for Â iron Â acquisition Â in Â C. Â neoformans. Â Therefore, Â presence Â of Â additional Â levels Â of Â iron Â regulation Â that Â have Â not Â yet Â been Â identified Â in Â C. Â neoformans Â is Â possible. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 146 Â Â Â Importantly, Â evidence Â from Â my Â transcriptional Â analysis Â by Â RNA-­âseq Â indicated Â that Â Grx4 Â is Â required Â for Â the Â repression Â of Â many Â genes Â that Â encode Â iron-­âdependent Â proteins, Â especially Â those Â involved Â in Â the Â TCA Â cycle, Â respiration, Â and Â Fe-­âS Â cluster Â assembly Â in Â iron-­âreplete Â conditions. Â RNA-­âseq Â profiling Â revealed Â and Â confirmed Â that Â Grx4 Â is Â essential Â for Â iron Â starvationâtriggered Â down Â regulation Â of Â genes Â in Â iron-­âdependent Â pathways Â and Â iron-­âtriggered Â down Â regulation Â of Â genes Â for Â iron Â acquisition Â functions. Â Thus, Â a Â key Â role Â of Â Grx4 Â in Â iron Â homeostasis Â of Â C. Â neoformans Â is Â to Â shut Â down Â iron-­âconsuming Â processes, Â such Â as Â respiration, Â amino Â acid Â metabolism, Â and Â heme Â biosynthesis, Â when Â iron Â becomes Â limiting. Â Collectively, Â these Â results Â show Â an Â impact Â of Â Grx4 Â in Â repression Â of Â iron-­âdependent Â pathways Â and Â also Â suggest Â the Â essential Â role Â of Â Grx4 Â in Â fungal Â adaptation Â to Â iron Â starvation Â conditions. Â Â Â Â The Â role Â of Â Grx4 Â in Â iron Â homeostasis Â is Â further Â emphasized Â by Â my Â phenotypic Â analyses. Â In Â this Â context, Â phleomycin Â is Â an Â antibiotic Â that Â damages Â nucleic Â acids Â in Â the Â presence Â of Â iron Â (K. Â R. Â Fox Â et Â al., Â 1987) Â (J. Â Kross Â et Â al., Â 1982). Â The Â grx4 Â mutant Â exhibited Â increased Â sensitivity Â to Â the Â drug Â presumably Â as Â a Â consequence Â of Â high Â levels Â of Â iron Â uptake Â activity. Â I Â also Â demonstrated Â that Â the Â grx4 Â mutant Â has Â elevated Â cell Â surface Â reductase Â activity. Â This Â was Â observed Â by Â the Â enhanced Â reduction Â of Â TTC Â to Â 1,3,5-­âtriphenyl-­âformazan, Â which Â appears Â as Â a Â pinkâred Â color Â colony Â on Â media Â with Â 0.1% Â TTC. Â It Â has Â been Â shown Â that Â loss Â of Â Cir1 Â results Â in Â elevated Â cell Â surface Â metalloreductase Â activity, Â likely Â as Â a Â consequence Â of Â lack Â of Â transcriptional Â downregulation Â of Â iron Â acquisition Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â I Â also Â observed Â that, Â similar Â to Â the Â cir1 Â mutant, Â the Â grx4 Â mutant Â displayed Â sensitivity Â to Â elevated Â iron Â levels Â in Â plate Â assays. Â Â Â Â I Â also Â studied Â the Â growth Â phenotype Â of Â the Â grx4 Â mutant Â in Â the Â presence Â of Â iron Â in Â Â Â Â Â Â Â Â Â Â Â Â Â Â 147 Â liquid Â cultures Â by Â growth Â curves Â analysis. Â In Â this Â analysis Â I Â found Â that Â iron-­âstarved Â grx4 Â mutants Â did Â not Â respond Â to Â iron. Â This Â suggested Â that Â iron Â starvation Â might Â trigger Â a Â non-­âresponsive Â state Â in Â grx4 Â mutant. Â Remarkably, Â I Â found Â that Â supplementation Â with Â GSH Â seemed Â to Â restore Â the Â capacity Â of Â grx4 Â mutant Â to Â perceive Â iron Â in Â high Â iron Â medium. Â This Â suggests Â that Â Grx4 Â is Â necessary Â for Â efficient Â growth Â of Â C. Â neoformans Â in Â response Â to Â iron Â Â Â Â Importantly, Â I Â demonstrated Â that Â loss Â of Â the Â conserved Â C-­âterminal Â Grx4 Â domain Â in Â a Â grx4 Â mutant Â lead Â to Â elaboration Â of Â the Â similar Â virulence-­ârelated Â phenotypes Â to Â cir1 Â mutants Â shown Â by Â Jung Â et Â al. Â 2006 Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Â It Â was Â previously Â shown Â that Â cir1 Â deletion Â mutants Â have Â a Â defect Â in Â capsule Â formation, Â altered Â melanin Â production, Â reduced Â growth Â at Â 37°C, Â and Â are Â avirulent Â in Â a Â mouse Â model Â (W. Â H. Â Jung, Â et Â al., Â 2006). Â Given Â the Â role Â of Â iron Â in Â regulating Â the Â expression Â of Â virulence Â factors Â in Â C. Â neoformans, Â it Â is Â likely Â that Â the Â perturbed Â iron Â homeostasis Â in Â grx4 Â mutant Â is Â the Â underlining Â reason Â for Â impaired Â virulence Â expression Â in Â this Â mutant, Â especially Â because Â grx4 Â mutants Â were Â unable Â to Â colonize Â and Â kill Â mice. Â This Â the Â first Â study Â that Â clearly Â demonstrates Â the Â role Â of Â a Â monothiol Â Grx Â as Â a Â virulence Â determinant Â in Â a Â pathogenic Â fungus. Â Taken Â together Â these Â results Â showed Â the Â role Â of Â Grx4 Â in Â both Â iron Â homeostasis Â and Â virulence Â n Â C. Â neoformans, Â and Â suggest Â that Â Grx4 Â may Â play Â a Â key Â role Â in Â transcriptional Â reprogramming Â under Â severe Â iron Â limitation Â encountered Â in Â the Â host. Â Overall, Â deregulation Â of Â genes Â required Â for Â metabolic Â adaptation Â to Â iron Â deficiency Â may Â account Â for Â the Â impaired Â ability Â of Â grx4 Â mutant Â to Â proliferate Â in Â the Â host. Â My Â findings Â also Â suggest Â that Â Grx4 Â might Â be Â involved Â in Â sensing Â iron Â levels Â in Â the Â host Â and Â expression Â of Â iron Â starvationâinduced Â virulence Â factors Â during Â growth Â of Â C. Â neoformans Â in Â the Â host. Â Further Â characterization Â of Â influence Â of Â Grx4 Â on Â virulence Â targets Â will Â provide Â new Â insights Â into Â the Â conserved Â role Â of Â the Â Grx4-­â Â Â Â Â Â Â Â Â Â Â Â Â Â 148 Â mediated Â iron Â homeostasis Â in Â cryptococcal Â pathogenicity Â in Â humans. Â Â 4.3 Â GSH Â is Â involved Â in Â stress Â response Â and Â virulence Â in Â C. Â neoformans Â Â Pathogenic Â fungi Â share Â the Â requirement Â to Â survive Â within Â an Â infected Â host. Â C. Â neoformans Â survives Â exposure Â to Â stresses Â during Â growth Â in Â the Â human Â host, Â including Â oxidative Â and Â nitrosative Â stress, Â high Â temperature, Â hypoxia, Â and Â nutrient Â deprivation Â by Â employing Â several Â strategies Â during Â infection Â (S. Â M. Â Brown Â et Â al., Â 2007). Â By Â definition, Â stress Â is Â a Â disturbance Â in Â the Â pro-­âoxidant-­âantioxidant Â balance Â resulting Â from Â the Â increased Â production Â of Â ROS Â or Â exhaustion Â of Â antioxidant Â reservoirs Â (H. Â Sies, Â 1991) Â (B. Â Halliwell Â and Â J. Â M. Â Gutteridge, Â 1995) Â (B. Â Halliwell Â and Â J. Â M. Â Gutteridge, Â 1992). Â Antioxidant Â mechanisms Â and Â restoration Â of Â redox Â balance Â is Â essential Â to Â avoid Â cell Â damage, Â loss Â of Â functional Â biomolecules, Â necrosis, Â and Â apoptosis Â (B. Â Halliwell Â and Â J. Â M. Â Gutteridge, Â 1995) Â (B. Â Halliwell Â and Â J. Â M. Â Gutteridge, Â 1992) Â (P. Â Jansen-­âDurr Â and Â H. Â D. Â Osiewacz, Â 2002). Â Eukaryotic Â cells Â remove Â cell-­âdamaging Â ROS Â by Â induction Â of Â enzymes Â such Â as Â SOD, Â catalase Â and Â GSH/Grx/Trx Â (R. Â Mittler Â and Â E. Â Tel-­âOr, Â 1991) Â (L. Â Kawasaki Â et Â al., Â 1997). Â This Â is Â further Â controlled Â by Â signal Â transduction Â cascades Â that Â activate Â the Â expression Â of Â genes Â involved Â in Â stress Â response Â pathways Â and/or Â alteration Â in Â GSH. Â (H. Â Dumond Â et Â al., Â 2000) Â (L. Â Kawasaki Â and Â J. Â Aguirre, Â 2001) Â (E. Â O. Â Garrido Â and Â C. Â M. Â Grant, Â 2002) Â (B. Â Halliwell Â et Â al., Â 1992). Â Â Â Â Glutathione Â is Â an Â essential Â metabolite Â in Â eukaryotes Â and Â is Â present Â in Â high Â concentration Â up Â to Â 10 Â mM Â in Â yeasts Â and Â filamentous Â fungi Â (R. Â C. Â Fahey Â and Â A. Â R. Â Sundquist, Â 1991) Â (A. Â Meister Â and Â M. Â E. Â Anderson, Â 1983). Â The Â classical Â function Â of Â GSH Â in Â oxidative Â stress Â defense Â mechanisms Â includes Â response Â to Â starvation, Â heat, Â cold, Â osmotic Â shock, Â and Â exposure Â to Â ROS Â (D. Â J. Â Jamieson, Â 1998) Â (V. Â I. Â Lushchak, Â 2012). Â In Â this Â context, Â Â Â Â Â Â Â Â Â Â Â Â Â Â 149 Â GSH Â synthetases Â including Â Gsh1 Â and Â Gsh2, Â Glr, Â Gsts, Â Gpx Â and Â Grxs Â are Â directly Â involved Â in Â the Â elimination Â of Â oxidative Â compounds Â in Â yeast Â and Â other Â fungi Â (D. Â J. Â Jamieson, Â 1998) Â (P. Â Moradas-­âFerreira Â and Â V. Â Costa, Â 2000). Â It Â has Â been Â proposed Â that Â the Â glutathione Â system Â is Â critical Â for Â resistance Â to Â oxidative Â and Â nitrosative Â stress Â in Â C. Â neoformans Â (T. Â A. Â Missall, Â et Â al., Â 2004a). Â Cooperation Â between Â glutathione Â system Â and Â the Â thioredoxin Â system Â in Â defense Â against Â oxidative Â stress Â has Â also Â been Â proposed Â (T. Â A. Â Missall, Â et Â al., Â 2004a). Â However, Â the Â role Â of Â GSH Â in Â cell Â physiology Â and Â metabolism Â of Â C. Â neoformans Â has Â not Â been Â explored Â to Â date. Â Â Â The Â role Â of Â GSH Â is Â not Â only Â restricted Â to Â cellular Â redox Â cycle, Â but Â is Â also Â required Â for Â maintenance Â of Â iron Â homeostasis, Â mitochondrial Â structure, Â and Â membrane Â integrity. Â In Â fungi Â GSH Â is Â also Â used Â as Â a Â nutritional Â supply Â under Â sulfur Â or Â nitrogen Â starvation. Â In Â this Â context, Â it Â has Â been Â shown Â that Â GSH Â is Â required Â for Â adaptation Â to Â carbon, Â sulfur, Â and Â nitrogen Â starvation Â stress Â in Â fungi. Â These Â conditions Â trigger Â induction Â of Â GSH-­âdependent Â elements Â of Â the Â antioxidant Â defence Â system, Â leading Â to Â an Â increased Â tolerance Â to Â oxidative Â stress Â (M. Â T. Â Elskens Â et Â al., Â 1991) Â (D. Â Mendoza-­âCozatl Â et Â al., Â 2005) Â (J. Â Amich Â et Â al., Â 2013, Â G. Â A. Â Marzluf, Â 1997, Â I. Â Pocsi Â et Â al., Â 2004). Â GSH Â is Â also Â involved Â in Â detoxification Â of Â harmful Â intermediates Â such Â as Â methylglyoxal, Â which Â is Â a Â by-­âproduct Â of Â glycolysis Â by Â the Â glyoxalase Â pathway Â in Â yeast Â (A. Â M. Â Martins Â et Â al., Â 2001). Â GSH-­âdependent Â detoxification Â processes Â are Â also Â required Â for Â elimination Â of Â xenobiotics Â and Â antifungal Â drugs Â through Â chemical Â reaction Â between Â GSH Â and Â the Â antifungal Â drugs Â (B. Â Maras, Â et Â al., Â 2014). Â Â Â One Â approach Â to Â determine Â the Â role Â of Â GSH Â is Â utilization Â of Â GSH-­âdepleting Â drugs, Â or Â alternatively Â deletion Â of Â genes Â in Â GSH Â synthesis Â pathways Â using Â genetic Â methods, Â followed Â by Â analysis Â of Â the Â consequences Â arising Â from Â GSH Â deficiency. Â In Â the Â study Â outlined Â in Â Â Â Â Â Â Â Â Â Â Â Â Â Â 150 Â chapter Â 3 Â of Â my Â thesis, Â GSH Â deficiency Â was Â obtained Â by Â deletion Â of Â GSH2 Â in Â C. Â neoformans, Â and Â the Â role Â of Â GSH Â biosynthesis Â in Â C. Â neoformans Â was Â studied. Â Â Â Phenotypic Â analysis Â of Â gsh2 Â mutants Â grown Â under Â non-­âstressed Â conditions Â in Â YPD Â and Â YNB Â revealed Â that Â GSH2 Â is Â dispensable Â for Â growth Â under Â normal Â conditions, Â despite Â significantly Â low Â levels Â of Â GSH Â in Â these Â mutants. Â I Â speculate Â this Â is Â most Â likely Â due Â to Â accumulation Â of Â product Â of Â GSH1 Â (dipeptide Â Î³-­âL-­âglutamyl-­âL-­âcysteine). Â It Â is Â also Â possible Â that Â GSH Â is Â not Â required Â under Â non-­âstressed Â conditions Â because Â Trxs Â are Â potentially Â overproduced Â to Â compensate Â for Â GSH. Â Importantly, Â a Â functional Â link Â between Â the Â GSH/Grx Â and Â Trx Â systems Â in Â yeast Â under Â normal Â growth Â conditions Â has Â been Â shown. Â On Â this Â note Â in Â normal Â growth Â conditions Â accumulation Â of Â ROS Â in Â cells Â is Â considered Â low Â and Â not Â detrimental Â (T. Â Draculic Â et Â al., Â 2000) Â (E. Â W. Â Trotter Â and Â C. Â M. Â Grant, Â 2003) Â (E. Â G. Â Muller, Â 1996). Â Â Â Overall, Â growth Â of Â gsh2 Â mutants Â under Â non-­âstressed Â conditions Â clearly Â demonstrate Â that Â GSH Â is Â not Â essential Â under Â these Â conditions Â given Â the Â very Â low Â levels Â of Â GSH Â measured Â in Â these Â mutants. Â However Â my Â studies Â on Â gsh2 Â mutants Â that Â underwent Â iron Â starvation Â led Â to Â a Â deeper Â understanding Â of Â the Â role Â of Â Gsh2 Â via Â GSH Â in Â C. Â neoformans. Â I Â found Â that Â iron-­âstarved Â gsh2 Â mutants Â were Â auxotrophic Â for Â GSH Â and Â only Â supplementation Â of Â GSH Â and Â not Â iron, Â restored Â growth Â in Â these Â phenotypes. Â Â In Â this Â context, Â it Â had Â previously Â been Â shown Â that Â iron Â starvation Â leads Â to Â ROS Â production Â and/or Â depletion Â of Â intracellular Â GSH Â pool Â in Â A. Â thaliana Â (L. Â Ramirez, Â et Â al., Â 2013). Â Iron Â is Â a Â major Â constituent Â of Â electron Â transport Â chain Â in Â mitochondria Â and Â chloroplasts, Â and Â so Â iron Â deficiency Â can Â lead Â to Â disruption Â of Â a Â normal Â electron Â transfer Â resulting Â in Â the Â overproduction Â of Â ROS. Â Consequently, Â antioxidant Â systems Â are Â required Â to Â combat Â the Â generated Â ROS. Â It Â was Â also Â shown Â that Â GSH Â treatment Â Â Â Â Â Â Â Â Â Â Â Â Â Â 151 Â counteracted Â iron Â deficiency-­âinduced Â ROS Â accumulation Â in Â A. Â thaliana Â leaves Â and Â led Â to Â restoration Â of Â ROS Â to Â basal Â levels Â in Â presence Â of Â GSH Â (L. Â Ramirez, Â et Â al., Â 2013). Â Similarly, Â my Â findings Â suggest Â that Â GSH Â is Â required Â to Â protect Â against Â detrimental Â effects Â of Â iron Â deficiency Â in Â C. Â neoformans. Â I Â also Â found Â that Â introduction Â of Â GSH Â to Â iron-­âstarved Â gsh2 Â mutants Â led Â to Â a Â statistically Â significant Â increase Â in Â GSH Â levels Â in Â gsh2 Â mutants. Â This Â suggests Â that Â GSH Â uptake Â system Â is Â present Â and Â functional Â in Â gsh2 Â mutants Â in Â C. Â neoformans. Â In Â this Â context, Â it Â has Â been Â shown Â that Â GSH Â is Â taken Â up Â from Â the Â environment Â by Â homologues Â of Â small Â oligopeptide Â transporters Â (OPT Â family Â of Â proteins) Â in Â fungi Â and Â plants. Â Assimilation Â of Â radioactively Â labeled Â forms Â of Â GSH Â from Â media Â led Â to Â the Â discovery Â of Â OPT Â homologues Â known Â as Â high Â affinity Â plasma Â membrane Â GSH Â transporter Â (Hgt1p) Â in Â yeast Â (T. Â Miyake Â et Â al., Â 1998) Â (A. Â Bourbouloux Â et Â al., Â 2000) Â (S. Â Sato Â et Â al., Â 1994). Â Hgt1 Â is Â a Â 799-­âamino Â acid Â polypeptide Â with Â a Â predicted Â molecular Â mass Â of Â 91.6 Â kDa Â with Â 12â14 Â transmembrane Â domains Â (A. Â Bourbouloux, Â et Â al., Â 2000) Â (T. Â Miyake Â et Â al., Â 2002). Â Â Homologues Â of Â Hgt1p Â are Â apparently Â restricted Â to Â plants Â and Â fungi, Â including Â C. Â albicans Â and Â S. Â pombe. Â It Â has Â been Â shown Â that Â hgt1 Â deletion Â mutants Â in Â yeast Â do Â not Â transport Â GSH, Â and Â hgt1 Â gsh1 Â double Â mutants Â are Â not Â viable Â (A. Â Bourbouloux, Â et Â al., Â 2000) Â (M. Â A. Â Lubkowitz Â et Â al., Â 1998, Â S. Â Sato, Â et Â al., Â 1994) Â (M. Â Hauser Â et Â al., Â 2000). Â Interestingly, Â C. Â neoformans Â genome Â encodes Â 5 Â small Â oligopeptide Â transporters Â with Â up Â to Â 60% Â of Â sequence Â identity Â to Â Hgt1 Â in Â yeast Â (encoded Â by Â CNAG_019654, Â CNAG_03013, Â CNAG_05790, Â CNAG_04617, Â CNAG_05737). Â Therefore Â analysis Â of Â the Â regulation, Â functions Â and Â contribution Â of Â these Â transporters Â in Â the Â WT Â and Â also Â in Â gsh2 Â mutants Â background Â is Â necessary Â to Â fully Â understand Â the Â role Â of Â GSH Â metabolism Â in Â virulence Â inside Â the Â host. Â In Â regards Â to Â virulence, Â here Â I Â demonstrated Â that Â gsh2 Â mutants Â were Â acapsular, Â non-­â Â Â Â Â Â Â Â Â Â Â Â Â Â 152 Â melanized Â and Â did Â not Â grow Â at Â 37°C, Â most Â likely Â due Â to Â lack Â of Â GSH Â in Â these Â mutants. Â It Â has Â previously Â been Â shown Â that Â elevated Â temperatures Â can Â lead Â to Â an Â increase Â in Â the Â frequency Â of Â mutations Â and Â interchromosomal Â DNA Â recombination, Â and Â damages Â mitochondrial Â DNA Â in Â yeast. Â Cytotoxic Â and Â genotoxic Â effects Â of Â elevated Â temperature Â are Â partially Â due Â to Â oxidative Â stress Â and Â intensified Â respiration, Â and Â it Â has Â been Â shown Â that Â GSH Â is Â required Â to Â counteract Â these Â events Â in Â yeast, Â For Â instance Â heat Â shock Â in Â S. Â cerevisiae Â leads Â to Â upregulation Â of Â GSH Â biosynthesis. Â (J. Â F. Â Davidson Â and Â R. Â H. Â Schiestl, Â 2001) Â (K. Â Sugiyama Â et Â al., Â 2000a) Â (J. Â Lee Â et Â al., Â 1997, Â K. Â Sugiyama Â et Â al., Â 2000b). Â Â Â In Â C. Â albicans Â it Â has Â been Â show Â that Â glutathione Â biosynthesis Â is Â essential Â in Â virulence Â and Â disruption Â of Â glutathione Â biosynthesis Â causes Â glutathione Â auxotrophy, Â elevated Â (ROS) Â levels Â and Â apoptosis Â (P. Â L. Â Fidel, Â Jr. Â et Â al., Â 1999) Â (M. Â A. Â Pfaller Â et Â al., Â 2010) Â (J. Â R. Â Wingard, Â 1995) Â (Y. Â U. Â Baek, Â et Â al., Â 2004) Â (A. Â K. Â Yadav Â et Â al., Â 2011). Â Â Overall Â in Â this Â study, Â the Â loss Â of Â main Â virulence Â traits Â in Â gsh2 Â mutants Â suggests Â the Â presence Â of Â a Â link Â between Â glutathione Â biosynthesis Â and Â virulence Â in Â C. Â neoformans. Â Although Â the Â role Â of Â GSH Â transporters Â is Â unknown Â at Â the Â moment Â to Â predict Â virulence Â of Â gsh2 Â mutants Â in Â animal Â models Â or Â in Â the Â host, Â it Â is Â reasonable Â to Â propose Â that Â C. Â neoformans Â exploits Â the Â host Â glutathione Â for Â its Â survival. Â I Â predict Â that Â despite Â the Â presence Â of Â the Â transporter, Â biosynthesis Â would Â still Â be Â essential Â for Â virulence Â of Â C. Â neoformans Â in Â the Â host Â depending Â on Â the Â niche Â that Â C. Â neoformans Â occupies Â in Â different Â stages Â of Â its Â life Â cycle Â in Â the Â host. Â For Â instance, Â the Â levels Â of Â GSH Â in Â the Â lining Â fluid Â of Â the Â lungs Â can Â reach Â 300 Â times Â the Â concentration Â fund Â in Â the Â plasma Â compared Â to Â blood Â plasma Â where Â levels Â of Â GSH Â are Â exceedingly Â low Â (L. Â J. Â Smith Â and Â J. Â Anderson, Â 1992). Â I Â speculate Â that Â GSH Â biosynthesis Â pathway Â will Â be Â most Â probably Â required Â for Â migration Â of Â C. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 153 Â neoformans Â in Â blood Â where Â GSH Â levels Â are Â low. Â In Â this Â case, Â even Â the Â presence Â of Â a Â GSH Â transporter Â will Â not Â be Â able Â to Â keep Â up Â with Â high Â glutathione Â demands Â of Â C. Â neoformans, Â and Â so Â the Â presence Â of Â a Â functional Â and Â efficient Â GSH Â biosynthesis Â pathway Â will Â become Â essential. Â Â Overall, Â given Â the Â pleotropic Â effects Â of Â GSH Â and Â the Â numerous Â cellular Â processes Â that Â can Â be Â influenced Â by Â GSH Â deficiency, Â studying Â the Â exact Â mechanisms Â that Â lead Â to Â the Â role Â of Â GSH Â in Â virulence Â in Â the Â host Â is Â important Â in Â further Â understanding Â of Â C. Â neoformans Â pathogenesis. Â Â 4.4 Â Conclusion Â Â Â Â Â In Â conclusion, Â the Â studies Â presented Â in Â my Â thesis Â demonstrated Â the Â critical Â role Â of Â Grx4 Â in Â iron Â homeostasis Â and Â virulence Â in Â the Â AIDS-­âassociated Â fungal Â pathogen Â C. Â neoformans Â var. Â grubii. Â I Â also Â found Â that Â glutathione Â has Â pleotropic Â functions Â with Â links Â to Â iron Â homeostasis Â and Â is Â required Â for Â elaboration Â of Â virulence Â traits Â in Â C. Â neoformans. Â The Â work Â presented Â in Â my Â thesis Â is Â novel Â because Â it Â is Â the Â first Â study Â to Â demonstrate Â the Â role Â of Â a Â monothiol Â Grx Â in Â virulence Â in Â a Â pathogenic Â fungus. Â Also, Â it Â is Â the Â first Â study Â to Â describe Â the Â role Â of Â glutathione Â in Â C. Â neoformans. Â Identifying Â additional Â components Â of Â iron Â homeostasis Â and Â deciphering Â their Â contributions Â to Â pathogenesis Â in Â C. Â neoformans Â will Â serve Â the Â ultimate Â goal Â of Â development Â of Â new Â strategies Â to Â combat Â and Â block Â cryptococcal Â infection. Â This Â goal Â could Â be Â achieved Â either Â by Â specifically Â blocking Â iron-­âacquisition Â systems Â of Â C. Â neoformans Â with Â antifungals, Â or Â by Â reducing Â the Â availability Â of Â iron Â in Â the Â intracellular Â niche Â occupied Â by Â C. Â neoformans Â during Â the Â infection. Â Â Â Â In Â the Â first Â part Â of Â my Â studies, Â in Â silico Â analysis Â of Â Grx4 Â revealed Â the Â presence Â of Â the Â conserved Â C-­âterminal Â Grx Â domain Â in Â this Â protein. Â The Â conserved Â CGFS Â motif Â in Â this Â domain Â Â Â Â Â Â Â Â Â Â Â Â Â Â 154 Â is Â well Â characterized Â in Â non-­âpathogenic Â fungal Â species Â including Â S. Â pombe Â and Â S. Â cerevisiae. Â Functional Â characterization Â of Â the Â grx4 Â mutant Â showed Â loss Â of Â GRX4 Â leads Â to Â elaboration Â of Â iron-­ârelated Â phenotypes Â including Â sensitivity Â to Â elevated Â levels Â and Â iron-­âresponsive Â drug Â phleomycin, Â also Â an Â increased Â cell Â surface Â reductase Â activity. Â Â These Â phenotypes Â suggested Â a Â perturbed Â iron Â homeostasis Â in Â absence Â of Â Grx4 Â in Â C. Â neoformans. Â Next, Â differential Â regulation Â of Â iron Â acquisition Â genes Â in Â the Â q-­âRT-­âPCR Â and Â RNA-­âseq Â analysis Â consolidated Â the Â role Â of Â Grx4 Â in Â iron Â homeostasis Â and Â showed Â that Â Grx4 Â is Â required Â for Â transcriptional Â remodeling Â of Â iron Â acquisition Â pathways Â in Â response Â to Â iron. Â Importantly, Â loss Â of Â GRX4 Â led Â to Â upregulation Â of Â iron Â acquisition Â components Â (including Â high Â affinity Â uptake Â system, Â siderophore Â transporters, Â and Â iron Â reductases) Â in Â iron-­âreplete Â conditions. Â Â These Â results Â highly Â suggest Â that Â Grx4 Â has Â an Â inhibitory Â influence Â on Â iron Â acquisition Â systems Â in Â response Â to Â iron. Â I Â also Â demonstrated Â that Â Grx4 Â controls Â the Â known Â major Â virulence Â factors Â of Â the Â pathogen Â including Â the Â capsule, Â the Â formation Â of Â the Â anti-­âoxidant Â melanin Â in Â the Â cell Â wall, Â and Â the Â ability Â to Â grow Â at Â host Â body Â temperature. Â Remarkably, Â Grx4 Â is Â a Â major Â contributor Â to Â the Â disease Â process, Â as Â confirmed Â by Â the Â avirulence Â of Â the Â grx4 Â mutant Â in Â mice. Â Â 4.5 Â Key Â areas Â for Â future Â directions Â Â 4.5.1 Â A Â Proposed Â model Â of Â Grx4-­âCir1 Â interaction Â Â The Â physical Â binding Â between Â Grx4 Â and Â Cir1 Â was Â clearly Â demonstrated Â in Â ALPHAScreen Â and Â Y2H Â analysis Â outlined Â in Â chapter Â 2. Â Given Â that Â Cir1 Â is Â the Â master Â iron Â regulator Â in Â C. Â neoformans Â (W. Â H. Â Jung, Â et Â al., Â 2006), Â association Â of Â Grx4 Â with Â Cir1 Â further Â highlights Â the Â significance Â of Â Grx4 Â in Â C. Â neoformans. Â Â The Â discovery Â of Â association Â of Â Grx4 Â with Â Cir1 Â resembles Â the Â scenario Â in Â S. Â pombe Â where Â Grx4 Â binds Â Fep1 Â and Â is Â required Â for Â Â Â Â Â Â Â Â Â Â Â Â Â Â 155 Â iron-­âdependent Â inhibition Â of Â Fep1 Â (M. Â Jbel, Â et Â al., Â 2011). Â The Â exact Â mechanisms Â of Â Grx4-­âCir1 Â binding Â is Â not Â clear Â at Â this Â point Â but Â given Â that Â grx4 Â mutant Â and Â cir1 Â mutant Â share Â similar Â iron-­â Â and Â virulence-­ârelated Â phenotypes, Â and Â also Â the Â fact Â that Â Cir1 Â was Â initially Â identified Â due Â to Â high Â homology Â to Â Fep1, Â it Â is Â possible Â that Â the Â interaction Â between Â Cir1 Â and Â Grx4 Â in Â C. Â neoformans Â follows Â similar Â principles Â as Â the Â mechanism Â of Â interaction Â in Â Grx4-­âFep1 Â in Â S. Â pombe. Â In Â addition Â to Â the Â role Â of Â Grx4 Â in Â Cir1-­âmediated Â iron Â regulation, Â I Â also Â predict Â a Â potential Â role Â for Â Grx4 Â in Â cellular Â iron Â trafficking Â and Â iron Â sensing Â by Â incorporating Â glutathione-­âcontaining Â Fe-­âS Â clusters. Â The Â conserved Â property Â of Â monothiol Â Grxs Â in Â binding Â glutathione-­âcontaining Â Fe-­âS Â clusters Â has Â been Â fully Â described Â in Â S. Â pombe Â and Â S. Â cerevisiae Â (J. Â Encinar Â del Â Dedo, Â et Â al., Â 2015, Â H. Â Li Â and Â C. Â E. Â Outten, Â 2012). Â Â Â Previous Â genetic Â and Â biochemical Â studies Â have Â revealed Â that Â regulation Â of Â iron Â metabolism Â in Â fungi Â is Â a Â multi-­âstep Â pathway Â involving Â proteins Â in Â the Â mitochondria, Â cytosol, Â and Â nucleus Â (D. Â H. Â Howard, Â 1999). Â The Â components Â of Â iron Â homeostasis Â in Â C. Â neoformans Â have Â been Â well Â characterized Â and Â it Â has Â previously Â been Â shown Â that Â Cir1 Â governs Â the Â regulation Â of Â iron Â homeostasis. Â Here Â I Â identified Â Grx4 Â as Â an Â additional Â piece Â of Â the Â iron Â homeostasis Â puzzle Â in Â C. Â neoformans Â that Â most Â likely Â exerts Â its Â function Â in Â iron Â homoeostasis Â through Â interaction Â with Â Cir1. Â I Â propose Â a Â model Â in Â which Â Cir1 Â is Â constitutively Â localized Â to Â the Â nucleus Â and Â Grx4 Â is Â required Â to Â regulate Â the Â activity Â of Â Cir1 Â through Â specific Â proteinâ Â protein Â interactions. Â In Â my Â proposed Â model, Â Grx4 Â is Â required Â to Â inhibit Â Cir1 Â from Â repressing Â its Â target Â iron Â acquisition Â genes Â under Â iron-­âdepleted Â conditions. Â In Â the Â absence Â of Â Grx4, Â Cir1 Â loses Â the Â ability Â to Â sense Â the Â iron-­âdeplete Â intracellular Â environment, Â which Â leads Â to Â constitutive Â repression Â of Â iron Â acquisition. Â In Â my Â proposed Â model Â iron-­âdependent Â modulation Â of Â Cir1 Â activity Â is Â exerted Â through Â Grx4 Â Â Â Â Â Â Â Â Â Â Â Â Â Â 156 Â binding Â the Â N-­âterminal Â DNA Â binding Â domain Â composed Â of Â a Â zinc Â finger Â and Â a Â cysteine-­ârich Â region Â (Figure Â 4.1). Â Â Â Â Â Â Â Â Â Â Â Â Â Â Figure Â 4.1: Â A Â schematic Â model Â of Â Grx4-­âCir1 Â interaction. Â Â Grx4 Â binds Â Cir1 Â in Â the Â nucleus Â and Â alters Â its Â activity Â by Â unknown Â mechanisms, Â in Â response Â to Â iron Â availability. Â Under Â low Â iron Â replete Â conditions, Â Grx4 Â is Â required Â for Â function Â of Â Cir1 Â in Â repression Â of Â iron Â acquisition Â genes. Â Fe-­âS Â or Â Fe-­âbinding Â capacity Â of Â Grx4, Â might Â play Â a Â critical Â role Â for Â its Â interaction Â with Â Cir1 Â and Â iron Â homeostasis. Â Whether Â and Â how Â FeâS Â or Â Fe Â is Â involved Â in Â this Â interaction Â is Â a Â challenging Â question Â to Â be Â tackled Â in Â the Â future. Â 4.5.2 Â Â A Â proposed Â crosstalk Â model Â for Â GSH Â functions Â Â Â In Â the Â second Â part Â of Â my Â studies Â I Â elucidated Â the Â roles Â of Â glutathione Â synthetase Â Gsh2 Â Â Â Â Â Â Â Â Â Â Â Â Â Â 157 Â and Â found Â that Â product Â of Â this Â gene, Â glutathione, Â remarkably Â affects Â a Â plethora Â of Â biological Â processes Â and Â functions Â with Â key Â implications Â in Â iron-­âstarvation Â and Â virulence Â in Â C. Â neoformans. Â Here, Â results Â from Â analysis Â of Â consequences Â of Â GSH Â depletion Â by Â deleting Â GSH2 Â suggested Â that Â GSH Â is Â required Â for Â growth Â of Â C. Â neoformans Â in Â iron-­ârestricted Â conditions Â representative Â of Â the Â conditions Â encountered Â by Â this Â pathogen Â in Â the Â host. Â I Â also Â showed Â that Â GSH Â is Â required Â for Â expression Â of Â major Â virulence Â traits Â including Â capsule Â formation, Â melanin Â production Â and Â growth Â at Â human Â body Â temperature Â at Â 37°C. Â Â Furthermore, Â I Â found Â that Â GSH Â is Â required Â for Â resistance Â to Â the Â major Â antifungal Â drugs, Â cell Â wall Â integrity Â and Â mitochondrial Â functions Â in Â C. Â neoformans. Â Â Â C. Â neoformans Â is Â subjected Â to Â numerous Â extreme Â conditions Â in Â the Â host Â including Â but Â not Â limited Â to, Â osmotic Â and Â thermal Â changes, Â exposure Â to Â ROS, Â nutrient Â deprivation Â (including Â particularly). Â An Â efficient Â and Â dynamic Â cross Â talk Â among Â cellular Â events Â and Â pathways Â that Â counteract Â the Â effects Â of Â harsh Â environment Â of Â the Â host Â is Â essential Â to Â facilitate Â the Â overall Â cellular Â integrity Â of Â C. Â neoformans Â and Â to Â enhance Â the Â capabilities Â of Â the Â C. Â neoformans Â to Â proliferate Â in Â the Â host. Â These Â pathways Â include Â iron Â homeostasis Â components Â and Â signaling Â pathways Â such Â as Â the Â calcineurin, Â high-­âosmolality Â glycerol, Â and Â PKC1 Â pathways, Â and Â numerous Â additional Â proteins Â (J. Â Kronstad, Â et Â al., Â 2012, Â J. Â W. Â Kronstad, Â et Â al., Â 2011). Â In Â the Â second Â part Â of Â my Â thesis, Â the Â findings Â on Â the Â pleotropic Â role Â of Â GSH Â and Â the Â number Â of Â cellular Â events Â suggested Â to Â require Â GSH, Â leads Â to Â the Â conclusion Â that Â GSH Â is Â a Â fascinating Â clue Â to Â cross Â talk Â among Â these Â pathways Â in Â C. Â neoformans. Â Â Â Glutathione Â is Â the Â major Â cellular Â antioxidant, Â detoxifier, Â and Â an Â important Â component Â both Â in Â prokaryotic Â and Â eukaryotic Â cells. Â Many Â of Â the Â roles Â of Â the Â GSH Â have Â been Â established Â in Â fungi Â as Â well Â (I. Â Pocsi, Â et Â al., Â 2004). Â In Â the Â last Â decade, Â the Â focus Â of Â Â Â Â Â Â Â Â Â Â Â Â Â Â 158 Â research Â on Â GSH Â has Â shifted Â from Â its Â prime Â role Â in Â thiol-­âredox Â control, Â towards Â its Â essential Â requirement Â in Â iron Â homeostasis. Â Emerging Â evidence Â has Â shown Â the Â association Â of Â GSH Â with Â monothiol Â Grxs Â via Â binding Â Fe-­âS Â clusters Â and Â serving Â as Â a Â vital Â component Â of Â iron Â sensing Â complexes Â in Â yeast Â and Â fission Â yeast Â (D. Â T. Â Mapolelo, Â et Â al., Â 2013) Â (J. Â Encinar Â del Â Dedo, Â et Â al., Â 2015) Â It Â is Â also Â established Â that Â GSH Â is Â essential Â for Â delivery Â and Â distribution Â of Â iron Â in Â eukaryotic Â cells Â and Â serves Â as Â a Â major Â cytosolic Â ligand Â for Â binding Â ferrous Â form Â of Â iron Â in Â the Â labile Â iron Â pool Â (M. Â Kruszewski, Â 2003) Â (C. Â C. Â Philpott Â and Â M. Â S. Â Ryu, Â 2014). Â Also, Â glutathionylation Â appears Â to Â be Â a Â common Â mode Â of Â post-­âtranslational Â modification Â of Â iron-­âdependent Â transcription Â factors Â in Â both Â prokaryotes Â and Â eukaryotes Â (C. Â L. Â Grek, Â et Â al., Â 2013). Â Furthermore, Â GSH Â depletion Â has Â been Â shown Â to Â cause Â an Â impairment Â of Â extra-­âmitochondrial Â Fe-­âS Â cluster Â assembly Â machinery Â (ISC) Â and Â activation Â of Â Aft1 Â in Â yeast Â (K. Â Sipos, Â et Â al., Â 2002, Â J. Â C. Â Rutherford, Â et Â al., Â 2005).These Â findings Â have Â established Â GSH Â as Â part Â of Â the Â mitochondrial Â ISC Â export Â process Â and Â a Â signal Â for Â regulation Â of Â iron Â homoeostasis Â in Â yeast Â (O. Â S. Â Chen Â et Â al., Â 2004, Â U. Â Muhlenhoff, Â et Â al., Â 2010). Â Furthermore, Â intracellular Â GSH Â hinders Â progression Â of Â heavy Â metal-­âinduced Â cell Â injuries Â by Â scavenging Â the Â metal Â ions Â themselves Â and/or Â by Â eliminating Â ROS Â (P. Â Perego Â and Â S. Â B. Â Howell, Â 1997, Â S. Â V. Â Avery, Â 2001, Â C. Â Cobbett Â and Â P. Â Goldsbrough, Â 2002). Â Â Based Â on Â the Â findings Â on Â the Â role Â of Â GSH Â in Â protection Â against Â iron-­âdeprivation Â induced Â oxidative Â stress Â and Â a Â plethora Â of Â cellular Â functions Â in Â C. Â neoformans, Â it Â is Â possible Â to Â propose Â a Â model Â in Â which Â GSH establishes a functional crosstalk between iron homeostasis and cellular function in C. neoformans (Figure 4.2). Given Â that Â GSH Â synthesis Â and Â Cir1 Â influence Â iron Â homeostasis Â and Â similar Â cellular Â functions, Â interplay Â between Â Cir1 Â and Â GSH Â synthesis Â is Â suggested Â in Â this Â model. Â Additionally, Â according Â to Â the Â mounting Â evidence Â on Â Â Â Â Â Â Â Â Â Â Â Â Â Â 159 Â role Â of Â GSH Â in Â coordinating Â Fe-­âS Â clusters, Â it Â is Â possible Â that Â GSH Â synthesis Â links Â Grx4 Â and Â Fe-­âS Â cluster Â coordination Â (W. Â Qi Â et Â al., Â 2012). Â As Â proposed Â in Â Figure Â 4.1 Â Grx4 Â most Â likely Â coordinates Â Fe-­âS Â clusters Â leading Â to Â its Â association Â with Â Cir1 Â through Â the Â GRX-­âlike Â domain. Â This Â interaction Â would Â induce Â conformational Â changes Â that Â subsequently Â modulate Â Cir1 Â activity. Â Â Â Taken Â together Â in Â this Â model, Â it Â is Â proposed Â that Â GSH Â serves Â as Â a Â core Â in Â maintenance Â of Â cellular Â homeostasis Â in Â C. Â neoformans. Â GSH Â physiological Â role Â is Â split Â between Â a Â function Â in Â iron Â homeostasis Â by Â coordinating Â Fe-­âS Â cluster, Â binding Â Grx4, Â and Â Â maintaining Â a Â vital Â thiol-­âredox Â balances Â needed Â for Â essential Â cellular Â functions. Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 160 Â Â Â Â Figure Â 4.2: Â Schematics Â of Â the Â proposed Â functional Â crosstalk Â among Â GSH, Â iron Â homeostasis Â and Â cellular Â functions Â in Â C. Â neoformans. Â Grx4 Â is Â required Â for Â iron Â homeostasis Â (blue Â line). Â Cir1 Â has Â an Â established Â role Â in Â iron Â homeostasis Â and Â cellular Â functions Â (pink Â line). Â Grx4 Â is Â proposed Â to Â form Â a Â complex Â with Â GSH Â and/or Â Cir1 Â by Â coordinating Â Fe-­âS Â clusters. Â The Â CGFS Â motif Â in Â the Â conserved Â C-­âterminal Â domain Â in Â Grx4, Â and Â CXXC Â motif Â in Â the Â DNA Â binding Â region Â of Â Cir1 Â are Â shown. Â Similar Â to Â Cir1 Â and Â Grx4, Â GSH Â is Â essential Â for Â key Â cellular Â pathways Â in Â C. Â neoformans. Â Â Â Further Â studies Â are Â required Â to Â understand Â the Â likely Â partnership Â between Â Grx4 Â Â Â Â Â Â Â Â Â Â Â Â Â Â 161 Â and Â GSH Â and Â its Â role Â in Â iron Â homeostasis. Â Â In Â a Â broader Â context, Â this Â will Â contribute Â to Â insights Â into Â the Â intricate Â links Â between Â redox Â imbalance, Â GSH Â depletion, Â alterations Â of Â iron Â homeostasis, Â oxidative Â stress Â and Â virulence Â in Â C. Â neoformans. Â This Â will Â provide Â concepts Â for Â new, Â more Â effective Â treatments Â for Â cryptococcosis, Â perhaps Â involving Â synergistic Â combinations Â of Â oxidant Â drugs, Â inhibitors Â to Â deplete Â glutathione Â or Â antifungals. Â 4.6 Â Future Â experiments Â 4.6.1 Â Â Study Â the Â interaction Â of Â Grx4 Â with Â Cir1 Â in Â C. Â neoformans Â Â Â Chapter Â 2 Â of Â this Â thesis Â demonstrated Â that Â Grx4 Â binds Â Cir1 Â in Â ALPHAScreen Â and Â Y2H Â assays. Â However Â the Â molecular Â events Â of Â this Â interaction Â in Â C. Â neoformans Â and Â whether Â iron Â levels Â have Â a Â role Â in Â this Â interaction Â was Â not Â determined. Â This Â could Â be Â investigated Â by Â in Â vivo Â co-­âimmunoprecipitation. Â This Â requires Â construction Â of Â strain Â that Â harbour Â tagged Â Cir1 Â and Â Grx4. Â For Â instance, Â CIR1::GFP Â fusion Â allele Â is Â already Â available Â and Â has Â already Â been Â constructed Â by Â Dr. Â Guanggan Â Hu. Â Generation Â of Â mCherry-­âGrx4 Â fusion Â for Â instance Â can Â be Â a Â good Â place Â to Â start. Â This Â will Â also Â involve Â preparation Â of Â lysates Â from Â the Â strains Â grown Â in Â low-­âiron Â medium Â or Â high-­âiron Â medium Â followed Â by Â use Â of Â anti-­âGFP Â and Â anti-­â Â dsRed Â antibodies Â in Â Western Â blot Â analysis. Â Alternatively, Â the Â generated Â strain Â can Â be Â used Â to Â investigate Â the Â Cir1-­âGrx4 Â interaction Â by Â confocal Â microscopy. Â Additionally, Â these Â strains Â could Â be Â used Â to Â determine Â the Â localisation Â of Â Grx4 Â in Â iron-­âdeplete Â or Â iron-­âreplete Â conditions Â by Â subcellular Â fractionation Â followed Â by Â Western Â blot. Â 4.6.2 Â Â Investigating Â Grx4 Â capacity Â to Â assemble Â Fe-­âS Â clusters Â Â As Â discussed Â in Â chapter Â 2, Â the Â function Â of Â monothiol Â Grxs Â with Â conserved Â CGFS Â motif Â in Â iron Â regulation Â has Â been Â extensively Â studied Â in Â S. Â cerevisiae Â and Â S. Â pombe. Â According Â to Â recent Â line Â of Â evidence, Â recombinant Â versions Â of Â bacterial, Â human, Â yeast Â and Â plant Â Â Â Â Â Â Â Â Â Â Â Â Â Â 162 Â monothiol Â Grxs Â orthologues Â produced Â in Â E. Â coli Â are Â able Â to Â incorporate Â FeâS Â clusters. Â This Â complex Â has Â a Â labile Â nature Â and Â detection Â of Â FeâS Â clusters Â bound Â to Â these Â CGFS Â Grxs Â require Â specific Â analytical Â and Â biochemical Â techniques, Â such Â as Â spectroscopic Â UV-­âvisible Â absorption/circular Â dichroism Â (CD), Â resonance Â Raman Â and Â Mössbauer Â analyses Â of Â anaerobically Â purified Â proteins Â or Â proteins Â repurified Â after Â in Â vitro Â Fe-­âS Â cluster Â reconstitution Â of Â apo-­âproteins. Â It Â will Â be Â important Â to Â investigate Â whether Â Grx4 Â assembles Â Fe-­âS Â cluster. Â If Â it Â is Â proven Â that Â Grx4 Â can Â undergo Â transition Â between Â a Â holo-­â Â and Â apo-­âGrx4, Â then Â this Â will Â ascertain Â a Â suggested Â role Â of Â Grx4 Â as Â the Â regulatory Â switch Â in Â the Â complex Â iron Â regulatory Â network Â of Â C. Â neoformans. Â Investigation Â of Â Fe-­âS Â cluster Â reconstitution Â on Â Grx4 Â will Â require Â advanced Â biochemical, Â spectroscopic Â and Â analytical Â techniques Â to Â examine Â binding Â of Â recombinant Â Grx4 Â to Â Fe-­âS Â clusters. Â This Â will Â also Â involve Â purification Â of Â recombinant Â Grx4 Â under Â anaerobic Â conditions Â to Â minimize Â loss Â of Â the Â Fe-­âS Â cluster. Â Also, Â reconstitution Â of Â Fe-­âS Â clusters Â on Â apo-­âGrx4 Â needs Â to Â be Â carried Â out Â under Â anaerobic Â conditions Â (O2 Â < Â 5 Â ppm) Â according Â to Â previously Â published Â protocols Â (R. Â Lill Â and Â U. Â Muhlenhoff, Â 2006) Â (H. Â Li, Â et Â al., Â 2009) Â (U. Â Muhlenhoff, Â et Â al., Â 2010, Â B. Â Zhang, Â et Â al., Â 2013) Â (P. Â Shakamuri Â et Â al., Â 2012). Â 4.6.3 Â Further Â understanding Â of Â the Â transcript Â structure Â of Â the Â GRX4 Â locus Â Â Â Re-­âanalysis Â of Â data Â obtained Â from Â RNA-­âsequencing Â outlined Â in Â chapter Â 2 Â indicated Â the Â presence Â of Â several Â transcript Â versions Â (with Â variation Â in Â N-­âterminal Â region) Â arising Â from Â the Â GRX4 Â locus. Â The Â identification Â of Â several Â transcripts Â from Â GRX4 Â locus Â in Â accumulating Â RNA-­âSeq Â data Â raised Â the Â possibility Â that Â there Â is Â more Â than Â one Â transcript Â from Â the Â GRX4 Â locus. Â Future Â work Â is Â required Â to Â thoroughly Â investigate Â the Â structure Â of Â the Â GRX4 Â locus. Â This Â analysis Â involves Â preparing Â cDNA Â from Â WT Â and Â grx4 Â mutant Â cells Â grown Â Â Â Â Â Â Â Â Â Â Â Â Â Â 163 Â under Â different Â conditions, Â using Â carefully Â designed Â primers Â at Â the Â boundaries Â of Â each Â predicted Â intron Â and Â exon Â to Â amplify Â sequences Â from Â cDNA Â and Â genomic Â DNA. Â This Â can Â be Â further Â evaluated Â either Â by Â Northern Â analysis Â using Â specific Â primer Â radiolabeled Â with Â [Î±-­â32P] Â dCTP, Â or Â by Â Western Â blot Â analysis Â with Â anti-­âGrx4 Â antibody Â and Â extracts Â from Â wild Â type Â and Â the Â grx4 Â mutant Â to Â understand Â the Â protein(s) Â expressed Â from Â the Â locus. Â Given Â that Â numerous Â efforts Â to Â delete Â the Â N-­âterminal Â region Â in Â C. Â neoformans Â reference Â strain Â H99 Â (haploid Â background) Â by Â biolistic Â transformation Â were Â unsuccessful, Â and Â taking Â into Â account Â that Â N-­âterminal Â region Â of Â the Â GRX4 Â locus Â is Â not Â essential Â in Â a Â stable Â diploid Â background, Â it Â would Â be Â interesting Â to Â determine Â whether Â there Â is Â a Â highly Â intricate Â regulation Â of Â gene Â expression Â in Â the Â GRX4 Â locus Â in Â particular Â in Â the Â N-­âterminal Â variable Â region, Â in Â C. Â neoformans Â It Â would Â also Â be Â interesting Â to Â find Â out Â whether Â these Â versions Â with Â variation Â in Â N-­âterminal Â region Â contribute Â to Â different Â functions Â under Â low Â iron Â or Â high Â iron Â condition Â or Â in Â different Â cellular Â compartments. Â Future Â work Â is Â required Â to Â determine Â whether Â transcript Â variability Â results Â from Â alternative Â splicing Â or Â alternative Â transcription Â start Â sites Â and Â if Â alternate Â isoforms Â are Â biologically Â relevant. Â 4.6.4 Â Â Â Investigation Â of Â reversible Â S-­âglutathionylation Â of Â Cir1 Â Â Â An Â increasing Â body Â of Â information Â supports Â the Â idea Â that Â monothiol Â Grxs Â regulate Â the Â activity Â of Â their Â targets Â by Â reversible Â glutathionylation. Â As Â discussed Â in Â chapter Â 2 Â of Â this Â thesis, Â one Â of Â my Â speculations Â on Â the Â mechanism Â Grx4-­âCir1 Â interaction Â was Â related Â to Â the Â possibility Â of Â Cir1 Â being Â post-­âtranslationally Â modulated Â by Â Grx4 Â de-­âglutathionylating Â activity. Â Future Â work Â is Â required Â to Â investigate Â the Â regulation Â of Â Cir1 Â by Â reversible Â S-­âglutathionylation. Â This Â will Â constitute Â an Â emerging Â and Â novel Â area Â of Â research; Â especially Â that Â since Â reversible Â S-­âglutathionylation Â in Â C. Â neoformans Â has Â never Â been Â reported. Â Â Â Â Â Â Â Â Â Â Â Â Â Â 164 Â Investigation Â of Â this Â phenomenon Â in Â regulation Â of Â Cir1 Â due Â to Â the Â established Â role Â of Â Cir1 Â as Â the Â Achilles Â heel Â of Â C. Â neoformans Â in Â regulation Â of Â iron Â acquisition Â and Â cellular Â functions Â including Â virulence Â factor Â expression Â adds Â to Â the Â value Â of Â this Â future Â experiment. Â 4.6.5 Â Â Â Construction Â and Â phenotypic Â characterization Â of Â GSH Â transporter Â deletion Â mutants Â Â Â Glutathione Â has Â key Â roles Â in Â a Â plethora Â of Â essential Â functions Â in Â fungi Â (I. Â Pocsi, Â et Â al., Â 2004). Â In Â general Â My Â findings Â in Â chapter Â 3 Â provide Â considerable Â insight Â and Â additional Â knowledge Â about Â glutathioneâs Â cellular Â and Â metabolic Â functions Â in Â C. Â neoformans. Â However, Â they Â are Â just Â the Â tip Â of Â the Â iceberg; Â future Â glutathione Â research Â will Â lead Â to Â novel Â discoveries Â in Â cryptococcal Â biology Â and Â pathogenesis. Â Â One Â of Â my Â key Â findings Â was Â that Â gsh2 Â mutants Â were Â acapsular, Â non-­âmelanized Â and Â did Â not Â grow Â at Â 37°C, Â and Â I Â speculated Â that Â this Â is Â most Â likely Â due Â to Â lack Â of Â GSH Â in Â these Â mutants. Â Future Â work Â is Â required Â to Â investigate Â the Â virulence Â capacity Â of Â gsh2 Â mutant Â in Â animal Â models. Â At Â present, Â the Â role Â of Â GSH Â transporters Â are Â unknown Â and Â therefore Â it Â is Â important Â to Â determine Â the Â role Â of Â these Â transporters Â in Â C. Â neoformans Â by Â generating Â deletions Â of Â their Â corresponding Â genes Â in Â the Â genome. Â Therefore Â analysis Â of Â the Â regulation, Â functions Â and Â contribution Â of Â these Â transporters Â in Â the Â WT Â and Â also Â in Â gsh2 Â mutants Â background Â is Â necessary Â to Â fully Â understand Â the Â role Â of Â GSH Â metabolism Â in Â virulence Â inside Â the Â host. Â Â 4.6.6 Â Â Investigation Â of Â Grx4 Â and Â GSH Â crosstalk Â in Â C. Â neoformans Â Â Collectively, Â my Â findings Â showed Â that Â both Â Grx4 Â and Â GSH Â have Â critical Â roles Â in Â iron Â homeostasis Â in Â C. Â neoformans. Â In Â chapter Â 4, Â I Â suggested Â possible Â crosstalk Â between Â Grx4 Â and Â GSH Â that Â influence Â functions Â in Â iron Â homeostasis Â and Â virulence Â in Â C. Â neoformans. 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Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 203 Â Appendices Â Appendix Â A Â Table Â A1: Â List Â of Â proteins Â identified Â in Â Cir1 Â binding Â assay Â identified Â by Â Mass-­âSpectrometry Â in Â an Â experiment Â performed Â by Â Dr. Â Horacio Â Bach. Â Broad Â ID Â Protein Â Domains Â found/ Â Comments Â CNAG_04158 Â Transcription Â co-­ârepressor Â -­âTup1-­âlike Â (enhances Â split, Â repression Â of Â chromatin Â by Â repression Â of Â histones Â genes) Â CNAG_03018 Â Nuclear Â protein Â (transcription Â regulation) Â -­â Â Fungal Â Zn(2)-­âCys Â (6) Â binuclear Â cluster Â -­â Â Fungal Â specific Â transcription Â factor Â domain Â CNAG_02426 Â Spliceosome Â complex Â protein Â -­â Â Tetratricopeptide Â repeat Â -­â Â HAT Â (half-­âA-­âTRP) Â important Â for Â complexes Â in Â RNA Â processing Â CNAG_03761 Â mRNA3âend-­âprocessing Â RNA Â 14 Â -­â Â PT Â repeat Â -­â Â Tetratricopeptide Â repeat Â (mediates Â protein-­â Â Â protein Â intractions) Â -­â Â Suppressor Â of Â forked Â protein Â (SUF) Â CNAG_03987 Â eRFS Â -­â Â Elongation Â factor Â Tu Â GTP Â binding Â domain Â -­â Â Elongation Â factor Â Tu Â domain Â 2 Â -­â Â Elongation Â factor Â Tu Â Câ Â terminal Â domain Â CNAG_00708 Â Pre-­âmRNA-­âsplicing Â factor Â SLT11 Â -­â Â Zinc Â finger Â C-­âX8-­âC-­âX5-­âC-­âX3 Â H Â type Â Â CNAG_06773 Â Protein Â Transport Â SEC Â 24 Â -­â Â Sec23/Sec24 Â Zinc Â Finger Â (COPII Â coated Â vesicles Â ER=>Golgi) Â Binds Â Sar1, Â Sec23/24, Â Sec13/31 Â in Â complex Â -­â Â Gelsolin Â repeat Â (binds Â to Â barbed Â ends Â of Â actin Â filaments Â and Â fibronectin) Â CNAG_02178 Â Cog3 Â -­â Â Sec Â 34-­âlike Â family Â CNAG_03582 Â pH-­âresponse Â regulator Â palA/RIM20 Â -­â Â BRO1-­âlike Â domain Â (endosomal Â targeting; Â ESCRT Â III Â subunit Â Snf7 Â binds Â to Â BRO1 Â for Â protein Â sorting Â function Â (VPS32) Â CNAG Â _03372 Â VPS-­âBRO1 Â -­â Â BRO-­âlike Â protein Â (in Â sorting Â multivesicular Â body) Â CNAG Â _04943 Â CBS Â domain-­âcontaining Â protein Â -­â Â DUF Â 21 Â -­â Â CBS Â domain Â pair Â (forms Â stable Â globular Â domains, Â binds Â adenosyl Â groups) Â Â Â Â Â Â Â Â Â Â Â Â Â Â 204 Â Broad Â ID Â Protein Â Domains Â found/ Â Comments Â CNAG_04303 Â E3 Â ubiquitin-­âprotein Â ligase Â BRE1 Â -­â Â DUF Â 1610 Â -­â Â Zn Â finger Â C3HC4 Â type Â (Ring Â finger, Â in Â ubiquitation Â pathway) Â -­â Â BRE Â E3 Â ubiquitin Â ligase Â transcriptional Â activator Â CNAG Â _05005 Â ULK/ULKProtein Â kinase Â -­â Â Tyrosine Â kinase Â -­â Â MIT Â (microtubule Â interacting Â and Â transport, Â binds Â ESCRT Â III Â endosomal Â sorting Â complexes Â CNAG_01655 Â Dynamin Â membrane Â transport Â CNAG_03319 Â Phosholipid Â binding Â protein Â -­â Â SH3 Â domain Â -­â Â SAM Â domain Â (sterile Â alpha Â motif, Â in Â protein-­âprotein Â interactions) Â -­â Â PH Â domain Â (pleckstrin Â homology, Â in Â intracellular Â signaling Â or Â cytoskeleton) Â CNAG_01185 Â Hsp70-­âlike Â protein Â -­â Â HEAT Â repeat Â -­â Â Nucleotide Â exchange Â factor Â FES1 Â CNAG_02950 Â Grx4 Â -­â Â Glutaredoxin Â domain Â CNAG_05730 Â Acyl Â protein Â thioesterase Â 1 Â -­â Â Phospholipase Â CNAG_03853 Â Small Â COPII Â coat Â GTPase Â SAR1 Â (see Â 6773) Â -­â Â ADP Â ribosylation Â factor Â family Â -­â Â MIRO-­âlike Â protein Â CNAG_01054 Â Phosphoprotein Â phosphatase Â -­â Â SSU72 Â domain Â (binds Â TFIIB, Â involved Â in Â polyadenylation Â and Â termination) Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 205 Â Figure Â A.1 Â Functional Â categorization Â of Â Cir-­â1 Â potential Â binding Â partners Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 206 Â Table Â A2: Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â grown Â under Â the Â iron-­âdeplete Â condition. Â Â Go terms* H99 Gene ID Gene annotation Fold change (Low-iron)a grx4Î vs WT oxidoreductase activity (GO:0016491) CNAG_01323 ubiquinol-cytochrome-c reductase 3.00 CNAG_01991 cytochrome c oxidase subunit V 3.00 CNAG_00462 oxidoreductase 3.01 CNAG_04417 2,4-dichlorophenoxyacetate alpha-ketoglutarate dioxygenase 3.02 CNAG_04471 FAD dependent oxidoreductase 3.03 CNAG_01229 L-mandelate dehydrogenase 3.04 CNAG_07911 streptomycin biosynthesis protein StrI 3.19 CNAG_03465 laccase 4.06 CNAG_05169 cytochrome b2 4.27 CNAG_04325 extradiol ring-cleavage dioxygenase 6.10 CNAG_02266 NADH ubiqionone oxidoreductase chain B 8.10 CNAG_01947 2,4-dienoyl-CoA reductase 8.11 CNAG_03663 L-lactate dehydrogenase 8.65 CNAG_04189 succinate dehydrogenase flavoprotein subunit 10.25 CNAG_06554 FAD dependent oxidoreductase superfamily 11.71 CNAG_01287 NADH-ubiquinone oxidoreductase 51 kDa subunit 12.11 CNAG_01470 NADH-ubiquinone oxidoreductase 24 kDa subunit 14.33 CNAG_00161 auxin-induced protein 16.05 CNAG_02489 mannitol-1-phosphate dehydrogenase 16.72 CNAG_01464 flavohemoglobin 26.45 CNAG_01577 glutamate dehydrogenase 26.67 CNAG_06556 oxidoreductase 27.35 CNAG_03629 NADH-ubiquinone oxidoreductase 32.84 CNAG_01846 flavoprotein 35.96 CNAG_00269 sorbitol dehydrogenase 38.54 CNAG_06524 conserved hypothetical protein 56.02 Â Â Â Â Â Â Â Â Â Â Â Â Â 207 Â Go terms* H99 Gene ID Gene annotation Fold change (Low-iron)a grx4Î vs WT CNAG_06976 ferric-chelate reductase -3.51 CNAG_02592 thioredoxin reductase GliT -7.83 Fe-S cluster binding (GO:0051536) CNAG_07177 NADH dehydrogenase Fe-S protein 3 3.48 CNAG_01137 aconitase 3.67 CNAG_04358 Fe-S clusters transporter ATM1 3.78 CNAG_02315 ubiquinol-cytochrome c reductase Fe-S subunit 3.84 CNAG_07908 aconitate hydratase 6.59 CNAG_04202 iron hydrogenase 15.51 CNAG_03226 succinate dehydrogenase Fe-S subunit 27.12 Metal cluster binding (GO:0051540) CNAG_01133 mitochondrial protein 3.00 CNAG_05179 ubiquinol-cytochrome c reductase complex core protein 2 3.38 CNAG_01915 ribonucleotide reductase subunit 3.52 CNAG_00022 holocytochrome-c synthase 4.22 CNAG_05909 electron transporter 5.83 CNAG_07862 fumarate reductase 8.49 CNAG_00716 electron carrier 18.22 CNAG_01464 flavohemoglobin 26.45 CNAG_05875 cytochrome c heme lyase -4.33 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 208 Â Table Â A3: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â in Â the Â grx4 Â mutant Â compared Â to Â WT Â cells Â grown Â under Â iron-­âreplete Â condition. Â Go terms* H99 Gene ID Gene annotation Fold change (High-iron)a Grx4Î vs WT oxidoreductase activity (GO:0016491) CNAG_04523 glyceraldehyde 3-phosphate dehydrogenase 3.00 CNAG_07865 ferro-O2-oxidoreductase 4.68 CNAG_06821 ferric reductase- Fre 2 5.52 CNAG_06976 ferric-chelate reductase- Fre6 5.76 CNAG_02958 ferroxidase 5.91 CNAG_06558 NADPH-ferrihemoprotein reductase 6.23 CNAG_02475 flavin-containing monooxygenase 6.61 CNAG_00269 sorbitol dehydrogenase 7.17 CNAG_01078 aldehyde dehydrogenase 7.47 CNAG_01947 2,4-dienoyl-CoA reductase 8.71 CNAG_03754 short-chain dehydrogenase/reductase SDR 9.27 CNAG_03199 FAD dependent oxidoreductase superfamily 9.48 CNAG_05316 inositol oxygenase 11.90 CNAG_02489 mannitol-1-phosphate dehydrogenase 12.36 CNAG_05258 glucose-methanol-choline oxidoreductase 12.84 CNAG_06556 oxidoreductase 99.22 CNAG_07836 NAD binding dehydrogenase family protein -6.39 CNAG_09012 cytochrome oxidase subunit II -19.10 CNAG_09010 NADH dehydrogenase subunit 2 -35.12 CNAG_02592 thioredoxin reductase GliT -3.02 transporter activity (GO:0005215) CNAG_07387 siderophore-iron transporter Str3 4.06 CNAG_02561 spermine transporter 4.59 CNAG_07751 siderophore iron transporter mirB 8.82 CNAG_00895 zinc transporter -5.7 CNAG_03398 zrt1 protein -6.21 CNAG_02959 high-affinity iron transporter 3.19 CNAG_07449 amino acid transporter 3.30 Â Â Â Â Â Â Â Â Â Â Â Â Â 209 Â Go terms* H99 Gene ID Gene annotation Fold change (High-iron)a CNAG_06329 high-affinity nicotinic acid transporter 3.74 CNAG_07448 urea transporter 3.78 CNAG_06415 copper ion transporter 3.79 CNAG_06119 spermine transporter 6.80 CNAG_03600 pheromone transporter 10.24 CNAG_06259 alpha-glucoside:hydrogen symporter 12.64 CNAG_03713 efflux protein EncT 22.59 CNAG_06557 membrane protein 23.12 CNAG_00235 ammonium transporter MEP1 31.29 CNAG_01960 efflux protein EncT 47.39 CNAG_06817 UAP1 82.41 antioxidant activity (GO:0016209) CNAG_05256 catalase 4.47 CNAG_03482 thioredoxin-dependent peroxide reductase 5.43 CNAG_03169 conserved hypothetical protein 6.69 CNAG_01138 cytochrome c peroxidase 7.31 CNAG_05015 catalase 4 8.34 CNAG_00575 catalase 3 11.14 CNAG_04268 DNA-(apurinic or apyrimidinic site lyase 3.37 Â CNAG_03637 damaged DNA binding protein 3.62 Â CNAG_01144 damaged DNA binding protein 3.64 Â Â CNAG_02549 conserved hypothetical protein 3.70 Â CNAG_02490 double-strand break repair protein MRE11 3.78 Â CNAG_03923 endonuclease 3.92 Â CNAG_00299 DNA repair protein RAD5 4.43 CNAG_06589 endoribonuclease L-PSP 4.65 CNAG_00720 recombinase 4.98 CNAG_01163 DNA supercoiling 5.01 CNAG_02771 DNA supercoiling 7.16 CNAG_07766 DNA polymerase lambda 7.38 CNAG_06724 DNA strand annealing 8.48 CNAG_07552 DNA repair protein rad8 8.78 CNAG_01465 5' flap endonuclease -3.59 CNAG_05201 conserved hypothetical protein -4.98 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 210 Â Table Â A4: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â similar Â patterns Â of Â expression Â in Â the Â grx4 Â and Â cir1 Â mutant Â mutant Â compared Â to Â WT Â cells Â grown Â under Â iron-­âdeplete Â condition. Â GO term H99 Gene ID Gene annotation Fold change (Low-iron)a grx4Î vs WT cir1Î vs WT oxidoreductase activity (GO:0016491) CNAG_04471 FAD dependent oxidoreductase superfamily 3.03 3.83 CNAG_03465 laccase 4.06 165.57 CNAG_01947 2,4-dienoyl-CoA reductase 8.11 5.33 CNAG_01078 aldehyde dehydrogenase 8.16 3.686 CNAG_06554 FAD dependent oxidoreductase superfamily 11.71 8.48 CNAG_00161 auxin-induced protein 16.05 17.89 CNAG_02489 mannitol-1-phosphate dehydrogenase 16.72 9.39 CNAG_01542 taurine catabolism dioxygenase TauD 24.47 18.45 CNAG_01577 glutamate dehydrogenase 26.67 11.21 CNAG_06556 oxidoreductase 27.35 18.69 CNAG_00269 sorbitol dehydrogenase 38.545 61.754 CNAG_06524 conserved hypothetical protein 56.02 30.61 CNAG_06764 short-chain dehydrogenase -3.50 -4.05 CNAG_00123 conserved hypothetical protein -4.02 -5.09 CNAG_05035 NADPH-adrenodoxin reductase -4.41 -5.12 CNAG_03272 conserved hypothetical protein -7.06 -3.97 transporter activity (GO:0005215) CNAG_05592 calcium-transporting ATPase 2.79 7.41 CNAG_04632 uracil permease 3.46 5.69 CNAG_05381 myo-inositol transporter 2 3.48 3.57 CNAG_04358 iron-sulfur clusters transporter ATM1 3.78 3.08 CNAG_06119 spermine transporter 4.09 3.560 CNAG_04210 sugar transporter 4.99 5.98 CNAG_06259 alpha-glucoside:hydrogen symporter 6.30 4.72 CNAG_04794 spermine transporter 6.63 3.08 CNAG_07693 high-affinity methionine permease 7.17 5.11 CNAG_00869 ATP-binding cassette 8.29 4.24 Â Â Â Â Â Â Â Â Â Â Â Â Â 211 Â GO term H99 Gene ID Gene annotation Fold change (Low-iron)a grx4Î vs WT cir1Î vs WT CNAG_05324 sugar transporter 10.29 4.28 CNAG_02561 spermine transporter 14.18 14.18 CNAG_04758 ammonium transporter 21.30 13.41 CNAG_07367 amino acid transporter 31.16 48.73 CNAG_04704 carboxylic acid transporter 34.64 4.67 CNAG_00235 ammonium transporter MEP1 42.45 27.18 CNAG_06817 UAP1 56.38 20.70 CNAG_03398 zrt1 protein -3.46 -97.47 CNAG_00979 copper uptake transporter -3.17 -10.01 CNAG_04872 mitochondrial protein -3.05 -5.80 CNAG_04015 amino acid transporter -5.87 -3.27 CNAG_04092 sugar transporter -5.41 -3.04 metal cluster binding (GO:0051540) CNAG_01915 ribonucleotide reductase subunit 3.52 2.54 CNAG_01464 flavohemoglobin 26.45 3.67 CNAG_03226 succinate dehydrogenase iron sulfur subunit 27.12 2.46 CNAG_01846 flavoprotein 35.96 4.96 CNAG_05011 mitochondrial iron uptake protein -2.24 -3.00 CNAG_01133 mitochondrial protein -3.40 -2.23 antioxidant activity (GO:0016209) CNAG_05015 catalase 4 5.754 4.22 CNAG_01138 cytochrome c peroxidase 143.91 98.48 CNAG_06287 glutathione peroxidase -2.69 -2.26 zinc ion binding GO:0008270 CNAG_04733 DNA repair protein RAD5 3.55 5.91 CNAG_01948 nuclear protein 3.98 5.35 CNAG_01890 5-methyltetrahydropteroyl triglutamate-homocysteine S-methyltransferase 4.41 4.68 CNAG_06052 galactose-1-phosphate uridylyltransferase) 5.40 4.68 CNAG_02603 early growth response protein 1 8.74 9.41 CNAG_0093 conserved hypothetical protein -6.25 -3.62 others CNAG_05303 isocitrate lyase 4.892 3.38 Â Â Â Â Â Â Â Â Â Â Â Â Â 212 Â GO term H99 Gene ID Gene annotation Fold change (Low-iron)a grx4Î vs WT cir1Î vs WT CNAG_01672 RAS1 -4.404 -3.08 CNAG_00183 alternative cyclin Pcl12 -5.00 -5.75 CNAG_01653 cytokine inducing-glycoprotein -6.66 -3.00 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 213 Â Table Â A5: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â similar Â patterns Â of Â expression Â in Â the Â grx4 Â and Â cir1 Â mutant Â mutant Â compared Â to Â WT Â cells Â grown Â under Â iron-­âreplete Â condition. Â GO term H99 Gene ID Gene annotation Fold change (High-iron)b grx4Î vs WT cir1Î vs WT Oxidoreductase CNAG_03464 Cu-oxidase 2.65 3.00 CNAG_06976 ferric-chelate reductase 3.78 3.00 CNAG_05329 NAD-binding Rossmann fold oxidoreductase family protein 3.82 3.50 CNAG_01955 alcohol dehydrogenase 3.92 15.43 CNAG_02958 ferroxidase 5.91 5.14 CNAG_01078 aldehyde dehydrogenase 7.47 5.04 CNAG_01947 2,4-dienoyl-CoA reductase 8.71 6.72 CNAG_01714 sulfonate dioxygenase 10.72 10.52 CNAG_02489 mannitol-1-phosphate dehydrogenase 12.36 14.21 CNAG_06374 malate dehydrogenase 14.04 7.65 CNAG_04325 extradiol ring-cleavage dioxygenase 15.81 7.51 CNAG_06554 FAD dependent oxidoreductase superfamily 19.92 8.35 CNAG_00735 succinate-semialdehyde dehydrogenase 63.96 16.61 CNAG_06556 oxidoreductase 99.230 38.511 antioxidant activity (GO:0016209) CNAG_0138 cytochrome c peroxidase 7.316 21.394 CNAG_05015 catalase 4 8.347 5.926 CNAG_05252 chaperone regulator 8.557 5.031 Transporter activity (GO:0005215) CNAG_04210 sugar transporter 3.103 6.461 CNAG_07448 urea transporter 3.78 4.96 CNAG_06415 copper-exporting ATPase 3.79 6.53 CNAG_07387 siderophore-iron transporter Str3 4.06 2.03 CNAG_01964 oligopeptide transporter 4.25 8.96 CNAG_07799 ABC transporter 4.38 6.75 CNAG_01118 amino acid transporter 5.13 4.30 CNAG_05324 sugar transporter 6.06 6.53 CNAG_07751 iron transporter mirB 8.82 4.14 Â Â Â Â Â Â Â Â Â Â Â Â Â 214 Â Â Â Â Â Â Â Â Â Â Â GO term H99 Gene ID Gene annotation Fold change (High-iron)b grx4Î vs WT cir1Îvs WT CNAG_06259 alpha-glucoside:hydrogen symporter 12.64 7.10 CNAG_04758 ammonium transporter 13.45 31.34 CNAG_04704 carboxylic acid transport protein 21.20 18.13 CNAG_00235 ammonium transporter MEP1 31.29 23.26 CNAG_07693 high-affinity methionine permease 45.462 3.426 CNAG_06817 UAP1 82.416 41.955 CNAG_04015 amino acid transporter -3.74 -4.18 CNAG_03398 zrt1 protein -6.21 -131.21 oothers CNAG_04733 DNA repair protein RAD5 5.548 5.050 CNAG_05838 GTPase activating protein 26.137 48.612 CNAG_05606 mitochondrial DNA repair PIF1 -2.001 -3.048 CNAG_09008 ATP synthase subunit 6 -6.070 -14.599 CNAG_07651 DEAD-box ATP-dependent RNA helicase 26 -15.514 -2.393 Â Â Â Â Â Â Â Â Â Â Â Â Â 215 Â Table Â A6: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â distinct Â patterns Â of Â expression Â in Â the Â grx4 Â or Â cir1 Â mutants Â compared Â to Â WT Â cells Â grown Â under Â iron-­âdeplete Â condition. Â Â Molecular function H99 Gene ID Gene annotation Fold change (Low-iron)a grx4Î vs WT cir1Î vs WT -holocytochrome-c synthase -heme binding -mitochondrial respiratory chain complex III; CNAG_01323 ubiquinol-cytochrome-c reductase) 3.00 -3.00 CNAG_01991 cytochrome c oxidase subunit V 3.00 -3.15 CNAG_05169 cytochrome b2 4.27 No change CNAG_00022 holocytochrome-c synthase 4.27 -3.00 CNAG_04029 cytochrome P450 -3.40 No change CNAG_05875 cytochrome c heme lyase 9.37 -3.00 Transporter CNAG_02777 phosphate transporter 7.01 -2.34 CNAG_05914 MFS maltose permease MalP -3.71 3.41 GPI anchor biosynthesis CNAG_02892 GPI mannosyltransferase 3 3 No change CNAG_05881 mannosyltransferase 3.52 No change CNAG_07527 Â alpha-­â1,6 Â mannosyltransferase Â 4.07 -2.11 CNAG_06551 carnitine O-acetyltransferase 3.00 -2.06 CNAG_02852 aminotransferase 3.83 -3.17 Others CNAG_06593 rhamnogalacturonan lyase 4.96 No change CNAG_00036 Sec14 cytosolic factor -5.38 No change Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 216 Â Table Â A7: Â Â Categories Â of Â differentially Â expressed Â genes Â based Â on Â GO Â terms Â associated Â with Â molecular Â function Â with Â distinct Â patterns Â of Â expression Â in Â the Â grx4 Â or Â cir1 Â mutants Â compared Â to Â WT Â cells Â grown Â under Â iron-­âreplete Â condition. Â Â Molecular function H99 Gene ID Gene annotation Fold change (High-iron)b grx4Î vs WT cir1Î vs WT DNA repair CNAG_00178 MUS42 5.36 no change CNAG_00299 DNA repair protein RAD5 4.43 -2.73 CNAG_00720 recombinase 4.98 -2.59 CNAG_01144 damaged DNA binding protein 3.64 -2.19 CNAG_01163 DNA supercoiling 5.01 no change CNAG_02561 spermine transporter 4.59 no change CNAG_02771 DNA supercoiling 7.16 -3.95 CNAG_02771 DNA supercoiling 3.62 no change CNAG_03923 endonuclease 3.92 no change CNAG_04220 Ku70 protein 3.57 no change CNAG_06589 endoribonuclease L-PSP 4.65 no change CNAG_06724 DNA strand annealing 8.48 -2.31 CNAG_07552 DNA repair protein rad8 8.78 no change CNAG_07766 DNA polymerase lambda 7.38 -2.06 Others CNAG_00036 Sec14 cytosolic factor -4.20 no change CNAG_04556 endoplasmic reticulum receptor 3.01 -3.00 Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 217 Â Appendix Â B Â Â Â Â Figure Â B.1: Â Deletion Â of Â GSH2 Â in Â gDNA Â extracted Â from Â 3 Â individual Â gsh2Î Â mutants Â and Â re-­âintegration Â of Â GSH2 Â in Â locus Â in Â gDNA Â extracted Â from Â gsh2Î::GSH2 Â in Â C. Â neoformans Â H99 Â background Â was Â confirmed Â by Â PCR. Â Primers Â amplified Â a Â 650 Â bp Â region Â inside Â GSH2 Â gene Â in Â C. Â neoformans. Â This Â region Â was Â amplified Â from Â gDNA Â extracted Â from Â the Â WT Â strain Â and Â and Â the Â complemented Â gsh2Î::GSH2 Â strain Â , Â no Â band Â was Â amplified Â when Â gDNA Â was Â extracted Â Â from Â 3 Â individual Â gsh2Î Â mutants Â Â Â Â
Thesis/Dissertation
eng
Microbiology and Immunology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Analysis of the roles of a monothiol glutaredoxin and glutathione synthetase in the virulence of the AIDS-associated pathogen Cryptococcus neoformans
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