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The extracellular mannoprotein Cig1 functions in iron acquisition from heme in the fungal pathogen Cryptococcus… Cadieux, Brigitte 2013

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The extracellular mannoprotein Cig1 functions in iron acquisition from heme in the fungal pathogen Cryptococcus neoformans  by  Brigitte Cadieux  B.Sc., University of Ottawa, 1998 M.Sc., McGill University, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April, 2013  © Brigitte Cadieux, 2013  Abstract Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, a life-threatening disease which affects an estimated 1 million people worldwide annually. Iron acquisition is an important but poorly understood aspect of the pathogenesis of C. neoformans. In particular, no heme uptake system has thus far been characterized in this fungus, although it has been shown to utilize heme as an iron source. A previous study identified the transcript for the extracellular mannoprotein CIG1 as the most abundant message in iron-starved cells with marked down-regulation by iron repletion, thus suggesting a possible iron-related role for Cig1. In the current study, it was found that deletion of CIG1 resulted in an extended lag phase in low iron medium with heme added as the sole iron source. Additionally, the cig1Δ mutant was more resistant to toxic heme analogs than the wild-type or complemented strains implying a role for Cig1 in heme uptake. Western blot analysis and immunofluorescence microscopy identified Cig1 at the cell surface and in association with extracellular vesicles. A heme pulldown experiment, absorbance spectroscopy and isothermal calorimetry also demonstrated that Cig1 is a potential heme-binding protein.  Importantly, deletion of CIG1 led to attenuated  virulence in a mouse infection model in absence of the high-affinity iron uptake system. More detailed studies on Cig1 revealed that the length of the lag phase of a cig1Δ mutant in low iron medium supplemented with heme was dependent on the inoculum size in support of a cell density-dependent heme acquisition system. Similarly, growth at acidic pH rescued the heme defect of a cig1Δ mutant indicating the presence of a Cig1-independent pathway at low pH. The transcription factor Rim101 may function in this pathway.  Finally, expression of a Cig1  truncated polypeptide established a role for Cig1 in secretion and cell wall integrity. In this context, a strain overexpressing CIG1 produced an enlarged capsule and secreted more extracellular vesicles than the wild-type strain. Overall, the data presented in this thesis have ii  contributed to a better understanding of heme uptake and secretion in C. neoformans and the results may facilitate the development of new strategies to treat cryptococcosis.  iii  Preface Relative contributions of all collaborators: Some of the work presented in this thesis resulted from collaborative efforts. The people who have contributed to the different experiments have been identified in each of the respective sections of the Materials and Methods. The details of their contributions are as follows:  Most of the work presented in Chapter 2 and a portion of the work from Chapter 3 have been published in the manuscript entitled “The mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans” in the Journal of Infectious Diseases on January 15th, 2013. Tian Lian and Carmelo Biondo contributed to this study by preparing some of the strains as indicated. Dr. Guanggan Hu constructed the E. coli strain expressing Cig1::GST and purified the recombinant fusion protein.  Joyce Wang  performed the virulence assay and determined the fungal loads in mouse tissue, while Dr. A. Louise Creagh did the isothermal titration calorimetry experiments. Victor Liu provided some technical assistance and Dr. Michael E. Murphy suggested the idea to test the toxicity of noniron metalloporphyrins as an indicator for heme uptake. I was responsible for all the remaining experiments and data collection. The manuscript was written by myself and my supervisor, Dr. James W. Kronstad, except for some details about the collaborative work included in the Materials and Methods, which were written by Dr. Guanggan Hu, Joyce Wang and Dr. A. Louise Creagh, respectively.  Another publication with the preliminary title “The fungal pathogen Cryptococcus neoformans casts a heme binding net”, is in preparation for publication of the rest of the work  iv  included in Chapter 3. Tian Lian, Carmelo Biondo and Dr. Emma J. Griffiths prepared some of the strains used in the study, as indicated, while Dr. A. Louise Creagh performed the isothermal titration calorimetry experiments. I was responsible for all the remaining experiments and data collection. The manuscript will be written by myself and my supervisor, Dr. James W. Kronstad.  Finally, a portion of the work presented in Chapter 4 was previously started by Tian Lian. Specifically, Tian had constructed some of the strains, as indicated, and tested the susceptibility of these strains to secretion inhibitors and agents that challenge the cell wall. All the other data included in this chapter resulted from my work.  Publications arising from graduate work: -Cadieux B, Lian T, Hu G, Wang J, Biondo C, Teti G, Liu V, Murphy ME, Creagh AL, Kronstad JW. (2013). The mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans. J Infect Dis. (Epub ahead of print). -Hu G, Caza M, Cadieux B, Chan V, Liu V, Kronstad J. (2013). Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence. Infect Immun. 81, 292-302. -Kim J, Cho YJ, Do E, Choi J, Hu G, Cadieux B, Chun J, Lee Y, Kronstad JW, Jung WH. (2012). A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs. Fungal Genet Biol. 49, 955-66. -Kronstad J, Saikia S, Nielson ED, Kretschmer M, Jung W, Hu G, Geddes JM, Griffiths EJ, Choi J, Cadieux B, Caza M, Attarian R. (2012). Adaptation of Cryptococcus neoformans to mammalian hosts: integrated regulation of metabolism and virulence. Eukaryot Cell. 11, 109-18.  v  -Kronstad JW, Attarian R, Cadieux B, Choi J, D'Souza CA, Griffiths EJ, Geddes JM, Hu G, Jung WH, Kretschmer M, Saikia S, Wang J. (2011). Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box. Nat Rev Microbiol. 9, 193-203.  University of British Columbia Ethics Board approval: The protocol for the virulence assays was approved by the University of British Columbia Committee on Animal Care (Certificate Number: A08-0586).  vi  Table of contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of contents ......................................................................................................................... vii List of tables................................................................................................................................ xiv List of figures ................................................................................................................................xv List of abbreviations ................................................................................................................ xviii Acknowledgements ......................................................................................................................xx Dedication .................................................................................................................................. xxii Chapter 1. Introduction ...............................................................................................................1 1.1. Cryptococcus neoformans .....................................................................................................1 1.1.1. Cryptococcosis ..............................................................................................................1 1.1.2. Virulence traits..............................................................................................................2 1.1.2.1. Polysaccharide capsule.............................................................................................3 1.1.2.2. Melanin ....................................................................................................................4 1.1.2.3. Growth at 37°C ........................................................................................................5 1.1.2.4. Other virulence traits ................................................................................................6 1.1.3. Secretion in C. neoformans...........................................................................................8 1.1.3.1. Extracellular vesicles ...............................................................................................9 1.1.3.2. Secreted mannoproteins .........................................................................................12 1.2. Iron and microbial pathogenesis ..........................................................................................14 1.2.1. Iron uptake in bacteria ................................................................................................14 vii  1.2.1.1. Gram-negative bacteria ..........................................................................................15 1.2.1.2. Gram-positive bacteria ...........................................................................................17 1.2.1.3. Heme acquisition in bacteria ..................................................................................18 1.2.2. Iron uptake in C. neoformans and other fungi ............................................................21 1.2.2.1. The reductive high-affinity iron uptake pathway ...................................................21 1.2.2.2. Low-affinity iron uptake pathway..........................................................................22 1.2.2.3. Transport of siderophore-bound iron .....................................................................23 1.2.2.4. Acquisition of iron from heme ...............................................................................25 1.2.3. Regulation of iron uptake ...........................................................................................26 1.3. Cytokine-inducing glycoprotein (CIG1) .............................................................................29 1.4. Rationale and aims of study ................................................................................................30 1.4.1. Hypotheses ..................................................................................................................30 1.4.2. Research objectives ....................................................................................................30 Chapter 2. Role of Cig1 in heme acquisition in C. neoformans var. grubii............................32 2.1. Introduction .........................................................................................................................32 2.2. Materials and Methods ........................................................................................................34 2.2.1. Strains and growth conditions ....................................................................................34 2.2.2. In silico protein analysis .............................................................................................36 2.2.3. Construction of strains ................................................................................................36 2.2.3.1. Construction of the cig1Δ mutant and complemented strains ................................38 2.2.3.2. Construction of the cig2Δ and cig1Δ cig2Δ mutants, and complemented strains ..39 2.2.3.3. Construction of the cig3Δ and cig1Δ cig2Δ cig3Δ mutants ...................................40  viii  2.2.3.4. Construction of the cig1Δ cfo1Δ mutant and the cig1Δ cfo1Δ CIG1 complemented strain ........................................................................................................................41 2.2.3.5. Construction of an HA::Cig1 fusion allele.............................................................42 2.2.3.6. Construction of an E. coli strain expressing Cig1::GST ........................................43 2.2.4. Purification of the recombinant Cig1::GST fusion protein ........................................43 2.2.5. Quantitative real time PCR .........................................................................................44 2.2.6. Protein extraction and Western blot analysis..............................................................44 2.2.7. Vesicle preparation .....................................................................................................46 2.2.8. Fluorescence microscopy............................................................................................47 2.2.9. Uptake of metalloporphyrins ......................................................................................48 2.2.10. Heme binding assays ..................................................................................................48 2.2.11. Mass spectrometry ......................................................................................................49 2.2.12. Absorption spectrophotometry ...................................................................................50 2.2.13. Isothermal titration calorimetry experiments..............................................................50 2.2.14. Virulence assays and determination of fungal loads in mouse tissue .........................51 2.3. Results .................................................................................................................................52 2.3.1. Characterization and expression of the CIG1 gene in C. neoformans ........................52 2.3.2. Cig1 is required for heme utilization ..........................................................................56 2.3.3. CIG2 and CIG3 do not contribute to iron utilization ..................................................60 2.3.4. The reductive high-affinity iron uptake system does not contribute to Cig1-mediated heme utilization ..........................................................................................................61 2.3.5. Cig1 is involved in heme uptake ................................................................................63 2.3.6. Localization of Cig1 ...................................................................................................66  ix  2.3.7. Analysis of Cig1 binding to heme ..............................................................................72 2.3.8. Identification of other potential heme-binding proteins in C. neoformans ................75 2.3.9. Cig1 is required for the full virulence of C. neoformans ............................................79 2.4. Discussion............................................................................................................................82 Chapter 3. Further characterization of heme uptake in C. neoformans ................................90 3.1. Introduction .........................................................................................................................90 3.2. Material and Methods ..........................................................................................................92 3.2.1. Strains and growth conditions ....................................................................................92 3.2.2. Construction of strains ................................................................................................93 3.2.2.1.Construction of the galactose-regulated CIG1 strains ............................................94 3.2.3. Quantitative RT-PCR..................................................................................................95 3.2.4. Aggregate formation ...................................................................................................96 3.2.5. Preparation of conditioned media ...............................................................................96 3.2.6. Isolation of exopolysaccharides ..................................................................................96 3.2.7. Absorption Spectrophotometry ...................................................................................97 3.2.8. Isothermal titration calorimetry experiments..............................................................97 3.3. Results .................................................................................................................................99 3.3.1. Prior adaptation of the cig1Δ mutant to growth in LIM supplemented with heme does not alter subsequent growth in presence of this iron source .......................................99 3.3.2. The role of Cig1 in heme uptake is pH dependent ...................................................100 3.3.3. Rim101 also contributes to heme acquisition ...........................................................104 3.3.4. Rim101 plays a role in iron and heme utilization at physiological and alkaline pH ..................................................................................................................................107  x  3.3.5. Cells form aggregates in the early stages of growth in LIM supplemented with heme ..................................................................................................................................109 3.3.6. Heme uptake via the Cig1-independent pathway is dependent on cell density ........113 3.3.7. Addition of conditioned media allowed the cells to grow faster ..............................115 3.3.8. Addition of exogenous polysaccharides allowed the cells to grow faster ................118 3.3.9. Binding of GXM to heme .........................................................................................121 3.4. Discussion .........................................................................................................................124 Chapter 4. Cig1 plays a role in heme utilization, as well as in secretion and maintenance of cell wall integrity in C. neoformans var. neoformans. ............................................................130 4.1. Introduction .......................................................................................................................130 4.2. Materials and Methods ......................................................................................................133 4.2.1. Strains and growth conditions ..................................................................................133 4.2.2. Construction of strains ..............................................................................................133 4.2.2.1.Construction of the cig1Δ mutant and complemented strains ...............................135 4.2.2.2.Construction of the cig1DΔstop strain ..................................................................137 4.2.2.3.Construction of the galactose-regulated CIG1D strain .........................................138 4.2.3. Quantitative RT-PCR................................................................................................138 4.2.4. Capsule assay ............................................................................................................139 4.2.5. Growth in LIM supplemented with heme .................................................................139 4.2.6. Plate assays ...............................................................................................................140 4.2.7. In silico analysis .......................................................................................................140 4.2.8. Total protein secretion assay ....................................................................................141 4.2.9. Protease assay ...........................................................................................................141  xi  4.2.10. Secretion of extracellular vesicles ............................................................................141 4.3. Results ...............................................................................................................................143 4.3.1. Complementation of the cig1DΔ mutation resulted in an enlarged capsule and overexpression of CIG1 ............................................................................................143 4.3.2. Construction of additional strains with mutated alleles of CIG1 in C. neoformans var. neoformans ...............................................................................................................145 4.3.3. Cig1 also plays a role in heme acquisition in C. neoformans var. neoformans ........145 4.3.4. Disruption of CIG1 results in increased susceptibility to secretion inhibitors in C. neoformans var. neoformans, but not in var. grubii .................................................146 4.3.5. Disruption of CIG1 results in a defect in cell wall integrity in C. neoformans var. neoformans but not in var. grubii .............................................................................148 4.3.6. Evidence that expression of a truncated Cig1 peptide influenced susceptibility to secretion inhibitors, agents that challenge the cell wall, heat and osmotic stress ....150 4.3.7. Cig1 may influence the export of total proteins, proteases and extracellular vesicles ..................................................................................................................................154 4.4. Discussion .........................................................................................................................159 Chapter 5. Discussion ...............................................................................................................163 5.1. Heme uptake ......................................................................................................................163 5.2. A novel Cig1-dependent heme uptake pathway in C. neoformans ...................................166 5.3. Multiple heme uptake pathways in C. neoformans ...........................................................168 5.4. Expression of the Cig1 truncated polypeptide alters secretion and leads to loss of cell wall integrity ..............................................................................................................................173 5.5. Key areas for future work..................................................................................................175  xii  5.5.1. Cig1 binding to heme................................................................................................175 5.5.2. Identifying the mechanisms involved in Cig1-dependent heme uptake pathway ....175 5.5.3. Identifying other proteins involved in the Cig1-independent heme uptake pathway(s) ..................................................................................................................................177 5.5.4. Identifying the QS-like molecule involved in cell density-dependent heme uptake ..................................................................................................................................178 5.5.5. Further characterization of the link between Cig1 and secretion .............................179 5.6. Conclusion .........................................................................................................................181 References ...................................................................................................................................182 Appendices ..................................................................................................................................211 Appendix A ............................................................................................................................211 Appendix B ............................................................................................................................212 Appendix C ............................................................................................................................214 Appendix D ............................................................................................................................217 Appendix E ............................................................................................................................218 Appendix F ............................................................................................................................219  xiii  List of tables  Table 2.1. Strains used in this study ..............................................................................................35 Table 2.2. Primers used for strain construction .............................................................................37 Table 2.3. Primers used for quantitative RT-PCR .........................................................................44 Table 2.4. Potential secreted heme-binding proteins .....................................................................78 Table 3.1. Strains used in this study ..............................................................................................93 Table 3.2. Primers used for strain construction .............................................................................93 Table 3.3. Primers used for quantitative RT-PCR .........................................................................95 Table 4.1. Strains used in this study ............................................................................................133 Table 4.2. Primers used for strain construction ...........................................................................134 Table 4.3. Primers used for quantitative RT-PCR .......................................................................139 Table 4.4. Primers used for amplifying and sequencing the DNA ..............................................140 Table 4.5. Summary of phenotypes observed with the different strains of C. neoformans var. neoformans ...................................................................................................................................158 Table C.1. Potential heme-binding proteins identified in the culture supernatants of cells grown in LI-YNB ....................................................................................................................................214 Table C.2. Proteins binding to agarose beads identified in the culture supernatants of cells grown in LI-YNB ....................................................................................................................................215 Table C.3. Potential heme-binding proteins identified in the culture supernatants of cells grown in LI-YNB + FeCl3 ......................................................................................................................216 Table C.4. Proteins binding to agarose beads identified in the culture supernatants of cells grown in LI-YNB + FeCl3 ......................................................................................................................216  xiv  List of figures  Figure 1.1 Secretion of virulence traits via extracellular vesicles in C. neoformans....................11 Figure 2.1 Confirmation of mutants...............................................................................................42 Figure 2.2 Cig1 is an iron-regulated mannoprotein in C. neoformans .........................................53 Figure 2.3 Cig1 is conserved in the different varieties of C. neoformans ....................................54 Figure 2.4 CIG2 and CIG3 are putative paralogs of CIG1 ............................................................55 Figure 2.5 Expression of CIG1 ......................................................................................................56 Figure 2.6 Cig1 is required for growth in LIM supplemented with heme ....................................58 Figure 2.7 Further examination of growth in LIM supplemented with heme ...............................59 Figure 2.8 Cig2 and Cig3 are not required for growth in LIM supplemented with heme .............61 Figure 2.9 Cfo1 is required for growth in LIM supplemented with FeCl3 but not with heme .....62 Figure 2.10 Cig1 influences susceptibility to non-iron MPs ........................................................65 Figure 2.11 Ga-PPIX has fungistatic activity against C. neoformans ...........................................66 Figure 2.12 Production of anti-Cig1 antibodies ............................................................................68 Figure 2.13 Cig1 is found at the cell surface ................................................................................70 Figure 2.14 Cig1 is associated with extracellular vesicles ...........................................................72 Figure 2.15 Vesicles containing Cig1 and purified recombinant Cig1 bind to heme-agarose beads ........................................................................................................................................................73 Figure 2.16 Recombinant Cig1::GST binds to heme .....................................................................75 Figure 2.17 CIG1 is required for virulence in absence of CFO1...................................................80 Figure 2.18 Determination of fungal loads in mice tissue .............................................................81 Figure 3.1 Galactose-regulated CIG1 strains .................................................................................95  xv  Figure 3.2 Prior adaptation does not rescue the growth defect of the cig1Δ mutant in LIM supplemented with heme..............................................................................................................100 Figure 3.3 Cig1 is not required for growth in LIM supplemented with heme at acidic pH ........102 Figure 3.4 The role of Cig1 in heme uptake is highly dependent on pH .....................................104 Figure 3.5 Rim101 also contributes to heme utilization ..............................................................105 Figure 3.6 Rim101 makes a Cig1-independent contribution to heme uptake..............................107 Figure 3.7 Rim101 plays a role in heme uptake at physiological pH and in iron uptake at alkaline pH.................................................................................................................................................109 Figure 3.8 The cells and heme form aggregates during growth ..................................................112 Figure 3.9 Cig1-independent heme uptake is dependent on cell density .....................................115 Figure 3.10 Addition of CM allows the cells to grow faster .......................................................117 Figure 3.11 Addition of exogenous polysaccharides allows the cells to grow faster ..................121 Figure 3.12 GXM does not appear to bind heme .........................................................................123 Figure 4.1 Construction and confirmation of mutants .................................................................135 Figure 4.2 Complementation of the cig1DΔ mutation results in enlarged capsule and overexpression of CIG1 ...............................................................................................................144 Figure 4.3 Cig1 in C. neoformans var. neoformans is required for growth in LIM supplemented with heme .....................................................................................................................................146 Figure 4.4 Disruption of CIG1 results in susceptibility to secretion inhibitors in C. neoformans var. neoformans but not in var. grubii .........................................................................................148 Figure 4.5 Disruption of CIG1 results in susceptibility to agents that challenge the cell wall in C. neoformans var. neoformans but not in var. grubii .....................................................................150 Figure 4.6 The cig1DΔ disruption mutants express a truncated polypeptide ..............................151  xvi  Figure 4.7 Expression of a Cig1 truncated polypeptide results in increased susceptibility to secretion inhibitors, agents that challenge the cell wall, heat and osmotic stress ........................154 Figure 4.8 Cig1 influences secretion of proteins and extracellular vesicles ................................156 Figure 5.1. Proposed model for different heme uptake pathways in C. neoformans ..................170 Figure A.1. The HA::Cig1 strain has a growth defect when grown in LIM supplemented with heme .............................................................................................................................................211 Figure B.1. Cig1 does not appear to bind polysaccharides ..........................................................213 Figure D.1. Cas1 may be involved in iron acquisition ...............................................................217 Figure E.1. Elaboration of the capsule by additional cig1DΔ mutants in C. neoformans var. neoformans ...................................................................................................................................218 Figure F.1. All cig1DΔ mutants in C. neoformans var. neoformans grow more slowly than wildtype in LIM supplemented with heme .........................................................................................219  xvii  List of abbreviations 3HAA AAs ABC transporters AIDS ATP BFA BLAST BSA cDNA CFEM Cfo1 Cft1 CFU CIG1 Cir1 CM Ct C-terminal Da DNA DOMON DTT EDTA ER ESCRT Fre Fur GalXM Ga-PPIX GFP GPI GST GXM HA HEPES HIS HIV HYG IgG IL-10 IPTG ITC Kd L-DOPA  3-hydroxyanthranilic acid amino acids ATP-binding cassette transporter acquired immune deficiency syndrome adenosine triphosphate brefeldin A basic local alignment search tool bovine serum albumin complementary deoxyribonucleic acid eight cysteine-containing domain Cryptococcus ferroxidase 1 C. neoformans iron permease 1 colony forming units cytokine-inducing glycoprotein 1 Cryptococcus iron regulator 1 conditioned media cycle threshold carboxyl-terminal Dalton deoxyribonucleic acid dopamine β-monooxygenase N-terminal dithiothreitol ethylenediaminetetraacetic acid endoplasmic reticulum endosomal sorting complex required for transport ferric iron reductases ferric uptake repressor galactoxylomannan gallium protoporphyrin green fluorescent protein glycosylphosphatidylinositol glutathione S-transferase glucuronoxylomannan hemagglutinin 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid histidine human immunodeficiency virus hygromycin immunoglobulin G interleukin-10 isopropyl beta-D-1-thiogalactopyranoside isothermal titration calorimetry dissociation constant L-3,4-dihydroxyphenylalanine xviii  LIM LI-YNB Mn-PPIX MPs mRNA MS n NAT NC NEAT NEM NEO NMR N-terminal NY PBS PCR Plb1 PSMs QS Qsp1 RNA RNA-seq rRNA RT-PCR SD SDS SDS-PAGE Ser/Thr Sit1 Sod1 sp. TBS TGF-β TLC TNF-α TTC U.S.A. UV var. WT YNB YPD YPG  low iron medium low iron yeast nitrogen base manganese protoporphyrin metalloporphyrins messenger ribonucleic acid mass spectrometry number of replicates nourseothricin North Carolina near iron transporter N-ethyl maleimide neomycin nuclear magnetic resonance amino-terminal New York phosphate-buffered saline polymerase chain reaction phospholipase B peptide spectrum match quorum sensing quorum sensing peptide 1 ribonucleic acid ribonucleic acid sequencing ribosomal ribonucleic acid real time polymerase chain reaction standard deviation sodium dodecyl sulfate sodium dodecyl sulfate polyacrylamide gel electrophoresis serine/threonine siderophore iron transporter superoxide dismutase species tris-buffered saline transforming growth factor beta thin layer chromatography tumor necrosis factor-alpha 2,3,5-triphenyltetrazolium chloride United States of America ultraviolet variety wild-type yeast nitrogen base yeast extract peptone dextrose yeast extract peptone galactose  xix  Acknowledgements I would like to thank all the current and past members of the Kronstad lab, including Anita Sham, Iris Liu, Po-Yan Cheng, Melanie Scofield, Won Hee Jung, Guanggan Hu, Emma Griffiths, Sanjay Saikia, Joyce Wang, Matthias Kretschmer, Jennifer Geddes, Rodgoun Attarian, Horacio Bach, Jaehuyk Choi, Melissa Caza, Erik Nielson, Scott Lambie, Cletus D’Souza, and Debora de Oliveira. You guys have been my colleagues and my friends, but most importantly, I consider you all to be part of my Vancouver family. You were by my side to celebrate the best of times and you were there to support me through the worst of times. I will forever be grateful to all of you. The times we shared together, inside and outside the lab, will always bring back fond memories. I will miss you dearly. Special thanks to Guanggan Hu for being so helpful, and for providing me with excellent advice and encouragement. Thank you for being patient with me over the past several years and for answering my many questions. I would like to extend my utmost appreciation to my supervisor Dr. Jim Kronstad for giving me the opportunity to work on such an exciting project. The continuous support and advice that you have offered me over the years have been instrumental in my scientific development. I would also like to acknowledge the members of my supervisory committee, Dr. Rachel Fernandez, Dr. John Smit, and Dr. Christopher Loewen for challenging me and providing helpful suggestions. I offer my gratitude to Tian Lian for starting some of the work presented in this thesis, as well as Catherine Gaudin, Sarah Thackray, Louise Creagh, Victor Liu, Lakshmi Miller-Vedam,  xx  Chun Mei Li, Joyce Wang, and Nikolay Stoynov for technical assistance. I would also like to thank Dr. Michael Murphy for his expertise and his insight on the work involving heme binding. Thanks to Jan Schwerdtner for driving me to work so many times late at night or on the weekend to measure the growth of my cells. Last but not least, I would like to thank my family and friends from back home who always believed in me. You guys have been by my side every step of the way. You have encouraged me to push harder and aim higher. You will never know how much your love and support was appreciated throughout this journey. I am very lucky to be surrounded by such amazing people.  xxi  Dedication  To my parents who have inspired me in more than one way.  À mes parents qui m’ont inspirée à plusieurs niveaux.  xxii  Chapter 1. Introduction 1.1. Cryptococcus neoformans 1.1.1. Cryptococcosis C. neoformans is an encapsulated fungal pathogen which causes cryptococcosis. The yeast cells and spores of C. neoformans are ubiquitous in the environment and are often associated with soil, vegetative debris or avian guano (Buchanan and Murphy, 1998; Govender et al., 2011). Cryptococcosis, a life-threatening disease, is acquired by inhaling C. neoformans spores which then cause an initial infection of the lungs that manifests itself as pneumonia (Williams et al., 2011). The infection can be cleared by the host’s immune system or alternatively can become a latent infection. If the host’s immunity is compromised, the dormant cells can become active and proliferate. Eventually, the cells cross the blood-brain barrier and progress to the central nervous system and brain resulting in meningoencephalitis (Dromer and Levitz, 2011). C. neoformans is the most common cause of fungal meningoencephalitis (Perfect and Casadevall, 2011). Although the pathogen can cause disease in immunocompetent individuals, it has a propensity to attack immunocompromised individuals, and cryptococcal meningitis is one of the most frequent causes of death among persons with AIDS (Warkentien and CrumCianflone, 2010; Park et al., 2011). The incidence of cryptococcosis exploded globally with the AIDS epidemic and as a consequence of organ transplantation and the use of corticosteroids. The highest incidence is found in sub-Saharan regions of Africa, which also have the greatest burden of HIV on a global scale (Park et al., 2011). The worldwide burden of cryptococcosis is estimated at 1 million cases per year with approximately 625,000 deaths, although fatality rates of cryptococcal meningitis can approach 100% in some areas (Park et al., 2009).  1  Antifungal drugs, such as amphotericin B and fluconazole, suppress but do not necessarily cure cryptococcosis and can be toxic to the human host (Jarvis et al., 2011). Combination antifungal therapy is a more successful approach in the treatment of cryptococcosis; however, access to the drugs is often limited, especially in many parts of the developing world. C. neoformans is classified into three different varieties, named variety grubii (serotype A), var. gattii (serotypes B and C), and var. neoformans (serotype D), based on antigenic differences of the polysaccharide capsule and distinct DNA fingerprinting patterns (Bhattacharjee et al., 1984; Janbon and Doering, 2011; Kwon-Chung et al., 2011).  C. neoformans var. grubii  represents the major clinical serotype, while C. neoformans var. gattii is emerging as a new and distinct pathogenic species. Since 1999, an outbreak of cryptococcosis has been ongoing on Vancouver Island infecting predominantly immunocompetent individuals (Stephen et al., 2002). The outbreak has also been spreading to the mainland and the Pacific Northwest area of the U.S.A. (Kidd et al., 2004; MacDougall et al., 2007; Bartlett et al., 2008). In British Columbia alone, 281 infections were reported from 1999-2010 and 19 individuals died from the disease (Bartlett et al., 2012). In the U.S.A., 60 cases were reported between 2004 and 2010 resulting in 20 deaths (Centers for Disease Control and Prevention, 2010).  In this context, a detailed  understanding of the virulence mechanisms for C. neoformans is needed to underpin new strategies to treat cryptococcosis.  1.1.2. Virulence traits The identification of virulence factors associated with C. neoformans and the corresponding genes which encode the virulence factors is a very complex task. It is likely that many subtle virulence traits are required for the pathogen’s ability to cause disease. So far, three  2  major virulence traits, as well as several secreted sugars and enzymes, have been identified to play a role in pathogenesis.  1.1.2.1. Polysaccharide capsule The key virulence trait of C. neoformans is its polysaccharide capsule which attaches to and extends from the cell wall. The capsule is composed primarily of two polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), as well as mannoproteins, described in section 1.1.3.2 (Reiss et al., 1985; Murphy et al., 1988; Bose et al., 2003; Zaragoza et al., 2009). GXM, which makes up 90% of the capsule mass, is a long, unbranched polymer (1,700-7,000 kDa) composed of alpha-1,3-linked mannose residues which are O-acetylated and decorated with xylose and glucuronic acid (McFadden et al., 2006; Zaragoza et al., 2009). GalXM is smaller than GXM (275,000 Da) and has an alpha-1,6-galactan backbone with xylosylated side chains of mannose and galactose (Cherniak et al., 1980; Zaragoza et al., 2009). The capsule of C. neoformans is a highly dynamic structure: physiological conditions (i.e., low iron, 5% CO2) induce capsule synthesis and the cell can modulate capsule thickness in response to the host tissue environment, i.e., the capsule is larger on fungal cells in the brain (Granger et al., 1985; Vartivarian et al., 1993; Zaragoza et al., 2003). It has also been shown that acapsular strains are avirulent in a mouse model of cryptococcosis (Kwon-Chung and Rhodes, 1986; Chang and Kwon-Chung, 1994; 1998; 1999; Chang et al., 1996). The capsule is extremely important for evasion of the host immune system. For example, the capsule can protect the cells from phagocytosis (Garcia-Rodas and Zaragoza, 2012). Interestingly, large amounts of capsular polysaccharides are also shed from the fungal cell into the extracellular environment and can thereby deplete the complement proteins of the host (Bose et al., 2003; Zaragoza et al., 2009). In  3  fact, GXM is the major cryptococcal component in body fluids of infected patients and can be a measure of the severity of the disease (Vecchiarelli, 2000). Additionally, the capsule interferes with the host immune response by altering antigen presentation, inhibiting leukocyte migration to infected tissues, and modulating cytokine production (Vecchiarelli, 2000; Zaragoza et al., 2009; Vecchiarelli et al., 2011). The capsule can also scavenge reactive oxygen intermediates and confer resistance to the cells against oxidative stress (Zaragoza et al., 2008). Therefore, if fungal cells are engulfed, the capsule allows C. neoformans to survive intracellularly and replicate (Tucker and Casadevall, 2002).  Although there has been steady progress in  understanding the mechanisms of capsule biosynthesis in the past decade, important questions remain unanswered, including where and how is the capsule synthesized.  1.1.2.2. Melanin Another virulence trait identified for C. neoformans is its ability to synthesize melanin. The enzyme laccase, which is localized in the cell wall, converts diphenolic compounds to melanin, a dark brown or black pigmented polymer, through a series of reactions and in combination with auto-oxidation (Polacheck et al., 1982; 1988; 1991; Zhu et al., 2001; Waterman et al., 2007; Eisenman and Casadevall, 2012). The detailed chemical structure of melanin is not known but it has been shown to have a granular structure, and melanin granules are deposited in the inner layer of the C. neoformans cell wall (Eisenman et al., 2005; Eisenman and Casadevall, 2012). Melanin production in this organism necessitates the availability of exogenous diphenolic substrates (e.g., catecholamines) and is regulated by glucose, iron and copper levels, as well as by temperature and the available nitrogen source (Nurudeen and Ahearn, 1979). Interestingly, large amounts of natural catecholamines are found in the brain,  4  which may explain the neurotropism of C. neoformans (Polacheck et al., 1982; Nosanchuk et al., 2000).  Laccase expression and melanin synthesis have been correlated with virulence in  numerous studies (Kwon-Chung et al., 1982; Rhodes et al., 1982; Polacheck and Kwon-Chung, 1988; Salas et al, 1996; Zhu and Williamson, 2004; Trofa et al., 2011). Specifically, it has been shown that the enzyme laccase is required for dissemination of C. neoformans from the lungs to other organs, and cells which are unable to synthesize melanin are generally attenuated for virulence (Kwon-Chung et al., 1982; Salas et al., 1996; Gomez and Nosanchuk, 2003; Noverr et al., 2004). Additionally, melanin inhibits phagocytosis of fungal cells and interferes with the normal function of phagocytic cells (Wang et al., 1995). For example, the host immune cells kill foreign cells by exposing them to free radicals, which will react with DNA and proteins leading to death of the cells. Melanin protects C. neoformans from oxidative and nitrosative challenges by macrophages by reacting with free radicals to neutralize them (Wang et al., 1995). Melanin also provides an increased negative charge to the cryptococcal cells which may explain, at least in part, the role of melanin in phagocytosis inhibition (Blasi et al., 1995; Wang et al., 1995; Nosanchuk and Casadevall, 1997). Furthermore, melanin modulates the immune system by altering cytokine levels and activating the complement system (Rosas et al., 2002; Mednick et al., 2005; Trofa et al., 2011).  1.1.2.3. Growth at 37ºC To cause infection in humans and animals, C. neoformans must be able to proliferate at 37ºC.  While it may seem obvious, growth at 37ºC is an important virulence trait for C.  neoformans as most fungi do not grow optimally above 30ºC. Several functions have been shown to influence growth at elevated temperature, including components of signaling pathways.  5  For example, Odom et al. (1997) showed that an intact calcineurin pathway is essential for thermotolerance. Specifically, a copy of the gene encoding the catalytic A subunit of calcineurin is required for growth at 37ºC (Odom et al., 1997). The CNB1 gene encoding the calcineurin regulatory subunit is also required for thermotolerance (Fox et al., 2001). Calcineurin is a Ca2+/calmodulin-activated serine/threonine-specific phosphatase involved in stress response and is structurally and functionally conserved from yeast to humans (Hemenway and Heitman, 1999). Perfect (2006) listed 19 additional genes that have been validated to be necessary for high-temperature growth and pathogenesis of C. neoformans. Two themes emerged from this list: the apparent protective properties of the sugar, trehalose, and the requirement for antioxidant protection for growth at human body temperature. Both trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2) were found to be required for growth at 37ºC and for virulence of C. neoformans (Petzold et al., 2006). Based on studies in Saccharomyces cerevisiae, it is thought that accumulation of trehalose can help prevent denaturation of proteins and aid heat-shock protein chaperones in renaturation by preventing aggregation of denatured proteins (Crowe, 2007). Additionally, the superoxide dismutase (Sod2) and the thiol-specific antioxidant (Tsa1) are both required for resistance to oxidative and nitrosative stresses and are necessary for high-temperature growth of C. neoformans (Giles et al., 2005; Missall et al., 2004).  1.1.2.4. Other virulence traits Other possible virulence traits include production of mannitol, superoxide dismutase, protease, phospholipase B, urease and acid phosphatase. However, these virulence traits have not been characterized to the same extent as capsule and melanin production.  6  It is thought that production of large amounts of mannitol by the enzyme mannitol dehydrogenase increases the osmolality of C. neoformans’ extracellular environment which may prevent oxidative damage to the fungal cells while inside the host (Perfect et al., 1996; Karkowska-Kuleta et al, 2009). Additionally, large amounts of mannitol may lead to brain edema and facilitate development of meningoencephalitis. Production of mannitol may also help cells resist other environmental stresses including heat stress and osmotic stress (Chaturvedi et al, 1996). C. neoformans also secretes multiple extracellular enzymes thought to play a role in virulence, including a role in nutrition of the fungal cell and damage to the host tissue. Secreted proteases have been demonstrated to degrade host tissues and destroy immunologically important proteins produced by the host in other bacterial and fungal pathogens (Loomes et al., 1993; Jansen et al., 1995; Kaminishi et al., 1995).  In C. neoformans,  extracellular proteinase activity has been detected and it was shown that the fungal pathogen could degrade immunologically important proteins, including immunoglobulin G and complement factor 5 (Chen et al., 1996). Phospholipase B is involved in fungal invasion by disrupting mammalian cell membranes to allow C. neoformans to penetrate host tissues (Djordjevic, 2010). For example, purified phospholipase B (Plb1) hydrolyses lung surfactant and facilitates adherence of C. neoformans to lung epithelial cells (Santangelo et al., 1999; Ganendren et al., 2006). Plb1 also facilitates the entry of C. neoformans into lung interstitial macrophages. Additionally, Plb1 is essential for dissemination of C. neoformans from the lung to the brain via the blood, and may also be required for cryptococcal cells to exit macrophages and thereby avoid killing by the macrophage’s immune defense mechanisms (Cox et al., 2001; Noverr et al., 2003; Santangelo et al., 2004; Djordjevic, 2010).  Cox et al. (2001) demonstrated that disruption of the gene  7  encoding phospholipase B resulted in attenuation of virulence in mouse and rabbit models of infection, and the mutant exhibited a growth defect in a macrophage-like cell line. Superoxide dismutase is thought to protect the cells against oxidative stress and allow survival within the phagocytes. Specifically, deletion of SOD1, a gene encoding the enzyme superoxide dismutase, resulted in reduction in the expression of a number of virulence traits (e.g., laccase, urease, and phospholipase) (Narasipura et al., 2003). Additionally, the sod1Δ mutant showed a marked attenuation of virulence in a mouse model and was significantly susceptible to in vitro killing by human neutrophils (Narasipura et al., 2003). Urease production is also important for pathogenesis as demonstrated by the fact that disruption of the urease gene, URE1, resulted in mutants attenuated for virulence in a mouse model of cryptococcosis (Cox et al., 2000). Urease is thought to facilitate yeast sequestration within microcapillaries and disruption of endothelial cells (Olszewski et al., 2004). Thus, urease plays a role in allowing the fungal cells to cross the blood-brain barrier.  Finally, acid  phosphatase is thought to contribute to pathogenesis by playing a role in adhesion of fungal cells to host epithelial cells (Collopy-Junior et al., 2006).  1.1.3. Secretion in C. neoformans As mentioned above, the ability of C. neoformans to influence the intracellular environment of macrophages during fungal proliferation, expulsion and transfer between cells is likely dependent to a great extent on exported fungal factors. Secretion is clearly necessary for cell surface delivery of known virulence traits such as the polysaccharide capsule, the enzyme laccase and the enzymes phospholipase B and urease.  Although the molecular details of  secretion in C. neoformans largely remain a mystery, it has been shown that many of these  8  factors are delivered, at least in part, by membrane-bound, extracellular vesicles that have been observed to traverse the cell wall (Eisenman et al., 2009; Rodrigues et al., 2007; 2008a). These vesicles are described in more detail in the following section.  1.1.3.1. Extracellular vesicles Recently, it has been shown that C. neoformans produces vesicles which vary in size between 30 and 400 nm, are surrounded by lipid bilayer membranes and somehow cross the cell wall to reach the extracellular environment (Rodrigues et al., 2007; 2008a; Yoneda and Doering, 2006; Eisenman et al., 2009). Newly synthesized capsular polysaccharides were shown to be trafficked within these vesicles and secreted via exocytosis (Yoneda and Doering, 2006). Rodrigues et al. (2007) went on to demonstrate that the extracellular vesicles also contain lipids that may contribute to virulence and serve as structural components (e.g., glucosylceramide and sterols). Finally, a proteomic analysis was performed to identify the proteins present in these vesicles (Rodrigues et al. 2008a). They identified 76 proteins including several proteins related to virulence (e.g., laccase, urease, acid phosphatase and enzymes linked to capsule synthesis) and protection against oxidative stress (e.g., heat shock proteins, superoxide dismutase). Furthermore, these extracellular vesicles have different enzymatic activities including laccase, urease and acid phosphatase activity, all known to play a role in virulence (Rodrigues et al., 2008a). The biological activity of the vesicles was confirmed by showing that they were capable  of  melanization  when  incubated  with  the  melanin  precursor  L-3,4-  dihydroxyphenylalanine (L-DOPA) (Eisenman et al., 2009). Interestingly, four main groups of extracellular vesicles were observed in C. neoformans based on their morphology and electron density (Rodrigues et al., 2008a). It is conceivable that the different types of vesicles contain  9  different proteins and virulence traits, some of which may be essential for pathogenesis while others may be required for other activities such as cell maintenance. In fact, only a portion of the vesicle population carried capsular polysaccharides (Wolf et al., 2012). This indicates that C. neoformans may use the vesicles as part of a general secretion mechanism for trans-cell wall transport and delivery of virulence traits and other cargo (e.g., enzymes) to the extracellular environment. Rodrigues et al (2008a) have thereby termed these vesicles “virulence-factordelivery bags”. The exact mechanism by which the extracellular vesicles are formed, how they cross the cell wall and how the virulence traits and other cargo are released from the vesicles remains unclear, although different models have been suggested (Figure 1.1). It was proposed that these vesicles are formed by a similar mechanism as described for exosomes (Nosanchuk et al., 2008; Rodrigues et al., 2008a; 2008b). Exosomes are found in maturing reticulocytes of different species (Johnstone, 2005). Their vesicular contents are released when the exosomes fuse with the plasma membrane suggesting that they could serve as a mechanism for shedding surface proteins (Harding et al., 1984; Pan et al., 1985). It is possible that C. neoformans utilizes a similar mechanism where multivesicular bodies fuse with the plasma membrane releasing the extracellular vesicles (Rodrigues et al., 2008a; 2008b). Alternatively, the virulence traits could be packaged in the vesicles, along with other cytosolic proteins, at the plasma membrane in a blebbing process (Doering, 2009). Finally, the virulence traits may be secreted via exocytosis. In an attempt to explain vesicular transport across the cell wall, Nosanchuck et al. (2008) and Rodrigues et al. (2008b) proposed the existence of pores in the C. neoformans cell wall which are large enough to allow the shuttling of the vesicles. It is also possible that the lipid bilayers of the vesicles are compressible to smaller diameters allowing transport through smaller pores.  10  Additionally, some motor proteins are most likely needed for efficient vesicular transport across the cell wall (Nosanchuck et al., 2008; Rodrigues et al., 2008b).  Figure 1.1 Secretion of virulence traits via extracellular vesicles in C. neoformans. The exact mechanism of trans-cell wall trafficking has not been identified although different models have been proposed, including fusion of the multivesicular bodies with the plasma membrane, blebbing from the plasma membrane, and exocytosis as illustrated. Figure prepared by B. Cadieux and Dr. J. Choi, and reproduced from Kronstad et al. (2011).  There are multiple potential advantages for C. neoformans to secrete proteins, lipids, enzymes and virulence traits in extracellular vesicles (Rodrigues et al., 2008a; 2008b). Primarily, this mechanism may be important for survival of the fungal cell in the host, and perhaps particularly inside phagocytic cells. More specifically, packaging virulence traits within vesicles may allow delivery of a concentrated toxic payload which would be far more damaging  11  to the host than if each virulence trait was secreted individually and had to reach the target cells by diffusion. Additionally, packaging fungal compounds in extracellular vesicles may increase the uptake efficiency by the host cells. It is also possible that packaging antioxidant proteins in the extracellular vesicles may help protect the fungal cell from the host immune response. The extracellular vesicles produced by C. neoformans have been shown to be important for pathogenesis and to influence the host immune response during infection. For example, the extracellular vesicles were produced by C. neoformans during infection of mouse macrophages, and the vesicle-associated proteins reacted with sera from cryptococcosis patients (Rodrigues et al., 2007; 2008a). The extracellular vesicles were internalized by mouse macrophages and stimulated phagocytic and microbicidal activities (Oliveira et al., 2010). The secreted vesicles also modulated the nitric oxide and cytokine production by macrophages (e.g., TNF-α, IL-10, and TGF-β). Finally, incubation of the extracellular vesicles with macrophages resulted in rapid disruption of the vesicles, enabling release of their cargo (Wolf et al., 2012). Similar outer membrane vesicles have been described in Gram-negative and Grampositive bacteria as a delivery mechanism for virulence factors or as a way to modulate the host defense response (Wai et al., 2003; Kuehn and Kesty, 2005; Lee et al., 2009). Additionally, other fungal species, including the human pathogen Histoplasma capsulatum, also secrete extracellular vesicles (Albuquerque et al., 2008).  1.1.3.2. Secreted mannoproteins Mannoproteins are a group of cell surface proteins and represent the majority of proteins secreted by C. neoformans (Mansour et al., 2002; Biondo et al., 2006; Eigenheer et al., 2007). Many mannoproteins share common structural features, such as an N-terminal signal sequence,  12  Ser/Thr rich regions, O-glycosylation and N-glycosylation sites, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (Mansour and Levitz, 2003). This GPI anchor serves to attach these mannoproteins to the cell membrane or to the β-1,6-glucans in the cell wall, however, cleavage of the GPI anchor results in secretion of the protein to the extracellular space (Djordjevic et al., 2005). Some mannoproteins are known to be regulated by iron, such as the FIT mannoproteins in S. cerevisiae (Protchenko et al, 2001). Several mannoproteins are also immunodominant cryptococcal antigens which play an important role in cell-mediated immunity against C. neoformans (Murphy, 1988; Zaragoza et al., 2009).  For example, many  mannoproteins are recognized by serum from patients with cryptococcosis (Biondo et al., 2005). Mannoproteins are also responsible for stimulating T-cell responses and can mediate partial protection in mice against C. neoformans (Levitz et al., 2001; Biondo et al., 2002; Huang et al., 2002). Finally, various studies have demonstrated that glycosylation of mannoproteins is critical for T-cell responses, enabling the mannose receptors on host cells to bind and internalize the cryptococcal mannoproteins (Mansour et al., 2002; Levitz and Specht, 2006; Specht et al., 2007).  13  1.2. Iron and microbial pathogenesis Iron is required by most organisms for growth and proliferation. Iron serves as a catalyst for many oxidative enzymes and is important in the synthesis of DNA as well as energy metabolism. It also serves as an essential co-factor in various cellular enzymes, oxygen carriers and electron transfer systems. The metal is particularly important in C. neoformans because its availability influences growth, capsule production and size, laccase expression and consequently pathogenesis (Jung et al., 2006). Specifically, capsule enlargement occurs in mammalian tissue, in part, due to the extremely low availability of iron (Zaragoza et al., 2009). Additionally, studies have shown that iron overload exacerbated virulence in a mouse model, facilitated colonization of the brain, as well as delayed and/or prevented a local cytokine response (Barluzzi et al., 2002). The iron-responsive regulator, Cir1, links iron sensing to virulence and iron uptake mechanisms (Jung et al., 2006; Jung and Kronstad, 2008). This link between iron uptake and virulence has been well established in bacteria. For example, Corynebacterium diphtheriae evolved a system where the expression of the diphtheria toxin, encoded by the tox gene, is induced at low iron levels (Tai et al., 1990). An iron-dependent negative-regulatory protein, DtxR, represses the diphtheria toxin gene, as well as siderophores and other components of the high-affinity iron uptake system, from being transcribed under high-iron conditions (Lee et al., 1997).  1.2.1. Iron uptake in bacteria Considering the fact that iron is essential for growth and proliferation of most organisms, the mammalian host protects itself from invading micro-organisms by sequestering most of its iron with iron-binding proteins (e.g., hemoglobin, transferrin, ferritin, lactoferrin), resulting in an  14  extremely limited level of free iron. The concentration of free iron in the mammalian host has been reported to be approximately 10-18 M (Bullen et al., 1978). This mechanism of sequestering free iron has a dual purpose, it also protects the host from the toxicity of free iron. Free iron is toxic to biological cells as it forms reactive oxygen species that rapidly destroy membranes, nucleic acids and proteins. In order to colonize and proliferate inside the host, pathogens must develop mechanisms to overcome the problem of iron unavailability. Gram-negative bacteria differ from Gram-positive bacteria based on their cell wall and membrane structures. Gramnegative bacteria have a cytoplasmic membrane and an outer membrane and the two membranes are separated by the periplasmic space. Gram-positive bacteria differ in that they do not have an outer membrane.  Instead, they have a thick peptidoglycan layer.  Bacteria must therefore  develop different uptake systems to allow passage of iron across these different cell wall and membrane structures. Most of these systems rely on cell surface receptors and transporters.  1.2.1.1. Gram-negative bacteria Important iron sources in the host include transferrin and lactoferrin. Transferrin is found in serum, while lactoferrin is present in lymph and mucosal secretions. Siderophores are small molecules which are produced and secreted by bacteria and fungi to chelate ferric iron. Siderophores have an extremely high affinity and can therefore steal iron from the host’s ironbinding proteins (i.e., transferrin and lactoferrin). To allow transport of iron inside the Gramnegative cell, bacteria possess highly specific outer membrane receptors (Braun et al., 2000). The receptors also serve as transporters and can translocate the iron across the outer membrane into the periplasmic space. Once the iron enters the periplasmic space, it is bound by ironbinding proteins, which protects the cell from the toxicity of free iron. In the final step, iron  15  transverses the cytoplasmic membrane via ATP-binding cassette (ABC) transporters in an energy-dependent manner. ABC transporters typically consist of one or two integral membrane proteins that act as permeases, coupled with two copies of one or two ATPases which provide the required energy. One example of a well characterized iron uptake system is the transport of the siderophore, ferrichrome, in Escherichia coli (Ferguson et al., 1998; 2000; Locher et al., 1998; Braun et al., 2000; Clarke et al., 2000). The system depends on the outer membrane receptor FhuA which also acts as the transporter (Ferguson et al., 1998; Locher et al., 1998). It is thought that the energy required for this process is provided by the protein complex, TonB, ExbB and ExbD, localized at the cytoplasmic membrane (Braun and Endriss, 2007). Once the ferrichrome reaches the periplasmic space, it is chelated by the periplasmic protein FhuD (Clarke et al., 2000). FhuD directly delivers the ferrichrome to the cytoplasmic membrane transport protein FhuB (Rohrbach et al., 1995). Ferrichrome is then transported across the cytoplasmic membrane via FhuB which acts as the permease, while the energy required for transport is provided by the ATPase, FhuC. Interestingly, outer membrane receptors are highly specific and usually different receptors are required for transportation of different iron sources. In contrast, the cytoplasmic transporters allow transport of different iron sources (Braun et al., 2000). For example, the outer membrane receptor FhuE transports the siderophore, coprogen, while IutA transports iron bound to the siderophore, aerobactin. However, both iron sources are transported across the cytoplasmic membrane via the FhuCDB transporter, the same transporter that is required for transport of ferrichrome. E. coli can also transport ferrous iron via the FeoABC iron transport system (Kammler et al., 1993). FeoA is a small protein thought to be localized in the cytosol. FeoB has been identified as a protein located at the cytoplasmic membrane that likely functions as the permease,  16  while FeoC may function as an iron-sulfur-dependent transcriptional repressor (Cartron et al., 2006). These proteins are also found in Salmonella enterica, Helicobacter pylori, and Vibrio cholera (Tsolis et al., 1995; Velayudhan et al., 2000; Wyckoff et al., 2006; Mey et al., 2008).  1.2.1.2. Gram-positive bacteria Gram-positive bacteria possess similar iron uptake systems as Gram-negative bacteria but have developed a different approach to circumvent the differences in their cell wall. In Grampositive bacteria, iron transport is mediated by an iron-binding protein anchored to the cytoplasmic membrane by lipids, and by ABC transporters (Schneider and Hantke, 1993; Braun, 2001). The iron-binding protein acts as a receptor and delivers the iron to the associated ABC transporter which will allow transport of iron across the cytoplasmic membrane. However, the exact mechanism for transport across the cell wall remains unclear. Transport of siderophore-bound iron is a major iron uptake mechanism in Gram-positive bacteria. The transport of siderophore-bound iron has been characterized in different bacteria including Staphylococcus aureus, Listeria monocytogenes, and Bacillus anthracis (Hammer and Skaar, 2011; Honsa and Maresso, 2011; Klebba et al., 2012). Specifically, S. aureus synthesizes at least two different siderophores, staphyloferrin A and staphyloferrin B. Both siderophores can scavenge iron from the host’s transferrin and lactoferrin. Once the iron is coupled to the siderophore, it is transported across the cell wall to the specific ABC transporter on the cytoplasmic membrane. The lipoprotein HtsA serves as the receptor for staphyloferrin A and is coupled with the membrane permease HtsBC (Beasley et al., 2009; Grigg et al., 2010). Staphyloferrin B-bound iron is acquired in a similar fashion although the lipoprotein SirA acts as the receptor and the membrane permease consists of SirBC (Dale et al., 2004; Grigg et al.,  17  2010). Finally, S. aureus is also able to take up iron bound to siderophores produced by other organisms. The lipoprotein receptors FhuD1 and FhuD2 and the permease FhuBG are involved in the transport of these xenosiderophores (Sebulsky et al., 2000; 2003; 2004). The energy required for transport of iron bound to all these different siderophores is thought to be generated by the ATPase, FhuC (Beasley et al., 2009; Speziali et al., 2006). Other bacteria, such as L. monocytogenes, are unable to synthesize siderophores and rely exclusively on uptake of iron bound to siderophores produced by other organisms (Klebba et al., 2012). Alternatively, Gram-positive bacteria can also extract iron from mammalian iron-binding proteins, including transferrin.  For example, a Tpn transferrin-binding protein has been  identified in S. aureus and Staphylococcus epidermidis. The Tpn protein is a glyceraldehyde-3phosphate dehydrogenase that is localized at the cell wall and that binds transferrin to facilitate iron release.  The released iron can then be transported into the cytoplasm via the ABC  transporters (Modun et al., 1998). Some bacteria, such as Bacillus cereus, have also been shown to utilize ferritin (Daou et al., 2009).  1.2.1.3. Heme acquisition in bacteria Heme is a prosthetic group that contains iron and is commonly found as a component of hemoglobin or other hemoproteins (e.g., myoglobin, cytochrome). Heme comprises the largest iron pool in the mammalian body, and therefore, can serve as an important iron source for many bacterial pathogens (Tong and Guo, 2009; Nobles and Maresso, 2011). Heme refers to the molecule in which the prosthetic group contains iron in the ferrous state (Fe2+), while hemin is used when the prosthetic group contains oxidized iron (Fe3+). The two terms are often used interchangeably and, for the sake of simplicity, heme will be used throughout the remainder of  18  this thesis. Bacteria employ specific heme transporters and receptors, and some bacteria also secrete heme-binding proteins known as hemophores (Tong and Guo, 2009; Nobles and Maresso, 2011; Wandersman and Delepelaire, 2012). Similarly to the uptake of other iron sources, the heme transport systems in Gram-negative bacteria are also composed of an outer membrane transport protein, a periplasmic heme-binding protein and an ABC transporter localized at the cytoplasmic membrane to allow transport of heme into the cell (Braun, 2001; Tong and Guo, 2009). For example, in Pseudomonas aeruginosa, PhuR acts as the single outer membrane heme receptor, while PhuT binds heme in the periplasm and delivers it to the ABC transporter which is composed of PhuUVW (Ochsner et al., 2000; Tong and Guo, 2009). Some bacteria, including Serratia marcescens, P. aeruginosa, Yersinia pestis, and Haemophilus influenzae, also secrete hemophores into the extracellular space to release the heme from hemoglobin and deliver it to the outer membrane receptor (Hanson et al., 1992; Letoffe et al., 1994; 1998; 1999; 2000; Jarosik et al., 1995; Cope et al., 1998; Arnoux et al., 1999; 2000; Wandersman and Stojiljkovic, 2000; Rossi et al., 2001; Tong and Guo, 2009). In Gram-positive bacteria, heme uptake relies on multiple cell surface proteins that form a relay system across the cell wall to ultimately deliver heme to an ABC transporter at the cytoplasmic membrane. One of the best described systems is the one present in S. aureus (Hammer and Skaar, 2011; Nobles and Maresso, 2011). A multi-gene operon, termed the ironregulated surface determinants (Isd) locus, encodes seven heme binding proteins (IsdA, IsdB, IsdC, IsdD, IsdE, IsdG, and IsdI). The operon also encodes sortases (SrtA, SrtB) which anchor the Isd proteins, specifically IsdA, IsdB, and IsdC, to the cell wall. IsdB is the most surface exposed protein, followed by IsdA and then by IsdC. Heme is passed along these proteins to finally be directed to the ABC transporter (IsdDEF), which receives the heme from IsdC. IsdD  19  functions as the permease, IsdE is a lipoprotein receptor, and IsdF is the ATPase. Finally, IsdH is also surface exposed and was shown to bind hemoglobin and haptoglobin. Interestingly, many Isd proteins (IsdA, IsdB, IsdC, and IsdH) contain near iron transporter (NEAT) domains and it has been demonstrated that heme binding occurs via the NEAT domains (Andrade et al., 2002; Grigg, et al., 2007; Hammer and Skaar, 2011; Nobles and Maresso, 2011). In the past few years, Gram-positive bacteria, including B. anthracis and L. monocytogenes, have also been found to secrete hemophores (Maresso et al., 2008; Wandersman and Delepelaire, 2012; Klebba et al., 2012; Honsa and Maresso, 2011). Specifically, B. anthracis possesses a heme uptake system similar to the Isd system in S. aureus, but also contains two additional secreted proteins, IsdX1 and IsdX2, which have been shown to both contain NEAT domains and to bind heme (Maresso et al., 2008). These hemophores acquire heme from hemoglobin and transfer the heme to IsdC. Hemophores have also been recently described in Mycobacterium tuberculosis (Tullius et al., 2011). Once inside the cell, the iron must be released from the heme porphyrin through oxidative degradation by the heme oxygenase which cleaves heme to produce biliverdin XI, carbon monoxide, and iron (Nobles and Maresso, 2011). The free iron can then be used directly by the cell in cellular functions or can be recycled.  One of the first heme oxygenases  characterized was the one found in C. diphtheriae (Schmitt, 1997), however, they have now been identified in many Gram-negative and Gram-positive bacteria. In fact, heme oxygenases are highly conserved among prokaryotes, fungi and higher eukaryotes (Wilks, 2002; Skaar et al., 2004; 2006; Wu et al., 2005).  Not surprisingly, the expression of heme oxygenases in  pathogenic bacteria is usually up-regulated in low-iron conditions and in the presence of heme (Schmitt, 1997; Reniere et al., 2010).  20  1.2.2. Iron uptake in C. neoformans and other fungi C. neoformans has also evolved multiple strategies to acquire iron while in the host, including a reductive high-affinity iron uptake pathway, a low-affinity iron uptake pathway, transport and use of siderophore-bound iron, and acquisition of iron from heme. These mechanisms will be described in detail in the following sections.  1.2.2.1. The reductive high-affinity iron uptake pathway The reductive high-affinity iron uptake pathway mediates iron utilization from inorganic iron sources and transferrin, which is an important iron source for C. neoformans during infection (Jung and Kronstad, 2008).  Components of this pathway are induced upon iron  limitation (Lian et al., 2005). In this pathway, iron must first be reduced, either by cell surface ferric iron reductases (Fre) or other reductants such as melanin and 3-hydroxyanthranilic acid (3HAA), and then be transported inside the cell by the iron permease/ferroxidase complex localized at the plasma membrane (Jacobson et al., 1997; Jung et al., 2008; 2009). Jung et al. (2008) demonstrated that C. neoformans possesses two high-affinity iron permeases, Cft1 and Cft2. They showed that deletion of CFT1 resulted in reduced growth and iron uptake in presence of low concentrations of ferric chloride and transferrin as sole iron sources. Additionally, the cft1Δ mutant was attenuated for virulence in a mouse model and resulted in a lower fungal burden in the brain when compared to infection with the wild-type strain. Interestingly, Cft2 played no apparent role in iron acquisition but was involved in virulence. Two multicopper ferroxidases were also found in C. neoformans, Cfo1 and Cfo2 (Jung et al., 2009). Similarly, Cfo1 was essential for growth in presence of low concentrations of ferric chloride and transferrin  21  as sole iron sources and resulted in attenuated virulence in a mouse model, while no phenotypes were associated with deletion of CFO2 (Jung et al., 2009). The reductive high-affinity iron uptake pathway was first described in S. cerevisiae (Lesuisse et al., 1987; Askwith et al., 1994; Stearman et al., 1996; Philpott, 2006). In this organism, the pathway also involves reduction of iron by extracellular ferric reductases (i.e., Fre1 and Fre2) and transport within the cell via the permease/multicopper ferroxidase complex, Ftr1/Fet3 (Askwith et al., 1994; Stearman et al., 1996). It was established that prior to being transported into the cell via Ftr1, the iron must first be oxidized by Fet3 (De Silva et al., 1995; 1997; Hassett et al., 1998; Wang et al., 2003). The reductive high-affinity iron uptake pathway has now been described in most pathogenic fungi (Kornitzer, 2009). In Candida albicans, two iron permeases are present, CaFtr1 and CaFtr2, although only CaFtr1 is required for growth in low-iron conditions and virulence in a mouse model (Ramanan and Wang, 2000). Additionally, a multicopper ferroxidase, CaFet3, is also required for growth in low iron environments, but is not essential for virulence (Eck et al., 1999). Similarly, the permease/multicopper ferroxidase complex (FetC and FtrA) is also found in Aspergillus fumigatus (Schrettl et al., 2004). However, deletion of FTRA does not result in any growth defect or attenuation in virulence.  1.2.2.2. Low-affinity iron uptake pathway When in abundance, iron can be taken up by the cell via the low-affinity iron uptake pathway in C. neoformans (Jacobson et al., 1998). This system relies on reduction of iron by reductants, including melanin, 3HAA or other secreted reductants, and eventual transport inside the cell via transporters, which have not been identified as of yet (Jacobson et al., 1997). The details and mechanism of this iron uptake pathway are still currently unknown.  22  A low-affinity iron uptake pathway has also been relatively well described in S. cerevisiae (Philpott, 2006; Sutak et al., 2008).  A non-ATP-dependent trans-membrane  transporter, Fet4, is the main component of the pathway (Dix et al., 1994). Similarly to C. neoformans, ferric iron must first be reduced to ferrous iron before being transported into the cell. The permeases involved in low-affinity iron uptake are not specific to iron but can also take up copper and zinc (Kaplan and Kaplan, 2009). For example, Fet4 has been implicated in transport of ferrous iron, zinc, copper and cadmium (Dix et al., 1994; 1997; Hassett et al., 2000). Other metal transporters described in S. cerevisiae are Smf1, Smf2, and Smf3 (Culotta et al., 2005). Smf1 and Smf2 are primarily manganese transporters although Smf1 can also transport ferrous iron (Cohen et al., 2000; Portnoy et al., 2000). Smf3 is involved in transfer of iron from the vacuolar stores to the cytosol (Portnoy et al., 2000). A similar low-affinity iron uptake system has not yet been characterized in C. albicans or A. fumigatus.  1.2.2.3. Transport of siderophore-bound iron C. neoformans does not produce siderophores, however, it can utilize iron bound to xenosiderophores. C. neoformans possesses siderophore iron transporters, one of which (Sit1) has been characterized by Tangen et al. (2007). They demonstrated that Sit1 is required for use of siderophore-bound iron and for growth in a low-iron environment. Deletion of SIT1 also resulted in altered melanin deposition and laccase activity, and changes in cell wall density. However, the importance of the siderophore in iron uptake during disease is not yet clear. Sit1 did not play a role in virulence, possibly because other iron uptake pathways were functional in the mutant and allowed acquisition of other iron sources in vivo, masking the role of the siderophore iron uptake pathway.  23  The siderophore non-reductive iron uptake pathway is very common among the different species of fungi. It was first described in S. cerevisiae but has now been characterized in several fungal species.  The siderophore-iron chelates are transported into the cell via Arn/Sit  transporters, which likely function as proton symporters energized by the membrane potential (Lesuisse et al., 1998). S. cerevisiae possesses four siderophore transporters (Kosman, 2003), each transporting a specific type of siderophore.  In C. albicans, only one siderophore  transporter, CaSit1/CaArn1, has been well characterized to date (Almeida et al., 2009). It is not specific to only one type of siderophore as it can transport a range of siderophores from other organisms as well as other iron complexes (Ardon et al., 2001; Heymann et al., 2002; Lesuisse et al., 2002; Bernier et al., 2005). Interestingly, transport of siderophores via CaSit1/CaArn1 has been linked to an endocytic pathway (Hu et al., 2002, Weissman et al., 2008). Although the CaSit1/CaArn1 siderophore transporter was not required for virulence in a mouse model, it appeared to be essential for epithelial invasion in a human cell model (Heymann et al., 2002; Hu et al., 2002). In A. fumigatus, uptake of iron by siderophore transporters appears to be the major iron uptake pathway (Haas, 2012). Ten putative SIT genes have been reported in this organism, although only two (MirA and MirB) have been characterized so far (Haas et al., 2003; 2008). A. fumigatus is also capable of synthesizing its own siderophores and biosynthesis has been linked to virulence. Specifically, deletion of the ornithine monooxygenase (SidA), an enzyme involved in siderophore biosynthesis results in loss of virulence in a mouse model (Schrettl et al., 2004; Hissen et al., 2005).  24  1.2.2.4. Acquisition of iron from heme Many pathogenic fungi can utilize heme as an iron source (Foster, 2002; Santos et al., 2003; Jung et al., 2008). However, very little is known about the mechanism involved in heme uptake in fungi. For example, H. capsulatum can grow on heme and it has been speculated that heme uptake is mediated by unidentified cell surface receptors (Foster, 2002). C. albicans can also grow on heme and the heme uptake mechanism relies on hemolytic factors to release the hemoglobin from the erythrocytes, and cell surface receptors (Manns et al., 1994; Pendrak et al., 2000). So far, two heme-binding proteins, Rbt5 and Rbt51, have been characterized as cell surface receptors for heme and hemoglobin utilization (Weissman and Kornitzer, 2004). Rbt5 and Rbt51 are both cell surface mannoproteins and carry a CFEM domain characterized by a set of eight conserved cysteine residues (Weissman and Kornitzer, 2004; Kulkarni et al., 2003). CFEM domains are common to many fungal surface proteins, however, their function is still unknown (Kulkarni et al., 2003). The expression of RBT5 was strongly induced in low-iron conditions and deletion of the gene resulted in growth defects in presence of heme or hemoglobin (Weissman and Kornitzer, 2004).  Additionally, expression of CaRbt51 in S.  cerevisiae enabled the cells to utilize hemoglobin as an iron source (Weissman and Kronitzer, 2004).  Furthermore, Weissman et al. (2008) demonstrated that Rbt5 facilitates the rapid  endocytosis of hemoglobin into the vacuole in C. albicans, and the pathway depends on the lumen acidification of late secretory vesicles and on the endosomal sorting complex required for transport (ESCRT).  ESCRT proteins are required to sort proteins to the lysosome for  degradation and they also participate in the formation of multivesicular bodies (Hurley, 2010). Interestingly, deletion of RBT5 did not result in attenuation of virulence in either a mouse or rabbit model of infection, possibly because other iron uptake pathways compensated for the loss  25  of the heme uptake pathway (Braun et al., 2000). Jung et al. (2008) have shown that C. neoformans can also utilize iron from heme, although the heme uptake system has not yet been characterized. Given that the majority of iron in mammals is present in heme, this source may be particularly important for C. neoformans. In the past year, work in our laboratory has shown that the ESCRT protein Vps23 plays a role in heme acquisition. Deletion of VPS23 resulted in a growth defect on heme, a capsule defect and attenuation of virulence in a mouse model (Hu et al., 2013). This study demonstrated that in C. neoformans, like in C. albicans, the ESCRT pathway may be involved in heme uptake.  Finally, C. neoformans has a putative heme  oxygenase, however, it has not yet been demonstrated to play a role in heme acquisition.  1.2.3. Regulation of iron uptake The uptake and utilization of iron need to be tightly regulated because of the highly toxic nature of iron. In C. neoformans, as in other fungi and bacteria, genes involved in the uptake, storage and utilization of iron are more highly expressed in iron deprived environments to allow the cell to survive and proliferate in such conditions (Yamaguchi-Iwai et al., 1995; 1996; Braun 2001; Jung et al., 2006; Nobles and Maresso, 2011). Iron regulation in pathogenic fungi appears to depend on the GATA-binding and CCAAT-binding classes of transcription factors (Kornitzer, 2009). For example, the GATA-binding transcription factor, Cryptococcus iron regulator (Cir1), was identified as the iron master regulator in C. neoformans (Jung et al., 2006) and was shown to integrate iron sensing with the expression of virulence traits. Specifically, microarray analysis revealed that Cir1 controls the expression of genes involved in iron acquisition, iron transport and iron homeostasis, and functions both as an activator or repressor (Jung et al., 2006). Cir1 also controls the expression of the genes encoding known virulence traits, including the capsule,  26  melanin synthesis and the ability to grow at 37°C. Upon deletion of the CIR1 gene, the mutant was defective in capsule formation and growth at 37°C but had increased laccase activity. Furthermore, the cir1Δ mutant was avirulent in a mouse model indicating a role for Cir1 in iron acquisition in the host (Jung et al., 2006). In C. albicans, the GATA-binding transcription factor Sfu1 was identified as the master iron regulator, regulating the expression of at least 149 genes, while SreA was identified to regulate the expression of at least 49 genes in A. fumigatus (Lan et al., 2004; Schrettl et al., 2008). The CCAAT-binding transcription factors in both C. albicans and A. fumigatus were also found to affect iron regulation (Kornitzer, 2009). Similarly, the expression of genes involved in iron uptake in many Gram-negative and Gram-positive bacteria, including E. coli and S. aureus, are regulated by the ferric uptake repressor (Fur) (Xiong et al., 2000; Hantke, 2001; Nobles and Maresso, 2011).  The Fur  transcription factor forms a dimer and binds to a palindromic DNA sequence known as a Fur box. When iron is present, it binds to Fur and results in the protein binding to the Fur box in the promoters of target genes, thus preventing the transcription of these genes.  In low iron  conditions, Fur dissociates from the box, permitting the RNA polymerase to initiate transcription of the target genes. HapX is another transcription factor involved in the regulation of iron acquisition in C. neoformans. For example, HapX plays a positive regulatory role in siderophore transporter expression as well as in expression of the Cir1 transcription factor (Jung et al., 2010). Deletion of HAPX resulted in a slight attenuation of virulence indicating that HapX plays a minimal role during infection of mammalian hosts (Jung et al., 2010). It is suggested that HapX may be more important as a regulator of environmental iron uptake functions. Interestingly, HapX is highly  27  conserved among most fungal species and orthologs have been identified in C. albicans and in A. fumigatus (Hortschansky et al., 2007; Baek et al., 2008). Rim101 is another highly conserved transcription factor among fungal species and is responsible for regulating gene expression in response to pH, thereby allowing the cells to adapt to changes in the environmental pH. However, Rim101 also regulates genes involved in iron acquisition in both C. neoformans and C. albicans (Bensen et al., 2004; Thewes et al., 2007; Baek et al., 2008; Nobile et al., 2008; Liang et al., 2009; O’Meara et al., 2010). Specifically, a microarray analysis in C. neoformans revealed a role for Rim101 in metal homeostasis and many proteins involved in iron transport were differentially regulated between the wild-type and rim101Δ strains (O’Meara et al., 2010). Phenotypically, the rim101Δ mutant had a capsule attachment defect, displayed a growth defect at alkaline pH or in presence of high salt, and had slower growth in low-iron conditions.  Interestingly, the rim101Δ mutant proved to be  hypervirulent in a mouse model, which could possibly be explained by the fact that the mutant survives better than the wild-type strain within macrophages.  28  1.3. Cytokine-inducing glycoprotein (CIG1) Previous work to identify iron uptake systems in C. neoformans characterized the transcriptome upon growth at different iron concentrations and in different iron sources (Lian et al., 2005; Jung et al., 2006; 2010). In addition to the reductive and siderophore uptake systems, these studies revealed that the CIG1 gene was the most abundant transcript in iron-starved cells of the C. neoformans var. neoformans strain B3501A. CIG1 was differentially expressed in response to iron and its expression was decreased by 10 fold when the cells were grown in ironreplete medium (Lian et al., 2005). The CIG1 gene encodes a predicted mannoprotein with a signal peptide which presumably allows the protein to be secreted to the cell surface (Levitz and Specht, 2006). It has been suggested that Cig1 is a cytokine-inducing glycoprotein as it was detected in a cell fraction containing several extracellular proteins that reacted with serum antibodies from AIDS patients infected with C. neoformans and it triggered cytokine production in cultured immune cells (Biondo et al., 2005; 2006; Murphy et al., 1993). Thus, Cig1 may represent one of the mannoproteins involved in modulating the immune response to C. neoformans. Disruption of the CIG1 gene in a C. neoformans var. neoformans causes a growth defect in low iron medium (LIM) and the loss of iron suppression of capsule size (Lian et al., 2005). Biondo et al. (2006) also identified Cig1 as one of 13 major secreted proteins. Additionally, CIG1 was recently identified to be regulated by the pH-responsive transcription factor Rim101 (O’Meara et al., 2010). The microarray analysis revealed that CIG1 was the most significantly differentially expressed transcript and was repressed over 470 fold in the rim101Δ mutant compared to wild-type strain.  29  1.4. Rationale and aims of study Iron acquisition and processing is essential for the pathogenesis of C. neoformans. As mentioned above, heme is the most abundant iron source in the mammalian host and C. neoformans has been shown to utilize heme as an iron source.  However, the heme uptake  pathway has not yet been characterized. Understanding the key players in the acquisition of this iron source could prove invaluable in identifying additional drug targets for more efficient therapy against cryptococcosis.  1.4.1. Hypotheses Given that the expression of CIG1 is strongly regulated by iron and that the protein is predicted to be localized extracellularly, the primary hypothesis for this study was that Cig1 functions in iron acquisition in C. neoformans. Additionally, iron is an essential component of pathogenesis leading to the second hypothesis that Cig1 plays a role in virulence. The analysis revealed that deletion of CIG1 resulted in delayed growth of C. neoformans in LIM supplemented with heme, however, the cig1Δ mutant was eventually able to grow, leading to the third hypothesis that an alternative pathway for heme acquisition must exist in C. neoformans. Finally, previous findings that disruption of CIG1 resulted in the loss of iron suppression of capsule size led to the fourth hypothesis that Cig1 may be involved in the regulation of capsule elaboration, possibly via secretion.  1.4.2. Research objectives The general objective of this study was to characterize the role of the CIG1 gene in both C. neoformans var. grubii and var. neoformans. The objective in Chapter 2 was to examine  30  whether Cig1 plays a key role in iron uptake. The specific goals were: 1) to determine if Cig1 is required for growth in different iron sources, 2) to establish the role of Cig1 in heme uptake, 3) to confirm the extracellular localization of Cig1, 4) to evaluate the ability of Cig1 to bind to heme, and 5) to test whether Cig1 contributes to virulence. For Chapter 3, the objective was to focus in more detail on establishing and elucidating alternative heme acquisition systems for C. neoformans. The specific goal was to examine the contribution of different factors which may be involved in Cig1-independent heme acquisition, including: 1) pH, 2) the pH master regulator Rim101, and 3) cell density. Finally, the goal in chapter 4 was to assess the link between Cig1 and the elaboration of the capsule. Specifically, the role of Cig1 in secretion and in maintaining cell wall integrity was evaluated.  31  Chapter 2. Role of Cig1 in heme acquisition in C. neoformans var. grubii 2.1. Introduction Iron is an essential nutrient for most organisms. As part of the immune defense system, the mammalian host protects itself against invading microorganisms by using iron-binding proteins to restrict the level of free iron. In order to survive and proliferate, C. neoformans has evolved multiple strategies to acquire iron while inside the host (Jacobson et al., 1997; 1998; Tangen et al., 2007; Jung et al., 2008; 2009; Jung and Kronstad, 2008). Iron availability is a particularly important aspect of cryptococcal disease because the metal influences the C. neoformans in mice (Jung et al., 2008; 2009) C. neoformans has also been shown to utilize heme as an iron source, however the mechanism for heme uptake is still unknown (Jung et al., 2008). In fact, very little is known about heme uptake in fungi, although heme acquisition systems have been well described in pathogenic bacteria (Weissman and Kornitzer, 2004; Weissman et al., 2008; Nobles and Maresso, 2011; Tong and Guo, 2009; Wandersman and Delepelaire, 2012). In general, heme uptake is mediated by cell surface heme receptors and transporters to traffic the molecule to the intracellular space (Nobles and Maresso, 2011; Tong and Guo, 2009).  In addition, some  microbes also secrete hemophores, which are high-affinity heme-binding proteins, to sequester heme from the environment and deliver it to the cell surface receptors (Wandersman and Delepelaire, 2012). Given that the majority of iron in mammals is present in heme, this source may be particularly important for C. neoformans. A previous study identified CIG1 as the most abundant transcript in C. neoformans var. neoformans cells grown in LIM. The CIG1 transcript was also found to be differentially expressed when the cells were grown in iron-replete conditions compared to LIM (Lian et al., 32  2005). The abundance and regulation of CIG1 led to the hypothesis that Cig1 may play an important role in C. neoformans and that it may be involved in iron acquisition. The goal of this study was therefore to characterize the role of Cig1 in iron acquisition. C. neoformans var. grubii represents the most important clinical serotype in individuals with AIDS, while C. neoformans var. neoformans is less virulent, based on a mouse model of infection, and is less clinically significant (Chaturvedi et al., 2002; Toffaletti et al., 2004; Barchiesi et al., 2005; Lin et al., 2007; Perfect and Casadevall, 2011). In this study, an ortholog of Cig1 was identified in the C. neoformans var. grubii strain H99, and this strain was chosen to examine the contribution of Cig1 in iron acquisition so that virulence could be assessed. The effects of deleting the CIG1 gene in C. neoformans and growing the cells in LIM supplemented with different iron sources were studied in order to test the hypothesis that Cig1 may be involved in iron acquisition. The cig1Δ mutant resulted in delayed growth in LIM supplemented with heme. The role of Cig1 in heme uptake was examined using toxic heme analogs. The localization of Cig1 was elucidated and the ability of Cig1 to bind to heme was also investigated. Finally, the contribution of Cig1 to virulence was assessed in a mouse model of infection.  33  2.2. Materials and Methods 2.2.1. Strains and growth conditions All strains used in this study are listed in Table 2.1. LIM was prepared as described (Vartivarian et al., 1993) and the pH was adjusted to 7.2. The water used for LIM was treated with Chelex-100 resin (Bio-Rad) to chelate iron. Alternatively, yeast nitrogen base (YNB; Difco) was also used. Low iron YNB (LI-YNB) was prepared with chelated water, as above, and 100 µM bathophenanthroline disulfonic acid (Sigma) was added to chelate any residual iron in the medium. Cells for growth assays were pre-grown overnight at 30°C with shaking in yeast extract peptone dextrose broth (YPD; Fisher). The cells were then washed twice with low iron water, inoculated into LIM at 4 x 106 cells/mL and grown at 30°C for two days to starve the cells for iron. After starvation, the cells were harvested, washed and inoculated in LIM alone or supplemented with heme (porcine hemin, Sigma), FeCl3 (Sigma), hemoglobin (Sigma), or ferrioxamine (Sigma) to a final concentration of 5 x 104 cells/mL. Cultures were incubated at 30°C and growth was monitored by measuring the optical density at 600 nm using a DU530 Life Science UV/Visible spectrophotometer (Beckman Instruments). Growth was also monitored by three other methods: 1) microscopically counting the number of cells; 2) determining the number of viable cells by colony forming units (CFU) and; 3) measuring the total reductase activity of the cells at each time point.  The CFU were  determined by diluting the cells in water and plating the dilutions on YPD: 1% agarose (Invitrogen) was added as the solidifying agent. The CFU were counted after incubating the plates for 3 days at 30°C. Total reductase activity was measured as an index of the number of cells based on metabolic viability (Conconi et al., 2000). Briefly, 850 µL of cells from each time point were washed and resuspended in 0.05 M sodium phosphate buffer (pH 7.5), and then 350  34  µL of a 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) solution were added to the cells. The samples were incubated at room temperature in the dark for 24 hours, and the cells were then washed in water. The formazan formed by the reductase activity was extracted from the cells by bead beating with glass beads in ethanol:acetone (1:1) for 5 minutes at maximum power. The cells were pelleted and the relative quantity of extracted formazan was determined by measuring the absorbance of the supernatant at 485 nm. To examine growth on solid media, iron-starved cells were diluted to 1 x 106 cells/mL in low iron water. Ten-fold serial dilutions were made in low iron water and 5 µL of each dilution was plated on LIM alone or supplemented with heme or FeCl3: 1% agarose was added as the solidifying agent. Plates were incubated for three days at 30°C and then photographed.  Table 2.1. Strains used in this study Strain Description H99 C. neoformans wild-type strain cig1Δ cig1 disruption mutant cig1Δ +CIG1 CIG1 complemented strain cig2Δ cig2 deletion mutant cig2Δ +CIG2 CIG2 complemented strain cig1Δ cig2Δ cig1 cig2 double deletion mutant cig1Δ cig2Δ +CIG1 CIG1 complemented in cig1Δ cig2Δ background cig3Δ cig3 deletion mutant cig1Δ cig2Δ cig3Δ cig1 cig2 cig3 triple deletion mutant cfo1Δ cfo1 deletion mutant cig1Δ cfo1Δ cig1 cfo1 double deletion mutant cig1Δ cfo1Δ +CIG1 CIG1 complemented in cig1Δ cfo1Δ background HA::Cig1 HA-tagged Cig1 strain Cig1::GST E. coli strain expressing recombinant GST-tagged Cig1  Provided by/ Prepared by Dr. Joseph Heitman Carmelo Biondo Tian Lian Tian Lian Brigitte Cadieux Tian Lian Brigitte Cadieux Brigitte Cadieux Brigitte Cadieux Dr. Wonhee Jung Brigitte Cadieux Brigitte Cadieux Tian Lian Dr. Guanggan Hu  35  2.2.2. In silico protein analysis The Cig1 amino acid sequence from the C. neoformans serotype D strain B3501A was used to search the C. neoformans serotype A genome sequence of the H99 strain (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans) as well as other serotypes of C. neoformans (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) to identify homologs of CIG1.  Multiple sequence alignments were performed using Clustal W  (http://www.ebi.ac.uk/clustalw/). The signal peptide for the Cig1 sequence was predicted using Signal P (http://www.cbs.dtu.dk/services/SignalP). Sites for glycosylation were predicted using NetNGlyc  (http://www.cbs.dtu.dk/services/NetNGlyc)  and  NetOGlyc  (http://www.cbs.dtu.dk/services/NetOGlyc) for N- and O- glycosylation, respectively. Big-PI Predictor (http://mendel.imp.ac.at/sat/gpi/gpi_server.html) was used to inspect sequences for the presence of putative GPI cleavage sites, while TMHMM was used to predict transmembrane regions  (http://www.cbs.dtu.dk/services/TMHMM).  HHpred  (http://toolkit.tuebingen.mpg.de/hhpred) was used to search for remote protein homology based on protein structure prediction, while Phyre (www.sbg.bio.ic.ac.uk/phyre) was used to predict protein folds.  2.2.3. Construction of strains Primers used for construction of all strains are listed in Table 2.2.  36  Table 2.2. Primers used for strain construction Allele Primer Primer sequence constructed identification cig1:NAT cig1-R TCAGTTACTACTCAGCATACATAG cig1-F ATGCGCGCTTATCACTTGCAGGC cig1-up TGGGTGAGTACATCCTTCGGTAG cig1-low TGGGTATGCCAGAAGATACAGACT CIG1:NEO cig1A-int-F AAACCGGACCACCACAATAA cig1A-int-R CACTTTGGGCTTCTCTGAGG CIG1:HYG cig1A-comp1 TCGGCCGACTTAGTTGACAT cig1A-comp2 AATTCCAGCACACTGGCGGCCGTTACTAGTGCCA CTGTCCGAGGAAATAA cig1A-comp3 AATTCTGCAGATATCCATCACACTGGCGGCAGAT GGTGCGAAGTATATAATAGGG cig1A-comp4 TCCACTTGACCAAAAGGTGT cig2:NEO TL2081 AAATGGCAGGCCAGCTAAGACCGA TL2082 AATTCCAGCACACTGGCGGCCGTTACTAGTAAGT ATAGAGGAAAGACGAGCCAG TL2083 AATTCTGCAGATATCCATCACACTGGCGGCGAGA AAGCACACGTTCGCACAGT TL2084 CTGCACCCACCCCCGATCATTCA TL2085 CCCCATTTGGGAAGCTGAT TL2086 CAGCATTCTTCAACACAACAACC TL2087 GACTAGGGGTTACCAATGC TL2088 CCGTGTTAATACAGATAAACCAA CIG2:HYG cig2A-comp1 AAGCGGAGTGTCTTCTGCAT cig2A-comp2 AATTCCAGCACACTGGCGGCCGTTACTAGTTTGG CGTCATGTTGTTCAGT cig2A-comp3 AATTCTGCAGATATCCATCACACTGGCGGCCCTCT TGTTTTGTACACATATTTCAAC cig2A-comp4 CCCCTATGTCTCGACCTCAA cig2A-int-F ACCGTTACCTGCTCCAACAC cig2A-int-R AGGGGTTTTGTGGTCAACAG cig3:HYG cig3A-1 AAGGAATGATTGTAGTTTCCCTTTC cig3A-3 AATTCTGCAGATATCCATCACACTGGCGGCGAAT GATGATGGGTGAAGATATAGG cig3A-4 AATTCCAGCACACTGGCGGCCGTTACTAGTAGAC ACTCACTTTGACGCTTTTAAT cig3A-6 AGTAACCCAAATTAACTCTCGCTTT cig3A-7 GTTCACGTTAGGAATGATTCTGG cig3A-8 GTTGCTTCTTATCTCGAGCACAT cfo1:NEO CFO1-KO1 GACGAAATCTCTCCCGAAAACTTTG CFO1-KO4 GGGATGTTACAGAACAGCTCTTC CFO1-KO5 CAGGGTATTCCCATCGCCTACC CFO1-KO6 GGTCGACTTGAAGTACTTGGACTTTTG CFO1-KO22 AATTCTGCAGATATCCATCACACTGGCGGCGAAG 37  Allele constructed HA::Cig1  Primer identification 311 300 303 312 301 302  Cig1::GST  NEO, HYG Transformant screen  BamH1-Cig1Not1-5 BamH1-Cig1Not1-3 TL2001 TL2002 colPCR-Rev  Primer sequence CAAAGGGCAATTGACACTAG CGTGACTATGTCGACTTTGAGTTGTA TCAGGAACATCGTATGGGTACTGTGTATCGATGA TCAGTC ACGTTCCAGATTACGCTGCTGACTTCGACACGAG TCCCG AAGAATGTTAACATTTGTAGCATCG GACTGATCATCGATACACAGTACCCATACGATGT TCCTGA CGGGACTCGTGTCGAAGTCAGCAGCGTAATCTGG AACGT CTCGTGGGATCCATCAGTGTTCAACGCAGAGCCC AGATC AGTCAGGCGGCCGCTCAGAGACGCTCCTTGGTGG GTATGCCAG ACTAGTAACGGCCGCCAGTGTGCTGGAATT GCCGCCAGTGTGATGGATATCTGCAGAATT GCCACTCGAATCCTGCATGCTTATG  2.2.3.1. Construction of the cig1Δ mutant and complemented strains To construct a cig1:NAT disruption allele in the serotype A strain H99, a 3.4Kb fragment of the CIG1 gene was obtained by amplification of genomic H99 DNA using primers cig1-R and cig1-F; this fragment was cloned into the TA vector (Invitrogen). The nourseothricin (NAT) resistance cassette was obtained by amplification from the vector pCH233 with the introduction of SnabI and ClaI restriction enzyme sites. The fragment was cloned into the SnabI and ClaI sites in the CIG1 open reading frame resulting in partial deletion of the gene. The 4.8 Kb cig1:NAT disruption allele was introduced into strain H99 by biolistic transformation by the method of Toffaletti et al. (1993) and transformants were screened by colony polymerase chain reaction (PCR) using primers cig1 up and cig1 low. Transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic H99 DNA using primers cig1A-comp3 and cig1A-comp4 (Figure 2.1A). To 38  complement the disruption mutation, the wild-type CIG1 gene was reintroduced to one of the mutants on the vector pHYG-kB1 that confers HYG resistance (Hua et al., 2000). Construction of the cig1Δ mutant and complemented strains was done by Carmelo Biondo and Tian Lian, respectively.  2.2.3.2. Construction of the cig2Δ and cig1Δ cig2Δ mutants, and complemented strains The cig2:NEO deletion allele was constructed by overlap PCR using the methods described by Davidson et al. (2002) and Yu (2004). In the first round of PCR, the 1,128 bp sequence upstream of the 5’ end of the CIG2 gene was amplified from genomic H99 DNA with primers TL2081 and TL2082, and the 1,080 bp sequence downstream of CIG2 was amplified with primers TL2083 and TL2084. The 1,970 bp sequence of the neomycin gene (NEO) was amplified from plasmid pJAF1 with primers TL2001 and TL2002. The nested primers TL2085 and TL2086 were then used to overlap the three products to yield the 4,096 bp cig2:NEO deletion allele. The construct was introduced into C. neoformans serotype A strain H99 and into the cig1Δ mutant by biolistic transformation to generate the cig2Δ and cig1Δ cig2Δ mutants, respectively. The resulting transformants were screened by colony PCR using primers TL2087 and TL2088. Transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic H99 DNA using primers cig2A-comp3 and cig2A-comp4 (Figure 2.1B). Construction of the cig2Δ and cig1Δ cig2Δ mutant strains was done by Tian Lian. To complement the cig2Δ mutation, the wild-type CIG2 gene was reintroduced into one of the mutants by biolistic transformation. The CIG2:HYG construct was prepared by overlap PCR. The left arm of the construct, including the open reading frame of the CIG2 gene, 696 bp  39  upstream of the start codon and 275 bp downstream of the stop codon, was amplified from strain H99 genomic DNA with primers cig2A-comp1 and cig2A-comp2. The right arm was amplified with primers cig2A-comp3 and cig2A-comp4 and included 725 bp downstream of the CIG2 gene. The 2,201bp sequence of the HYG marker was amplified from plasmid pJAF15 (kindly provided by Dr. J. Heitman, Duke University, Durham, NC, U.S.A.) with primers TL2001 and TL2002. Primers cig2A-comp1 and cig2A-comp4 were then used to overlap the three products to yield the 5,092 bp CIG2:HYG construct. Transformants were screened by colony PCR using primers cig2A-int-F and cig2A-int-R. A construct containing the CIG1 gene and the HYG resistance cassette was generated by overlap PCR to complement cig1Δ in the cig1Δ cig2Δ double mutant. In the first round of PCR, the wild-type CIG1 gene was amplified from genomic DNA of strain H99 using primers cig1Acomp1 and cig1A-comp2 and the construct included 687 bp upstream of the gene and 167 bp downstream of the gene. The HYG gene was amplified as described above. The third fragment of the construct included a 692 bp sequence downstream of the CIG1 gene and was amplified with primers cig1A-comp3 and cig1A-comp4. Primers cig1A-comp1 and cig1A-comp4 were then used to overlap the three products to yield the 4,751 bp CIG1:HYG construct which was then introduced into the cig1Δ cig2Δ mutant by biolistic transformation. Transformants were screened by colony PCR using primers cig1A-int-F and cig1A-int-R.  2.2.3.3. Construction of the cig3Δ and cig1Δ cig2Δ cig3Δ mutants The cig3:HYG deletion allele was constructed by overlap PCR. In the first round, the 855 bp sequence upstream of the 5’ end of the CIG3 gene was amplified from genomic H99 DNA with primers cig3A-1 and cig3A-3, and the 963 bp sequence downstream of CIG3 was  40  amplified with primers cig3A-4 and cig3A-6. The 2,201 bp sequence of the HYG gene was amplified as described above. The nested primers cig3A-7 and cig3A-8 were then used to overlap the three products to yield the 3,904 bp cig3:HYG deletion allele. The construct was introduced into the C. neoformans serotype A strain H99 and into the cig1Δ cig2Δ mutant by biolistic transformation to generate the cig3Δ mutant and the cig1Δ cig2Δ cig3Δ triple mutant, respectively. Transformants were screened by colony PCR using primers cig3A-1 and colPCRRev. Transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic H99 DNA using primers cig3A-4 and cig3A-6 (Figure 2.1C).  2.2.3.4. Construction of the cig1Δ cfo1Δ mutant and the cig1Δ cfo1Δ CIG1 complemented strain The cfo1:NEO construct was amplified using primers CFO1-KO5 and CFO1-KO6 from genomic DNA from the cfo1Δ cfo2Δ mutant kindly provided by Dr. W. Jung (Jung et al., 2009). The 3,874 bp cfo1:NEO construct was introduced into the cig1Δ mutant by biolistic transformation to yield the cig1Δ cfo1Δ. The resulting transformants were screened by colony PCR using primers CFO1-KO4 and colPCR-Rev. Transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic H99 DNA using primers CFO1-KO1 and CFO1-KO22 (Figure 2.1D). To complement the cig1Δ deletion mutation in the cig1Δ cfo1Δ strain, the CIG1:HYG construct described in Section 2.2.3.2 was introduced into the cig1Δ cfo1Δ double mutant by biolistic transformation. Reintroduction of the wild-type allele was confirmed by PCR using primers cig1A-int-F and cig1A-int-R.  41  Figure 2.1 Confirmation of mutants. Southern blot analyses were performed to confirm deletion of the CIG1 (A), CIG2 (B), CIG3 (C) and CFO1 (D) genes. In panel A, genomic DNA of the indicated strains was digested with NcoI and hybridized with the downstream arm of the CIG1:HYG construct. In panel B, genomic DNA from the indicated strains was digested with ScaI and hybridized with the downstream arm of the CIG2:HYG construct. In panel C, genomic DNA from the indicated strains was digested with SpeI and hybridized with the upstream arm of the cig3:HYG construct. In panel D, genomic DNA from the indicated strains was digested with EcoRV and SalI and hybridized with the downstream arm of the cfo1:HYG construct.  2.2.3.5. Construction of an HA::Cig1 fusion allele The HA::Cig1 strain, which expresses a triple copy of the HA epitope inserted at the 30th amino acid position of CIG1 (just downstream of the signal peptide) was constructed by overlap PCR. In the first round, the 5' end of the CIG1 gene was amplified with primers 311 and 300, the 3' end was amplified with primers 303 and 312, and the HA epitope was amplified with primers 301 and 302. The HA sequence was amplified from the plasmid pIS027-3xHA. Primers 311 and 312 were then used to overlap the three products to yield the 800 bp HA::Cig1 allele. The fragment from the overlapping PCR was cloned into the SalI and HpaI sites of the pJAF15 plasmid which confers HYG resistance. The plasmid was transformed into the cig1Δ mutant thereby potentially complementing the cig1Δ mutation. Integration of the HA epitope-tagged  42  allele was confirmed by PCR and expression was confirmed by Western blot analysis. Construction of the HA::Cig1 strain was done by Tian Lian.  2.2.3.6. Construction of an E. coli strain expressing Cig1::GST Total RNA was extracted using the RNeasy mini kit (Quiagen) from H99 cells cultured in LIM.  The RNA was then reverse transcribed into cDNA using SuperScript II reverse  transcriptase (Invitrogen). A CIG1 cDNA fragment (without the signal peptide) of 789 bp was amplified using primers BamH1-Cig1-Not1-5 and BamH1-Cig1-Not1-3, and the product was digested with BamHI and NotI, and cloned into BamHI/NotI digested pGEX-6P-3 (GE Healthcare, USA) to generate plasmid pCig1c. The plasmid pCig1c was then transformed into E. coli strain BL21 for protein expression.  Construction of the E. coli strain expressing  Cig1::GST was done by Dr. Guanggan Hu.  2.2.4. Purification of the recombinant Cig1::GST fusion protein Expression of the recombinant GST-tagged Cig1 fusion protein was induced by the addition of 0.5 mM IPTG to a culture in Luria-Bertania medium (LB; 1% tryptone, 1% sodium chloride, 0.5% yeast extract) followed by overnight growth at room temperature. The extraction and purification of the recombinant Cig1::GST protein was performed using a glutathione affinity resin kit (Qiagen), following the manufacturer’s recommendations. The concentration of purified Cig1::GST protein was determined by absorption at 280 nm using a calculated extinction coefficient for Cig1 (72770 M-1cm-1). Purification of the recombinant Cig1::GST fusion protein was performed by Dr. Guanggan Hu.  43  2.2.5. Quantitative real time PCR Quantitative real time (RT) PCR was used to examine gene expression. The cells were grown in LIM alone or LIM supplemented with FeCl3 or heme for 6 hours at 30°C with shaking. Total RNA was extracted using the RNeasy kit (Qiagen), treated with DNase (Qiagen) and cDNA was synthesized using the SuperScript First Strand System (Invitrogen) following the manufacturers’ recommendations.  PCR reactions were monitored using the 7500 system  (Applied Biosystems) as described previously (Tangen et al., 2007), while the primers used were designed using Primer Express software 3.0 (Applied Biosystems) and are listed in Table 2.3. The relative gene expression was quantified using the SDS software 1.3.1 (Applied Biosystems), based on the 2-ΔΔCT method (Livak and Schmittgen, 2001). The average cycle threshold value (Ct) of 18S rRNA was used for normalization.  Table 2.3. Primers used for quantitative RT-PCR Primer identification Primer sequence CIG1A-RT-F GGTGGTCCGGTTTCCTTCA CIG1A-RT-R GACTCGTGGTCGTGCATAACA CIG2A-RT-F GCCGTCTTCGGCTTTGC CIG2A-RT-R GGAGGGCATTACGGGAGAA CIG3A-RT-F CGCTACTATTGTTGCCTTCATCTC CIG3A-RT-R TGCCATTACTGGTAGTGTTGAAGTC 18S-RT-F AACAGGTCTGTGATGCCCTTAGA 18S-RT-R ACTCGCTGGCTCAGTCAGTGT  Target Gene CIG1 CIG1 CIG2 CIG2 CIG3 CIG3 18S rRNA 18S rRNA  2.2.6. Protein extraction and Western blot analysis Three types of C. neoformans protein fractions, whole-cell, cytosolic and membrane, were employed to examine the localization of Cig1 (Chen et al, 1999). Briefly, cells expressing HA::Cig1 fusion protein were grown for 24 hours at 30°C in 50 mL LI-YNB. The cells were washed twice with ice-cold, sterile distilled water and lyophilized overnight. Glass beads (a 0.5  44  mL volume of 4 mm diameter beads) were added to the pellets and the cells were disrupted by vortexing to generate a fine powder. The cell powder was suspended in ice cold lysis buffer containing phosphate-buffered saline (PBS; Gibco), 1 mM DTT (BioChemika), 1 mM ethylenediaminetetraacetic acid (EDTA; Sigma), 1% Triton X-100 (Sigma), and complete protease inhibitor cocktail (Roche). The disrupted cells were incubated at 37°C for one hour and approximately 90% of cells were confirmed to be broken by microscopic observation. Cell debris was removed by centrifugation (3,000 × g, 2 minutes, 4°C), and the supernatants, representing the total cell extracts, were placed in tubes. For the separation of cytosol and membrane fractions, these extracts were centrifuged in a Beckman Optima Ultracentrifuge with the TLA100.3 rotor (Beckman Instruments Inc.) at 100,000 × g for one hour at 4°C. The supernatant consisted of the cytosolic protein fraction. The membranes were resuspended in wash buffer containing PBS, 1 mM EDTA and protease inhibitors, and collected by centrifugation (100,000 × g, 30 minutes, 4°C).  Membrane proteins were extracted in PBS  containing 1 mM EDTA, 1% sodium dodecyl sulfate (SDS), and protease inhibitors by heating the mixture to 65°C for 20 minutes, and removing the cell debris by centrifugation (12,000 × g, 15 minutes, 4°C). Protein concentrations were determined by using the Bio-Rad Protein Assay (Bio-Rad). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the Bio-Rad Mini-Protean 3 system at 125 V in 25 mM Tris-HCl, 192 mM glycine, 0.1% SDS running buffer according to the method of Laemmli (1970). Transfer to nitrocellulose membranes was carried out using the Transblot SD semi dry transfer system (Bio-Rad) at 25 volts for 20 minutes with a buffer composed of 25 mM Tris-HCl, 192 mM glycine, 0.05% SDS, and 20% methanol. Membranes were blocked with 2% milk in Tris-buffered saline (TBS; 50 mM Tris, 150 mM  45  NaCl [pH 7.4]) for one hour at room temperature. Membranes were incubated with rabbit antiHA antibody (Sigma) diluted in TBS buffer with 1% milk to detect HA::Cig1. Alternatively, rabbit anti-Cig1 antibodies prepared against the recombinant Cig1::GST fusion protein (Pro-Sci Inc.) or anti-GST monoclonal antibodies produced in mice (GenScript) were used to detect wildtype Cig1 and Cig1::GST, respectively. After primary antibody incubation, membranes were washed three times with TBS and then incubated with horseradish peroxidase-conjugated goat antibody to rabbit IgG (Cedarlane) or peroxidase-conjugated goat antibody to mouse IgG (BioRad) at a dilution of 1:2,000 in TBS buffer with 1% milk. Membranes were washed again as described and the bands were visualized with Super Signal West Pico chemiluminescence substrate (Thermo Scientific). All antibody incubations were performed for one hour at room temperature.  2.2.7. Vesicle preparation Cells expressing HA::Cig1 fusion protein were grown in one liter of LI-YNB with or without 100 µM FeCl3 for four days at 30°C with shaking. The extracellular vesicles were isolated from the culture supernatant as described by Rodrigues et al., (2007). Briefly, the culture supernatant was harvested by centrifugation, concentrated 10 times using the stirred ultrafiltration cell with a 100,000 molecular weight cut-off membrane (Amicon).  The  concentrated supernatant was then ultracentrifuged and the pellet was resuspended in 1 mL of PBS. The concentration of proteins within the vesicle preparations was determined by using the Bio-Rad Protein Assay (Bio-Rad) and normalized. The proteins were then separated using SDSPAGE and the HA::Cig1 protein was detected by Western blot analysis, as described above.  46  2.2.8. Fluorescence microscopy The fluorescence of cells expressing HA::Cig1 fusion protein was examined after overnight growth at 30°C with shaking in 25 mL of LI-YNB.  The cells were harvested by  centrifugation, fixed with 3% paraformaldehyde in PBS for 30 minutes, and permeabilized with 1% Triton-X-100 in PBS for 10 minutes. The cells were incubated with rabbit anti-HA antibody diluted 1:50 in PBS with 1% Bovine Serum Albumin (BSA). The cells were then washed with 1% BSA-PBS and incubated with fluorescence-conjugated goat antibodies to rabbit IgG (Alexa 488, Molecular Probes) diluted 1:100 in 1% BSA-PBS. The cells were finally washed in 1% BSA-PBS and viewed on a Zeiss 510 Meta laser scanning confocal microscopy for fluorescence with the appropriate fluorescence filter. For visualizing the capsule, a monoclonal mouse antiGXM antibody diluted 1:100 was used in combination with a fluorescence-conjugated antibodies to mouse IgG (Alexa 568, Molecular Probes). The monoclonal mouse antibody was kindly provided by Dr. A. Casadevall (Albert Einstein College of Medicine, Bronx, NY, U.S.A.). The extracellular vesicles were also visualized by immuno-fluorescence microscopy using anti-HA antibody and fluorescence-conjugated secondary antibody, as described above. A ceramide dye (Bodipy TR ceramide complexed to BSA, Molecular Probes) was added at 1:100 with the primary and secondary antibodies as a marker for the vesicles. The vesicles were incubated with the dye and antibodies for 30 min at 4°C and one hour at room temperature. The washes were performed with PBS filtered through a 0.22 µm filter and the vesicles were collected from the washes by ultracentrifugation at 100,000 × g, for 1 hour at 4°C.  47  2.2.9. Uptake of metalloporphyrins To test for metalloporphyrin uptake, cells were grown overnight in YPD but not starved for iron. The cells were washed and 10-fold serial dilutions were prepared in low iron water. YPD or LIM plates with or without 10 µM heme or 100 µM FeCl3 were spread with 200 µl of 10 µM Ga-protoporphyrin (Ga-PPIX; Frontier Scientific), 100 µM Mn-protoporphyrin (Mn-PPIX; Frontier Scientific) or 10 µM GaCl3 (Sigma) prior to spotting 5 µl of the diluted cells. The plates were incubated for two days at 30°C. To determine whether Ga-PPIX had fungicidal or fungistatic activity, LIM alone or LIM supplemented with 100 µM FeCl3, or 10 µM heme and 100 µM Ga-PPIX was inoculated with 5 x 107 cells/mL. The cultures were incubated at 30°C for 24 hours. Following this incubation period, the cells were harvested by centrifugation (15,000 x g for 1 minute) using an Eppendorf centrifuge 5424, washed twice with water to remove any residual Ga-PPIX, diluted and plated on YPD agar plates to determine the number of viable cells. The plates were incubated at 30°C for 48 hours before counting the CFU.  2.2.10. Heme binding assays Heme binding to Cig1 was determined by batch adsorption to heme-agarose beads (bovine hemin-agarose beads; Sigma), as described by Tsutsui and Mueller (1982a) with some minor modifications. Briefly, 10 µg of an extracellular vesicle preparation derived from cells expressing HA::Cig1 fusion protein or 0.3 µg of the purified Cig1::GST fusion protein were incubated with 100 µl heme-agarose beads for one hour at 37°C. After three washes with 0.5 M NaCl, 10 mM sodium phosphate, pH 7.5, the proteins bound to the beads were eluted with 100 µl protein electrophoresis sample buffer and heated at 95°C for two minutes.  Sepharose 4B  48  (Sigma) agarose beads without the heme conjugate were used as a negative control to verify for non-specific binding to the agarose beads. Following adsorption to the beads, the proteins from different fractions were separated by SDS-PAGE and HA::Cig1 was detected by Western blot analysis using anti-HA antibodies as described above. Alternatively, recombinant Cig1::GST was detected using anti-GST antibodies.  2.2.11. Mass spectrometry Cells from the wild-type strain were grown in LI-YNB with or without 100 µM FeCl3 at 30°C with shaking. After 4 days, the culture supernatant was harvested by centrifugation (15, 000 × g, 15 minutes, 4°C) and concentrated 10 times using the stirred ultrafiltration cell with a 3,000 molecular weight cut-off membrane (Amicon).  A batch adsorption experiment was  performed using 20 mL of concentrated culture supernatant and 300 µl heme-agarose beads as described above (Tsutsui and Mueller, 1982a). Proteins were allowed to bind for 1.5 hours at 37°C and eluted with 150 µl protein electrophoresis sample buffer for 30 minutes at 37°C before electrophoretic separation on an SDS-PAGE gel. After electrophoresis, the gel was fixed with 40% ethanol; 10% acetic acid overnight, stained for 2 hours with a modified Coomassie Blue G250 stain (Candiano et al., 2004), and destained with water. The whole lane representing all the proteins eluted from the heme-agarose beads or from the agarose beads was excised from the gel and the proteins were in-gel digested with trypsin as described by Shevchenko et al., (1996). The trypsinized peptides were purified using the stop and go extraction tips as described by Rappsilber et al., (2003), and analyzed by tandem mass spectrometry (MS; LTQ Orbitrap Velos, Thermo Scientific). The tandem MS spectra were then used to search the C. neoformans H99 protein  sequence  database  49  (www.broadinstitute.org/annotation/genome/cryptococcus_neoformans). The mass spectrometry analysis (excluding the sample preparation) was performed by Nikolay Stoynov.  2.2.12. Absorption spectrophotometry Titrations of purified recombinant Cig1::GST protein with heme were monitored by absorption spectroscopy in the Soret range as previously described (Gaudin et al., 2011). Briefly, increasing amounts of heme dissolved in 0.1 M NaOH and diluted in 50 mM Tris, 100 mM NaCl buffer (pH 7.5) were added to 1 mL of 5 µM Cig1::GST in buffer. The preparation was incubated at room temperature for 5 minutes after adding heme to allow binding before measuring the absorbance spectra. Heme added to buffer without protein served as a reference. Spectra were measured using a Cary 50 Bio UV-visible spectrophotometer (Agilent Technologies) with an optical path length of 1 cm in a quartz cell.  2.2.13. Isothermal titration calorimetry experiments Isothermal titration calorimetry (ITC) was performed using a MicroCal iTC200 (GE Healthcare). Samples were in 50 mM Tris pH 7.5, 0.1 M NaCl or 10 mM phosphate pH 7 or 7.5, 0.5 M NaCl. Titrations were performed by injecting consecutive 2 µL aliquots of heme solution (0.4-0.7 mM) into the calorimeter (volume = 200 µL) containing Cig1::GST protein (4-50 µM). All solutions were degassed at room temperature before loading in the ITC. The heat of dilution data were obtained by titrating 2 µL injections of heme solution into Cig1-free buffer. All titrations were performed at 25 C. ITC experiments were performed by Dr. Louise Creagh.  50  2.2.14. Virulence assays and determination of fungal loads in mouse tissue The virulence of each cryptococcal strain was examined using female A/Jcr mice (4 to 6 weeks old) from the Jackson Laboratory (Sacramento, CA). The iron-starved fungal cells were washed twice and resuspended in PBS (Invitrogen, Canada). The A/Jcr mice in groups of 10 were anesthetized intraperitoneally with ketamine (80 mg/kg of body weight) and xylazine (5.5 mg/kg) in PBS and inoculated intranasally with 50 μL of the cell suspensions (5 × 104 cells). The number of viable cells in each inoculum was confirmed by CFU. The health status of the mice was monitored daily post-inoculation.  Mice reaching the humane endpoint were  euthanized by CO2 asphyxiation. Three mice from each group were used at the endpoint to assess fungal loads in the lungs and brains of the animals. Following euthanasia, the brains and the lungs were aseptically removed and immersed in PBS.  Organs were homogenized using an automated tissue  homogenizer (Retsch, PA, USA). The samples were serially diluted in PBS and plated on YPD supplemented with 35 µg/mL chloramphenicol. After two days of incubation at 30oC, the CFU were counted manually. The protocol for the virulence assays (protocol A08-0586) was approved by the University of British Columbia Committee on Animal Care. Statistical analysis of survival differences was performed by Kaplan-Meier survival curves and log rank tests. An unpaired, two-tailed t-test was used to determine the differences in mean CFU per organ (i.e., brain and lung) between mice inoculated with different cryptococcal strains. A P-value of 0.05 or less was considered significant. All statistical analyses were carried out using GraphPad Prism version 4 for Windows (GraphPad Software, San Diego, CA). The virulence assay and determination of fungal loads in mouse tissue was performed by Joyce Wang.  51  2.3. Results 2.3.1. Characterization and expression of the CIG1 gene in C. neoformans Previous analysis of transcriptome changes upon growth of C. neoformans var. neoformans in high and low iron concentrations identified the transcript of the CIG1 gene as the most abundant mRNA in cells grown in LIM, and a strong regulation of the transcript level by iron was also observed (Lian et al., 2005).  The amino acid sequence of Cig1 was further  examined to potentially give insight into its role. In silico analysis of the predicted 282 amino acid polypeptide sequence did not identify any known functional domains but revealed structural characteristics common to mannoproteins, such as a putative signal peptide, four potential sites for N-glycosylation, and a serine/threonine-rich region for potential O-glycosylation as shown in Figure 2.2 (Mansour & Levitz, 2003; Biondo et al., 2005). One trans-membrane region was also predicted near the C-terminus. Additionally, a chitin-binding fold was predicted by remote protein homology and this region shared 22% similarity to the chitin-binding protein found in S. marcescens. The predicted chitin-binding fold starts at the 57th amino acid of the Cig1 sequence and extends up to the 148th amino acid. A putative immunoglobulin-like beta-sandwich fold that overlaps the chitin-binding fold was also identified based on structure prediction (11% identity to E set domains of sugar-utilizing enzymes) and this fold extends from the 68th to the 159th amino acid of the Cig1 sequence.  52  Figure 2.2 Cig1 is an iron-regulated mannoprotein in C. neoformans. Diagram of the Cig1 polypeptide illustrating the predicted sites for post-translational modifications, domains and folds.  Orthologs of the CIG1 gene are present in C. neoformans var. grubii, the variety responsible for the majority of cases of cryptococcosis in AIDS patients, as well as in Cryptococcus gattii, a related species responsible for infections in immunocompetent individuals (Currie and Casadevall, 1994; Franzot et al., 1997; Viviani et al., 2006; Stephen et al., 2002). The amino acid sequences of the CIG1 orthologs share clear similarities as shown by the multiple sequence alignment (Figure 2.3). A BLASTp search identified a putative homolog of Cig1 in the fungal pathogen Trichosporon asahii which shares 35% identity with the C. neoformans protein.  This protein in T. asahii was also annotated as a cytokine-inducing  glycoprotein, probably based on its similarity with the C. neoformans protein. Finally, potential but distant homologs were found in various other fungal species (e.g., Gibberella species, Penicillium sp., Nectria sp., Aspergillus sp., Glomerella sp.); however, the predicted homologs were annotated as hypothetical proteins.  53  Figure 2.3 Cig1 is conserved in the different varieties of C. neoformans. Multiple sequence alignment of the Cig1 amino acid sequence from C. neoformans var. neoformans (B3501A), C. neoformans var. grubii (H99) and C. gattii (WM276).  Finally, two potential paralogs of CIG1 were found in C. neoformans var. grubii based on sequence homology, although the similarity between amino acid sequences was relatively low (27-29%) as seen in Figure 2.4. The abundance of the CIG1 transcript in cells grown in LIM and the conservation of the gene within different varieties of cryptococci and potentially among multiple fungi suggest that Cig1 plays an important role in C. neoformans under low iron conditions, such as those encountered in the mammalian host environment during cryptococcosis. Consequently, the role of Cig1 in virulence was further studied in the H99 strain 54  of C. neoformans var. grubii, a strain commonly used to investigate mechanisms of cryptococcal pathogenesis.  Figure 2.4 CIG2 and CIG3 are putative paralogs of CIG1. Multiple sequence alignment of the Cig1, Cig2, and Cig3 amino acid sequences from C. neoformans var. grubii strain H99.  The iron-regulation of CIG1 observed in var. neoformans was also observed in var. grubii strain H99 as demonstrated by quantitative PCR (Figure 2.5A). In this strain, CIG1 transcript levels were reduced more than four-fold in presence of inorganic (FeCl3) or mammalian-host-related (heme) iron sources, compared to the low iron condition. Based on the heme concentrations tested, the regulation of CIG1 did not vary in response to the amount of heme but rather in response to its absence or presence (Figure 2.5B). Specifically, a lower concentration (10 µM) of heme resulted in the same level of expression of CIG1 as a higher  55  concentration (100 µM) of heme.  Therefore, C. neoformans var. grubii responds to iron  deprivation in a similar fashion to var. neoformans by increasing the expression of CIG1.  Figure 2.5 Expression of CIG1. The wild-type cells were grown in LIM, LIM + 10 µM FeCl3, or LIM + 10 µM heme (A) or in LIM with different concentrations of heme (B). All experiments were repeated three times and the data are plotted as the average ± SD. Statistical analysis was performed by Student’s t test. The * represents significant differences at P < 0.05 relative to LIM alone.  2.3.2. Cig1 is required for heme utilization The regulation of CIG1 by iron suggests that the protein plays an iron-related role in C. neoformans.  Therefore, mutants lacking CIG1 were constructed for phenotypic analysis.  Complemented strains were also created by reintroducing the wild-type copy of CIG1 into the mutants. The complemented strains were included as controls in all experiments. To examine the role of Cig1 in iron utilization, the cig1Δ mutant was grown in LIM with the addition of different iron sources including FeCl3, heme, hemoglobin, and the iron-loaded siderophore, ferrioxamine. Growth of the cig1Δ mutant was measured spectrophotometrically and compared to the wild-type and complemented strains (Figure 2.6A-E). As expected, iron-depleted medium did not support growth of any of the strains confirming that iron is necessary for the growth of C. neoformans. The cig1Δ mutant grew as well as the wild-type and complemented strains in LIM supplemented with FeCl3 and ferrioxamine. However, when grown in LIM with heme added as 56  a sole iron source, the cig1Δ mutant remained in lag phase for significantly longer (60 hours) than the wild-type strain (36 hours). The cig1Δ mutant eventually did initiate exponential growth to reach stationary phase at a similar cell density as the wild-type strain. The reintroduction of CIG1 into the cig1Δ mutant restored growth, confirming that the phenotype of the mutant was due to deletion of CIG1. Similarly, the cig1Δ mutant also had a longer lag phase when grown in hemoglobin as a sole iron source, but at this time, it is not known how C. neoformans processes hemoglobin. Preliminary studies from our laboratory did not detect any hemolytic activity by C. neoformans on blood agar (Drs. W. Jung and G. Hu, personal communications). It is likely that the hemoglobin preparation used in this study was contaminated with free heme, and further work is therefore needed to investigate the use of hemoglobin. Overall, the data indicate that Cig1 is required for efficient utilization of heme as an iron source under low iron conditions but has no significant role in utilization of FeCl3 or ferrioxamine under the conditions tested. Growth was also tested on solid LIM containing various concentrations of heme (Figure 2.6F). Interestingly, the cig1Δ mutant displayed a growth defect at a lower heme concentration (10 µM) but not at higher heme concentrations (50-100 µM) suggesting a possible role for Cig1 in highaffinity heme acquisition. High-affinity uptake systems are usually important in conditions of low substrate concentration while low-affinity uptake systems play a role when the substrate is present in high concentrations.  57  Figure 2.6 Cig1 is required for growth in LIM supplemented with heme. Growth of ironstarved cells in LIM (A), LIM + 10 µM FeCl3 (B), LIM + 10 µM ferrioxamine (C), LIM + 10 µM heme (D), and LIM + 10 µM hemoglobin (E). Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD. (F) Ten-fold serial dilutions of iron-starved cells spotted onto LIM + 10 µM, 50 µM or 100 µM heme as indicated. The plates were incubated at 30°C for two days and photographed. The experiment was repeated three times and a representative photograph is shown. Figure reproduced and modified from Cadieux et al. (2013).  Aggregates of cells were detected during growth in LIM supplemented with heme, and this phenomenon will be discussed further in Chapter 3. However, the formation of these aggregates prompted a further examination of the growth in LIM supplemented with heme to confirm that the extended lag phase observed for the cig1Δ mutant was indeed a result of the cells not dividing, and not due to inaccurate measurements of optical density. Therefore, growth was assessed by three additional techniques including microscopically counting the cells, 58  determining the number of viable cells by plating and counting the CFU, and lastly, measuring metabolic activity by using the formation of formazan as an indicator of total reductase activity. The results from all three of these methods correlated well with the results obtained by measuring the optical density of the cells at different time points (Figure 2.7), thus validating the use of optical density as an indicator of cell growth. Considering that measuring optical density is the simplest and fastest method to determine cell numbers, this method was used throughout the remainder of the growth studies presented in this thesis.  Figure 2.7 Further examination of growth in LIM supplemented with heme. Growth of iron-starved cells in LIM + 10 µM heme as determined by measuring the optical density at 600 nm (A), by counting the cells microscopically (B), by counting CFU on YPD plates (C), and by measuring the total reductase activity of the cells using TTC (D). All experiments were repeated three times and the data are plotted as the average ± SD.  59  2.3.3. CIG2 and CIG3 do not contribute to iron utilization CIG2 and CIG3 were identified as potential paralogs of CIG1. Although CIG2 and CIG3 share a low level of similarity with CIG1 (27-29% identity), it is possible that they share functional redundancy. The expression of these genes in low iron and iron-replete medium was investigated by quantitative RT-PCR (Figure 2.8A). CIG2 appeared to be regulated in a similar fashion as CIG1 (i.e., down-regulated in the presence of FeCl3 and heme) and CIG3 was also similarly regulated in presence of FeCl3, but it was not differentially expressed in the presence versus absence of heme. Mutants lacking CIG2 or CIG3, as well as mutants lacking both CIG1 and CIG2, or even all three CIG genes were constructed to determine the contribution of each paralog in iron utilization. Complemented strains were also created by reintroducing the wild-type copy of CIG1 into the mutants. All strains were tested for growth in LIM supplemented with heme (Figure 2.8B). The cig2Δ and cig3Δ mutants did not exhibit any difference in growth compared to the wild-type or complemented strains in LIM supplemented with heme whereas the cig1Δ cig2Δ double mutant and the cig1Δ cig2Δ cig3Δ triple mutant showed similar growth defects as the cig1Δ single mutant. Based on these results, CIG2 and CIG3 do not appear to play an important role in heme utilization and are most likely not functionally redundant with CIG1.  60  Figure 2.8 Cig2 and Cig3 are not required for growth in LIM supplemented with heme. (A) Expression of CIG2 and CIG3 in the wild-type grown in LIM, LIM + 10 µM FeCl3, or LIM + 10 µM heme. All experiments were repeated three times and the data are plotted as the average ± SD. Statistical analysis was performed by Student’s t test. The * represents significant differences at P < 0.05 relative to LIM alone. (B) Growth of iron-starved cells in LIM + 10 µM heme was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD.  2.3.4. The reductive high-affinity iron uptake system does not contribute to Cig1-mediated heme utilization The reductive high affinity iron uptake system is required for uptake of inorganic iron and iron from transferrin (Jung et al., 2008; 2009). To determine whether this high-affinity uptake system contributes to the role of Cig1 in utilization of heme, a strain lacking the CFO1 gene was used. CFO1 encodes the essential ferroxidase component of the high-affinity uptake system (Jung et al., 2009). The CFO1 gene was also deleted in the cig1Δ mutant. The growth of these strains in LIM, FeCl3, ferrioxamine, and heme as sole iron sources was measured as described above and compared to the wild-type and complemented strains (Figure 2.9). As previously reported, the cfo1Δ mutant was unable to grow in LIM supplemented with FeCl3 but 61  grew as well as the wild-type strain in LIM supplemented with heme, confirming that the reductive high-affinity uptake system is not involved in heme acquisition. Similarly, when CFO1 was deleted in the cig1Δ mutant background, the strain was also unable to grow in LIM supplemented with FeCl3, while the cig1Δ cfo1Δ mutant showed a similar growth defect as the cig1Δ mutant when grown in LIM with heme. The growth defect for heme was restored when CIG1 was reintroduced in the cig1Δ cfo1Δ mutant confirming that the phenotype is due to the loss of CIG1 and not the loss of CFO1.  These results indicate that the role of Cig1 is  independent of Cfo1 and that the high-affinity iron uptake pathway does not contribute to Cig1mediated heme utilization.  Figure 2.9 Cfo1 is required for growth in LIM supplemented with FeCl3 but not with heme. Growth of iron-starved cells in LIM (A), LIM + 10 µM FeCl3 (B), LIM + ferrioxamine (C), and LIM + 10 µM heme (D). Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD. Figure reproduced and modified from Cadieux et al. (2013).  62  2.3.5. Cig1 is involved in heme uptake Non-iron metalloporphyrins (MPs), which are toxic heme analogs, were employed as reagents to indirectly investigate the potential role of Cig1 in heme uptake. Non-iron MPs, such as Ga-PPIX and Mn-PPIX, share structural similarities with heme and, therefore, gain entry into the cell via heme uptake systems, as shown in bacteria (Stojiljkovic et al., 1999). Non-iron MPs may stimulate production of reactive oxygen radicals leading to toxicity and displace heme in essential metabolic functions (Stojiljkovic et al., 1999). Growth of the strains was compared in the presence of Ga-PPIX or Mn-PPIX (Figure 2.10). When grown on YPD or LIM with added FeCl3, the wild-type, cig1Δ mutant and complemented strains all grew well in the absence or presence of Ga-PPIX indicating that the heme uptake system was not essential in these conditions (Figure 2.10A). Interestingly, the cfo1Δ mutant did not grow on YPD in the presence of Ga-PPIX, while the cig1Δ cfo1Δ mutant grew well, presumably because heme uptake systems are up-regulated in absence of the high-affinity iron uptake system. Reintroduction of CIG1 into the double mutant also resulted in susceptibility to Ga-PPIX although not to the level of the cfo1Δ mutant (possibly because of partial complementation). Additionally, the cfo1Δ mutants did not grow well in LIM supplemented with FeCl3 whether Ga-PPIX was absent or present. When heme was provided as the iron source, the wild-type, cfo1Δ mutant, and the complemented strains were unable to grow in the presence of Ga-PPIX while the cig1Δ mutant grew well. The toxicity of Ga-PPIX for the wild-type strain was eliminated by adding excess heme (Figure 2.10B). Interestingly, the cig1Δ cfo1Δ mutant had difficulty growing in LIM supplemented with heme plates in the presence or absence of Ga-PPIX; this phenotype is likely due to a contribution of Cfo1 to an additional mechanism of heme use and/or to high-affinity acquisition of any free iron contaminating the heme. Mn-PPIX has also been reported to be toxic for cells with a  63  functional heme uptake system although Mn-PPIX is less potent than Ga-PPIX in bacteria (Stojiljkovic et al., 1999). Accordingly, growth in the presence of Mn-PPIX also yielded similar results to Ga-PPIX for C. neoformans, although a 10-fold higher concentration of Mn-PPIX was required. Finally, all of the strains grew well in the presence or absence of GaCl3 thus supporting the idea that toxicity was due to uptake of Ga-PPIX rather than gallium. These results suggest that Cig1 contributes to the uptake of Ga-PPIX resulting in toxicity for the cells, but in absence of Cig1, the cells do not acquire Ga-PPIX and, consequently, are not affected. Instead, a separate heme uptake system may be present that allows the cig1Δ mutant to grow in LIM supplemented with heme as the sole iron source in the presence of Ga-PPIX. In this case, the other system may allow heme utilization in the absence of uptake (e.g., by surface reduction/extraction of the iron from the heme molecule and uptake of the released iron via the high-affinity reductive iron system), and therefore, not promote Ga-PPIX susceptibility.  64  Figure 2.10 Cig1 influences susceptibility to non-iron MPs. Ten-fold serial dilutions of strains grown in YPD were spotted onto YPD, LIM + 100 µM FeCl3 or LIM + 10 µM heme in absence or presence of 10 µM Ga-PPIX, 100 µM Mn-PPIX or 10 µM GaCl3 as indicated (A). Ten-fold serial dilutions of iron-starved wild-type cells were spotted onto LIM + 10 µM, 50 µM, and 100 µM heme in absence or presence of 10 µM Ga-PPIX (B). The plates were incubated at 30°C for two days and photographed. The experiments were repeated three times and a representative photograph is shown. Figure reproduced and modified from Cadieux et al. (2013).  To determine whether the toxicity of Ga-PPIX was fungistatic or fungicidal, the cells of the wild-type, cig1Δ mutant and complemented strains were exposed to a 10-fold higher concentration of Ga-PPIX than was employed in the experiments shown in Figure 2.10A. The cells were then washed and plated on YPD to count CFU and assess survival (Figure 2.11). The number of cells recovered after exposure was similar for all three strains and was not significantly different from the number of cells prior to exposure. Therefore, the survival rate  65  was high and equivalent for all three strains, suggesting a fungistatic effect of Ga-PPIX on C. neoformans.  Figure 2.11 Ga-PPIX has fungistatic activity against C. neoformans. Survival of iron-starved cells after incubating 5 x 107 cells/mL in LIM (A), LIM + 100 µM FeCl3 (B) or LIM + 10 µM heme (C) supplemented with 100 µM Ga-PPIX for 24 hours. The cells were washed prior to plating onto YPD. All experiments were repeated three times and the data are plotted as the average ± SD.  2.3.6. Localization of Cig1 The localization of the Cig1 protein was examined next. The amino acid sequence of Cig1 contains a putative signal peptide, one predicted trans-membrane domain near the Cterminal of the polypeptide, and no GPI anchor, suggesting that Cig1 is a secreted protein. Additionally, Biondo et al. (2006) identified Cig1 as a secreted protein in the culture supernatant of C. neoformans giving evidence for the expression of CIG1 and implying an extracellular localization for at least a portion of the protein. Primary antibodies against Cig1 were prepared  66  using the recombinant GST::Cig1 fusion protein expressed in E. coli to help evaluate the localization of Cig1. The specificity of the antibodies was assessed using different cellular fractions, including cell lysates, cytosolic fractions and cell wall/membrane fractions (Figure 2.12A).  Additionally, C. neoformans produces extracellular vesicles and, therefore, the  specificity of the antibodies was also tested using fractions containing the extracellular vesicles as seen in Figure 2.12B (Rodrigues et al., 2007; 2008a; Yoneda and Doering, 2006). Unfortunately, these antibodies proved to be uninformative, presumably because of the lack of affinity towards native Cig1 and particularly because of a lack of specificity. In fact, it was not possible to clearly identify the Cig1 protein among several protein bands identified on a Western blot containing proteins from the wild-type strain and mutants lacking the CIG1 gene.  67  Figure 2.12 Production of anti-Cig1 antibodies. Antibodies against recombinant Cig1::GST protein were used to detect Cig1 in different cell fractions from wild-type and mutant strains. (A) Cells from the designated strains were grown in LI-YNB and fractionated into cell lysate (L), cytosol (C) and membrane/cell wall (M). (B) Cells from the designated strains were grown as in (A) and extracellular vesicles were isolated from the culture supernatant. Samples were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. cig1: cig1Δ mutant; cig2: cig2Δ mutant; cig3: cig3Δ mutant; cig1/2: cig1Δ cig2Δ double mutant; cig1/2/3: cig1Δ cig2Δ cig3Δ triple mutant.  To further examine the localization of the Cig1 protein, an HA::Cig1 strain was constructed in which a triple copy of the HA epitope was inserted at the 30th amino acid, just downstream of the signal peptide. After growing the HA::Cig1 cells in LI-YNB, the proteins were isolated from the different cell fractions and analyzed by SDS-PAGE and Western blot, using antibodies specific to the HA tag. Cig1 was detected in the membrane/cell wall fraction, but not in the cytosol (Figure 2.13A).  68  Immuno-fluorescence microscopy was next used to localize Cig1 (Figure 2.13B). After growing the HA::CIG1 cells in low iron and iron-replete media, the cells were fixed and permeabilized, and Cig1 was detected using antibodies specific to the HA tag and fluorescenceconjugated secondary antibodies before viewing by epifluorescence. According to the resulting fluorescence, the Cig1 was localized only at the surface of the cells and in a punctate distribution. As expected, no Cig1 was detected in cells grown in iron-replete conditions. The association of Cig1 with the capsule was also examined. Co-localization of a portion of Cig1 with the capsule was shown using antibodies specific to GXM, the major capsular polysaccharide, while some Cig1 was found outside the capsule in the extracellular space. Overall, these results confirm the reported extracellular localization of Cig1 (Biondo et al., 2006). Unfortunately, insertion of the HA::Cig1 allele in the cig1Δ mutant did not complement for the loss of CIG1 and a lag phase longer than that of the wild-type and cig1Δ strains was observed when the cells were grown in LIM supplemented with heme (Appendix A). However, given that detection of the HA::Cig1 protein confirmed the reported localization of Cig1, the strain was used to further investigate localization.  69  Figure 2.13 Cig1 is found at the cell surface. (A) The C. neoformans strain expressing HA::Cig1 protein was grown in LI-YNB. Cells were fractionated into cell lysate (L), cytosol (C), and membrane/cell wall (M). Samples were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The HA::Cig1 protein was detected using antibodies against HA. (B) Immunofluorescence microscopy of cells grown in LI-YNB either alone or supplemented with 100 µM FeCl3. Antibodies against HA and against the capsular component GXM were used to detect HA::Cig1 (green) and the capsule (red), respectively. Bar = 5 µm.  As mentioned above, it has been shown that C. neoformans produces extracellular vesicles that somehow cross the cell wall to be released in the extracellular environment (Rodrigues et al., 2007; 2008a; Yoneda and Doering, 2006). The extracellular distribution of Cig1 demonstrated in Figure 2.13B suggested that Cig1 might be associated with extracellular 70  vesicles. To confirm this, HA::CIG1 cells were grown in low iron and iron-replete media until stationary phase and the extracellular vesicles were isolated from the culture supernatant by a series of centrifugation steps as described by Rodrigues et al. (2007). The proteins from the vesicle fractions were analyzed by Western blot, using antibodies specific to the HA tag to determine if Cig1 was associated with the extracellular vesicles (Figure 2.14A). Cig1 was detected in the isolated vesicle fraction but only when the cells were grown in LI-YNB and not when the cells were grown in iron-replete conditions. The vesicles were examined more closely using immuno-fluorescence microscopy. For visualization, the isolated vesicles were labeled with a ceramide dye to stain the glucosylceramide in the vesicular membrane and the HA-tagged Cig1 protein was detected with antibodies specific for the HA tag (Figure 2.14B). Fluorescence from HA::Cig1 was found to co-localize with the fluorescence from the ceramides in the vesicles. Again, no Cig1 was detected in the vesicles isolated from cells grown in iron-replete media and only fluorescence from the ceramide dye could be observed. Also, a sample of the vesicles isolated from the culture supernatant of cells grown in iron-replete media was spiked with vesicles isolated from the culture supernatant of cells grown in LIM. The sample was prepared as above for visualization of vesicles and HA-tagged Cig1 by immuno-fluorescence microscopy. As expected, the majority of the vesicles only emitted fluorescence from the ceramide dye while a fraction of the vesicles emitted fluorescence for both Cig1 and the ceramides in the vesicles; again, co-localization of fluorescence from Cig1 and from the ceramide dye was observed (Figure 2.14B). Taken together, these results demonstrate that Cig1 is localized at the cell surface and is associated, at least partially, with extracellular vesicles.  71  Figure 2.14 Cig1 is associated with extracellular vesicles. (A) Western blot analysis of the proteins found in the extracellular vesicle fraction isolated from cells grown in LI-YNB either alone or supplemented with 100 µM FeCl3. Antibodies against HA were used to detect HA::Cig1. (B) Immunofluorescence microscopy of the extracellular vesicle fraction. Antibodies against HA were used to detect Cig1 (green) and a ceramide dye was used to visualize the vesicular membranes (red).  2.3.7. Analysis of Cig1 binding to heme The extracellular location of Cig1 and the genetic evidence for a role in iron acquisition from heme suggested that Cig1 could directly interact with heme. To examine the ability of Cig1 to bind heme, extracellular vesicles with associated Cig1 were incubated with hemeagarose beads. Vesicles isolated from the HA-tagged Cig1 strain were used to be able to detect Cig1 using antibodies specific to the HA tag. The unbound, washed and eluted proteins were separated by gel electrophoresis and analyzed by Western blotting (Figure 2.15A). All of the detectable Cig1 was found in the eluted fraction suggesting an interaction between Cig1 and heme, or between heme and vesicles that contained or were associated with Cig1.  The  extracellular vesicle fraction was also incubated with agarose beads without the heme ligand and the separated proteins were analyzed by Western blotting to determine if Cig1 was interacting 72  non-specifically with the agarose beads. Cig1 was only detected in the unbound protein fraction which implies that the interaction is specific for the heme ligand. The same experiment was also performed using a recombinant Cig1::GST protein purified from cell lysates of E. coli. As above, the Cig1::GST protein was only detected in the eluted protein fraction when the protein was incubated with the heme-agarose beads and not upon incubation with agarose beads (Figure 2.15B). Together, these results suggest that Cig1 can bind heme.  Figure 2.15 Vesicles containing Cig1 and purified recombinant Cig1 bind to heme-agarose beads. The proteins within the extracellular vesicle fraction isolated from cells expressing HA::Cig1 (A) and purified recombinant Cig1::GST expressed in E. coli (B) were analyzed by Western blot after batch adsorption to heme-agarose beads or agarose beads. Lanes include the proteins in the start material (S), the unbound proteins (U) the proteins collected during the washes (W) and the eluted proteins (E). The beads (B) with no proteins added were also loaded on the gel as a control. The HA::Cig1 protein was detected using antibodies against HA, while the Cig1::GST protein was detected using antibodies against GST. Agarose beads were used as a control for non-specific binding as described in the text.  The potential interaction between Cig1 and heme was also investigated further by measuring the absorbance spectra of the purified Cig1::GST protein after the addition of increasing amounts of heme. Heme groups have characteristic absorbance bands that depend on 73  the ligation and conformational state of the chromophore allowing detection of heme-binding proteins by spectrophotometry. Titration of Cig1::GST protein with heme revealed a distinct absorption spectrum defined by a maximum Soret peak at 407 nm as well as β- and α- peaks at 570 nm and 600 nm, respectively, indicative of a heme-binding protein (Figure 2.16A). The heme binding curve of Cig1 yielded multiple inflection points but heme binding saturation was not detected for Cig1 even when a 10-fold molar ratio excess of heme was mixed with the protein (Figure 2.16B). Therefore, the complexity of the interaction between Cig1 and heme did not allow a dissociation constant (Kd) to be determined using this method. Although some Cig1GST degradation products were detected after purification, heme binding was not observed with GST alone (Figures 2.16C).  Another attempt to determine the Kd was carried out using  isothermal titration calorimetry (ITC). As with the spectrophotometric approach, weak binding was observed but saturation was not detected when heme was added to Cig1::GST protein, thus preventing determination of a Kd (Figure 2.16D). Overall, the data support the conclusion that Cig1 may be a heme-binding protein, although the interaction appears to be weak or transient. A putative chitin-binding fold was predicted to be present in Cig1 by remote protein homology.  The ability of Cig1 to bind various polysaccharides was evaluated, however,  preliminary results did not reveal any binding between Cig1 and the polysaccharides under the conditions tested (Appendix B). Further work will be necessary to fully explore the interaction between Cig1 and polysaccharides.  74  Figure 2.16 Recombinant Cig1::GST binds to heme. (A) Absorbance spectra of Cig1::GST and heme after addition of increasing amounts of heme (0 to 60 µM) to purified Cig1::GST (5 µM). The difference between the absorbance of the Cig1-heme complex and free heme at different heme concentrations is plotted against the measured wavelengths. The arrows represent the Soret peak as well as β- and α-peaks, respectively. (B) Heme binding curve of Cig1::GST. The difference between the absorbance of the Cig1-heme complex and free heme at 407 nm is plotted against the increasing concentration of heme. (C) Heme binding curve of GST. The difference between the absorbance of the GST-heme complex and free heme at 407 nm is plotted against the increasing concentration of heme. (D) Binding of heme to Cig1::GST. ITC of heme alone (red) or Cig1::GST plus heme (black) over the specified period of time. All assays were repeated three times and representative graphs are shown. Figure reproduced and modified from Cadieux et al. (2013).  2.3.8. Identification of other potential heme-binding proteins in C. neoformans The previous experiments described above demonstrated an interaction between Cig1 and heme. However, fusion proteins were used in these experiments and the addition of detectable tags could have modified the conformation of Cig1 and its ability to interact with other molecules, including heme.  Therefore, culture supernatants containing the wild-type Cig1 75  protein were used to further examine the potential for Cig1 to bind heme. Wild-type cells were grown for four days in LI-YNB, alone or supplemented with FeCl3, and a pull-down experiment using heme-agarose beads (as described in Section 2.3.7.) was performed with the concentrated culture supernatants. The eluted proteins were trypsinized and analyzed by tandem MS. A pulldown with agarose beads without the heme ligand was also performed in parallel to identify and eliminate any proteins which interact non-specifically with the beads. The raw data including the list of all proteins detected by MS from two independent replicates are presented in Appendix C. Upon data analysis, any proteins from supernatants of cells grown in either LI-YNB alone or in LI-YNB supplemented with FeCl3, and identified to bind to sepharose beads were removed from the list of potential heme-binding proteins.  The thirty most abundant proteins (as  determined by the MS score), which were found to bind only to the heme-agarose beads, are listed in Table 2.4. Cig1 was routinely detected as a heme-binding protein in culture supernatants of cells grown in LI-YNB but not in LI-YNB + FeCl3, which is consistent with the strong iron regulation of Cig1 observed throughout this study with the HA::Cig1 strain. Additionally, Cig1 did not bind to the sepharose beads indicating that the interaction was specific to heme. The MS data support the conclusion that wild-type Cig1 binds to heme-agarose beads. It also confirms the extracellular localization of Cig1 that was previously determined with the cells expressing the HA::Cig1 fusion protein. Another interesting putative heme-binding protein listed in Table 2.4 is the predicted transmembrane receptor (CNAG_03524). Although it was not detected at a very high abundance, it could be a receptor involved in heme uptake. Also, like Cig1, it was only detected in supernatants from cells grown in LI-YNB but not in LI-YNB + FeCl3, suggesting that the expression of this protein is iron-regulated.  The common regulation of the putative  76  transmembrane receptor and Cig1 raises the possibility of both proteins being part of the same pathway. Future work will be needed to determine if this candidate transmembrane receptor plays a role in heme utilization. During the MS analysis, other putative heme-binding proteins were also identified (Table 2.4 and Appendix C).  Functions related to polysaccharide and carbohydrate metabolism  emerged as common themes among the list of proteins detected. It is possible that the overrepresentation of these categories is due to the cells having been grown in low carbohydrate concentrations (i.e., 0.5% glucose). In such conditions, the cells could compensate by increasing the expression of genes involved in metabolizing carbohydrates or in breaking down complex carbohydrates/polysaccharides for eventual use. Although the data suggest that these proteins involved in carbohydrate metabolism might also be heme-binding proteins, it does not necessarily indicate a role for these proteins in heme utilization. Further work will be needed to determine if the proteins listed below play a role in heme acquisition and to confirm their interaction with heme. Lastly, enzymes involved in virulence of C. neoformans have also been identified as secreted putative heme-binding proteins, such as laccase, phospholipase B, acid phosphatase, and superoxide dismutase. However, it is unclear whether these proteins directly interact with heme or if they are involved in heme acquisition and, therefore, future work will be required.  77  Table 2.4. Potential secreted heme-binding proteins Accession CNAG_01239 CNAG_02030 CNAG_00407 CNAG_01854 CNAG_06501 CNAG_03465 CNAG_02860 CNAG_06081 CNAG_04373 CNAG_06291 CNAG_01653 CNAG_06835 CNAG_02966 CNAG_05138 CNAG_03525 CNAG_00799 CNAG_00919 CNAG_00150 CNAG_05799 CNAG_02189 CNAG_00587 CNAG_06085 CNAG_04524 CNAG_00250 CNAG_01405 CNAG_02850  Description  Score  FeCl3 in medium +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 +/- FeCl3 + +/- FeCl3 +/- FeCl3  chitin deacetylase 1004.92 glyoxal oxidase 773.78 glyoxal oxidase 575.23 heparinase II/III family protein 574.18 1,3-beta-glucanosyltransferase 510.53 laccase 497.78 endo-1,3(4)-beta-glucanase 379.03 glucose oxidase 315.21 alginate lyase 305.52 deacetylase 302.52 cytokine inducing-glycoprotein 298.55 glucosidase 243.33 carboxypeptidase D 224.07 exo-beta-1,3-glucanase 212.26 trehalase 199.52 cellulase 181.56 carboxypeptidase D 179.72 peptidase 178.36 chitin deacetylase 177.47 alpha-amylase 171.95 conserved hypothetical protein 152.94 secreted phospholipase B 120.49 zinc metalloprotease 120.28 conserved hypothetical protein 119.81 conserved hypothetical protein 108.08 glucan endo-1,3-alpha-glucosidase 107.75 agn1 CNAG_03120 alpha-1,3-glucan synthase 106.72 CNAG_05471 alpha-glucosidase 98.04 CNAG_03524 transmembrane receptor 90.30 CNAG_01562 conserved hypothetical protein 89.51 The score reflects how well the observed MS spectra matched to the amino acid sequence of the protein. A higher score indicates a more confident match. The score can also be used as a measure of protein abundance.  78  2.3.9. Cig1 is required for the full virulence of C. neoformans The virulence of the cig1Δ mutant was compared to that of the wild-type and complemented strains using a mouse inhalation model of cryptococcosis. The cig1Δ mutant was as virulent as the wild-type and complemented strains, and 100% of the mice exhibited symptoms of cryptococcosis and were euthanized by 23 days post-infection (Figure 2.17). This is likely because the cryptococcal cells are still able to utilize other iron sources besides heme, such as transferrin, which are available within the animal host. In addition, there may be multiple mechanisms of heme utilization. The virulence of the cfo1Δ and cig1Δ cfo1Δ mutants was also compared to further examine the role of Cig1 during infection. As previously reported, the cfo1Δ mutant was attenuated for virulence and the mice survived up to 35 days post-infection (Jung et al., 2009). Interestingly, the cig1Δ cfo1Δ mutant was more severely attenuated for virulence than the cfo1Δ mutant, with two of the ten mice still not showing symptoms by the end of the experiment (day 60). These results imply that Cig1 plays an important role in iron acquisition and virulence in the host in absence of the high-affinity iron uptake system or possibly when the iron sources acquired by the high-affinity iron uptake system are not available.  79  Figure 2.17 CIG1 is required for virulence in absence of CFO1. Ten female A/JCr mice were inoculated intranasally with each of the C. neoformans strains indicated. The survival of the mice was monitored daily up to 60 days. Statistical analysis was performed by log rank tests. The difference in survival between the wild-type strain and the cfo1Δ or cig1Δ cfo1Δ mutants was significant (P<0.0001). Additionally, the difference between the cfo1Δ mutant and the cig1Δ cfo1Δ mutant was significant (P<0.0001). Figure reproduced and modified from Cadieux et al. (2013).  The distribution of fungal cells in different tissues of the infected mice was examined to determine the ability of the cig1Δ mutant to colonize host tissue. The lungs and brains of the infected mice were collected when the mice reached the endpoint and the fungal burden was measured by determining the CFU (Figure 2.18). The wild-type, mutant and complemented strains were all equally abundant in the lungs. In addition, the wild-type, cig1Δ mutant and complemented strains were all capable of dissemination to the brain and the fungal burden was comparable for all these strains. However, the number of fungal cells in the brain was much lower for the cfo1Δ mutant as previously reported (Jung et al., 2009). Additionally, the cig1Δ cfo1Δ and cig1Δ cfo1Δ::CIG1 strains showed similar results to the cfo1Δ mutant and no specific contribution of Cig1 in the colonization of these organs was noted. Together, these results suggest that heme may not be the primary source of iron for C. neoformans in the mice lungs and brain, and that alternate sources of iron may be preferred in these host tissues.  80  Figure 2.18 Determination of fungal loads in mice tissue. The fungal burden in the lungs and brain of three mice from each group was determined by counting the CFU. The organs were collected at the endpoint, homogenized and the cells were plated onto YPD + 35 µg/mL chloramphenicol. Statistical analysis was performed using an unpaired, two-tailed t test. The * represents significant differences with a P < 0.05. Figure reproduced and modified from Cadieux et al. (2013).  81  2.4. Discussion C. neoformans uses heme as a sole iron source although the mechanisms of heme uptake and processing have not been characterized in this pathogen (Jung et al., 2008). In this study, the mannoprotein Cig1, but not Cig2 nor Cig3, was identified as a component of a novel heme uptake mechanism as demonstrated by 1) the extended lag phase of a cig1 mutant in presence of heme as a sole iron source; 2) the reduced susceptibility to Ga-PPIX and Mn-PPIX that require a heme uptake system for toxicity and; 3) heme binding by a recombinant Cig1::GST fusion protein, by an HA::Cig1 fusion protein associated with extracellular vesicles and by native Cig1 as detected by MS. Heme uptake systems have been well studied in pathogenic bacteria and they generally involve heme-binding cell surface receptors that bind heme with high affinity. Heme is then either transferred between binding domains of multiple surface proteins to a membrane transporter for internalization in the case of Gram-positive bacteria or transferred directly to the membrane transporter in Gram-negative bacteria (Braun, 2001; Nobles and Maresso, 2011). Once inside the cell, heme is degraded and iron is released into the cytoplasm. Some bacteria also secrete heme-binding proteins (hemophores) to the extracellular space to scavenge heme or extract heme from the host heme proteins and return it to the cell surface receptors (Cescau et al., 2007; Wandersman and Delepelaire, 2012). Cig1 was confirmed to be localized at the cell surface, secreted in the extracellular space and associated with extracellular vesicles, and therefore, Cig1 might fulfill a similar role as a hemophore or heme-interacting surface protein for C. neoformans. Other pathogenic fungi, including C. albicans, and H. capsulatum, are also capable of utilizing heme and/or hemoglobin as an iron source, although little is known about the key players and the mechanisms involved in uptake (Foster, 2002; Santos et al., 2003). For example,  82  Rbt5 and Rbt51 were identified as receptors for heme and hemoglobin and were localized at the plasma membrane in C. albicans (Weissman and Kornitzer, 2004). Rbt5 and Rbt51 are both mannoproteins and the expression of RBT5 is strongly induced by iron starvation. Similar to the results presented in this study, deletion of RBT5 significantly reduced the ability of C. albicans to use iron from heme. Weissman et al. (2008) later identified the ESCRT pathway as a possible endocytic mechanism for hemoglobin uptake in C. albicans and they showed that hemoglobin bound to Rbt5 was endocytosed into the vacuole. Presumably, Cig1 may also deliver heme to the cell surface of C. neoformans for subsequent internalization. Deletion of CIG1 resulted in an extended lag phase but eventual growth to a density comparable to that of the wild-type strain. It is possible that heme contains some contaminating inorganic iron that could be taken up by the high-affinity iron uptake system encoded by the permease Cft1 and the ferroxidase Cfo1 (Jung et al. 2008; 2009). This idea is supported by the fact that the double mutant, cig1Δ cfo1Δ, grew slightly slower than the cig1Δ single mutant, although the difference in growth was not dramatic. An alternative explanation is that one or more additional mechanism(s) for heme use could eventually be activated to compensate for loss of the Cig1 pathway. Non-iron MPs (e.g., Ga-PPIX and Mn-PPIX) are toxic heme analogs that are taken into bacterial cells via heme uptake pathways (Stojiljkovic et al., 1999). Enzymes that use heme as a co-factor can insert Ga-PPIX in their catalytic centers instead of heme resulting in inactivity because gallium is not able to undergo the same redox reactions as iron. Additionally, non-iron MP may stimulate production of reactive oxygen radicals leading to toxicity (Stojiljkovic et al., 1999). The toxicity of Ga-PPIX and Mn-PPIX for the wild-type strain but not the cig1Δ mutant supports a role for Cig1 in heme uptake. This phenotype was only apparent when cells were  83  forced to use heme as the sole iron source. Interestingly, the cig1Δ mutant was able to grow in LIM supplemented with heme as a sole iron source in the presence of Ga-PPIX perhaps because of some residual iron reserves (i.e., the cells were not pre-starved for iron). Moreover, as mentioned earlier, there may be a small amount of free iron contaminating the heme and/or the agarose in the medium to support some growth. The growth defect of the cig1Δ cfo1Δ mutant in LIM supplemented with heme partially supports this explanation although a separate mechanism for growth in LIM supplemented with heme may be present. In this case, the additional system may allow heme utilization in the absence of uptake (e.g., by surface reduction and/or extraction of the iron for uptake via the high-affinity system) and therefore not promote non-iron MP susceptibility. The growth defect of the cig1Δ cfo1Δ mutant in LIM supplemented with heme supports this hypothesis. Cig1 was observed to be localized at the cell surface and in association with the extracellular vesicles produced by C. neoformans. A strain expressing HA-tagged Cig1 protein was used in the experiments performed to study the localization of Cig1. Although the HA::Cig1 strain was valuable, the results should be interpreted carefully as the expression of HA::Cig1 did not functionally complement for the loss of CIG1. This is most likely due to the presence of the HA tag which could prevent proper folding of the HA::Cig1 protein, alter its conformation and/or interfere with its function. However, as seen by immunofluorescence microscopy, the HA::Cig1 is secreted to the cell surface and outside the cell as would be expected for the wild-type Cig1 protein. These observations confirm the findings of Biondo et al. (2006), which identified Cig1 as a secreted protein found in the culture supernatant of C. neoformans.  Additionally, the MS  data presented in this study, as well as other MS work done by J. Geddes (personal communications) provide further evidence for the extracellular localization of the wild-type Cig1  84  protein as it was consistently detected in the culture supernatant of wild-type cells grown in low iron conditions. A pull-down experiment with heme-agarose beads identified HA-tagged Cig1 as a potential heme-binding protein. It is important to note that heme is a hydrophobic molecule and therefore, some of the binding observed could be a result of non-specific interactions rather than heme binding. Specifically, using the extracellular vesicles as starting material could have led to non-specific interactions between the hydrophobic heme ligand and the membranes or lipids in the vesicles. However, binding of Cig1 to the heme-agarose beads was also detected when purified recombinant Cig1::GST protein from E. coli or wild-type Cig1 protein from concentrated culture supernatants of C. neoformans were used as starting material which provides added confidence in the results. Nonetheless, the interaction between Cig1 and heme was investigated further. Cig1 was demonstrated to bind heme as detected by spectrophotometric titration and ITC using Cig1::GST protein from E. coli. The absorbance spectra showed the appearance of distinct peaks in the soret, alpha and beta band areas characteristic of a heme-binding protein. Determination of the Kd was not possible because the heme binding curve of Cig1 yielded multiple inflection points and saturation of the binding interaction was not detected, even when more than 10 molar excess of heme was added to protein. Similar cases have been reported in the literature. For example, the heme-binding protein in rabbit has been reported to bind a 25-35 molar excess of heme (Tsutsui and Mueller, 1982b). It has also been reported that the IsdX2 protein isolated from B. anthracis required 20 times more heme to protein ratio to reach saturation and that multiple inflection points were detected (Maresso et al., 2008). It is also possible that the lack of saturation is caused by weak binding or even non-specific binding and  85  that heme could potentially bind to the Cig1 protein at multiple sites on the protein. Alternatively, expressing the Cig1::GST protein in E. coli, where post-translational modifications such as glycosylation do not occur, could alter the activity of the protein. Glycosylation of proteins plays an important role in their function. For example, glycosylation of a receptor involved in regulating natural killer cell responses has been shown to be required for binding to its ligands (Margraf-Schonfeld et al., 2011). Also, ablation of N-glycosylation of a plant peroxidase by site-directed mutagenesis influenced the folding of the protein, its thermostability and its catalytic ability (Lige et al., 2001). Finally, the presence of the GST tag may have altered the conformation of Cig1 or decreased the affinity of the protein for heme. Further work will be needed to fully understand the heme-binding activity of Cig1. Many heme-binding proteins contain one or multiple conserved heme-binding domains, such as the NEAT domains found in bacteria or possibly the CFEM domains found in the heme receptors from C. albicans (Andrade et al., 2002; Grigg et al., 2007; Weissman and Kornitzer, 2004). No conserved heme-binding domains were identified for Cig1 although a chitin-binding fold and a putative immunoglobulin-like β-sandwich fold were detected. It is possible that the chitin-binding fold may be involved in heme binding.  For example, the dopamine β-  monooxygenase N-terminal (DOMON) domains have been described in several secreted, or cell surface proteins from plants and animals (Aravind, 2001; Ponting, 2001; Lakshminarayan et al., 2007). These DOMON domains are rich in β-strands, adopt a β-sandwich-like fold, and have been shown to be involved in ligand binding, either as heme- or sugar- binding domains (Lakshminarayan et al., 2007).  Alternatively, the immunoglobulin-like β-sandwich fold could  also participate in heme binding. In fact, one of the two main regions that mediate heme binding, specificity in ligand and transfer functions within the NEAT domains contains an  86  immunoglobulin-like fold. This fold has been demonstrated to be the platform where the heme molecule sits once it is bound to the NEAT domain (Grigg et al., 2007; Sharp et al., 2007; Watanabe et al., 2008; Honsa and Maresso, 2011; Nobles and Maresso, 2011). The overlapping predicted chitin-binding and immunoglobulin-like folds will be a good starting point to identify the heme-binding domain (s) of Cig1 in future studies. Interestingly, a general theme of carbohydrate metabolism was revealed during the MS analysis. It is possible that carbohydrate metabolism was over-represented because the cells were grown in low glucose conditions. The proteins (mostly enzymes) identified were found to be secreted in the culture supernatant and to bind to the heme-agarose beads, however, they might not necessarily be involved in heme utilization. The interaction between the proteins and the beads could be non-specific due to the hydrophobic nature of heme, as discussed above. It is also possible that the proteins identified do not bind directly to the heme-agarose beads but bind to other proteins or to secreted polysaccharides that bind to heme. Nonetheless, a link between carbohydrate metabolism and heme uptake may exist. Specifically, the data from this study suggest that Cig1 is a heme-binding protein involved in heme acquisition, and although no known heme-binding domains were found in the Cig1 protein sequence, a fold similar to that of the chitin-binding domain was identified. Another example supporting the link between iron uptake and carbohydrate metabolism was reported in S. cerevisiae, where iron limitation resulted in metabolic rearrangement and increased glycerol production (Ansell and Adler, 1999). Finally, kinetic studies of a cellobiose oxidase, an enzyme involved in the break-down of cellobiose, revealed that the protein functions primarily as a ferric reductase in the white-rot fungus Phanerochaete chrysosporium (Kremer and Wood, 1992).  87  Other putative secreted heme-binding proteins identified by the MS analysis involved enzymes that play a role in virulence of C. neoformans, including laccase, phospholipase B, acid phosphatase, and superoxide dismutase (Rodrigues et al., 2008a). These proteins have been associated with the extracellular vesicles produced by C. neoformans which could explain their presence in the culture supernatants (Rodrigues et al. 2008a).  It is also possible that the  extracellular vesicles, which would remain intact in the concentrated culture supernatants, bind to the heme-agarose beads via Cig1, or another heme-binding protein.  Alternatively, the  vesicular membranes could bind non-specifically to the hydrophobic heme ligand of the agarose beads. In either case, the vesicles would be found among the eluted proteins, and the cargo proteins transported within the extracellular vesicles would be released upon heating of the samples prior to gel electrophoresis. Overall, future work will be required to determine if the proteins identified by MS analysis play a role in heme acquisition and to further understand their interaction with heme. The role of Cig1 in virulence reflects a growing appreciation for the multiple mechanisms of iron acquisition by C. neoformans. That is, deletion of CIG1 alone did not attenuate virulence in a mouse model of cryptococcosis but deletion of both CIG1 and CFO1 resulted in significant attenuation of virulence beyond that observed with deletion of CFO1 alone. Thus, the influence of Cig1 is evident only in the absence of the high-affinity iron uptake system (Cft1/Cfo1). Jung et al. (2008) reported that transferrin and heme are potential host-derived iron sources for C. neoformans, but it is not known whether the fungus preferentially uses one source or the other in vivo, and whether there are niche-specific sources and preferences. The results presented here support the conclusion that C. neoformans uses both iron sources to proliferate in mammalian hosts. Similar results were found in C. albicans, where only deletion of the iron permease,  88  CaFTR1, of the high-affinity iron uptake system has been shown to play a role in virulence (Ramanan and Wang, 2000). Deletion of the heme receptor Rbt5 did not reveal a role in virulence although a rbt5Δ mutation was not tested in the Caftr1Δ mutant background (Braun et al., 2000). In summary, Cig1 was identified as a component of a novel heme uptake system in the fungal pathogen C. neoformans, and Cig1 was hypothesized to act as a heme receptor, a transporter at the cell surface or a hemophore that scavenges heme in the extracellular environment. Further investigation will be needed to develop a more detailed understanding of the Cig1-mediated mechanism of heme uptake and to investigate the additional heme uptake mechanism(s) revealed by the characterization of Cig1.  89  Chapter 3. Further characterization of heme uptake in C. neoformans 3.1. Introduction The work presented in Chapter 2 identified Cig1 as a major player in heme acquisition in C. neoformans. However, given enough time, the cig1Δ mutant cells were eventually able to grow in LIM supplemented with heme as a sole iron source. This observation led to the hypothesis that C. neoformans may have more than one pathway for heme utilization and that a Cig1-independent pathway may be required for heme acquisition in absence of Cig1. The study presented in this chapter focuses on examining various factors that are potentially involved in Cig1-independent heme utilization, including pH, the pH regulator Rim101, and cell density. The solubility and bioavailability of iron is dependent on pH. For example, at neutral and alkaline pH, iron is mostly found in its ferric state (Fe3+) and is insoluble, making it less available to C. neoformans (Wilkins, 1991; Cotton et al., 1999). Transition between ferric iron (Fe3+) and ferrous iron (Fe2+) is pH-dependent, and therefore, iron is more soluble and more readily available at acidic pH. Given the effect of pH on iron bioavailability, changes in pH could contribute to eventual heme utilization by the cig1Δ mutant cells. Rim101 is the master regulator of the pH response and is highly conserved in many fungal species (Tilburn et al., 1995; MacCabe et al., 1996; Ramon et al., 1999; Lamb and Mitchell, 2003). A recent study identified Rim101 as a regulator of iron homeostasis in C. neoformans (O’Meara et al., 2010). Specifically, Rim101 was found to regulate the expression of CIG1 and the CIG1 transcript was more than 400-fold lower in the rim101Δ mutant compared to wild-type cells, implying a probable functional link between Rim101 and Cig1 with regard to heme use. Additionally, Rim101 regulated the expression of the siderophore iron transporter  90  (SIT1), and the ferric permease (CFT1) involved in the reductive high-affinity iron uptake pathway. Therefore, it is possible that Rim101 also regulates Cig1-independent heme uptake. Finally, the long lag phase of the cig1Δ mutant when grown in LIM supplemented with heme suggested that cell density and a quorum sensing (QS)-like mechanism could be required for Cig1-independent heme acquisition. Microbial cells often monitor the cell density of the population by releasing and receiving signaling molecules and thereby coordinating the population’s behaviour in order to adapt to an array of different and sometimes hostile environments (Bassler, 2002; Joint et al., 2007; Williams, 2007; Albuquerque and Casadevall, 2012). The secreted signals are usually small molecules and their concentration increases with the population size. Once the concentration surpasses a certain threshold, it induces the cells to cooperate in diverse behaviours, including uptake of nutrients, growth, biofilm formation, and elaboration of virulence factors. To test the hypothesis that C. neoformans evolved more than one pathway for heme utilization, this study examined potential factors contributing to Cig1-independent heme uptake. Specifically, the role of pH in heme acquisition and the link between the pH regulator Rim101 and heme utilization were evaluated. The effect of cell density on growth in LIM supplemented with heme was also tested and preliminary identification of a QS-like molecule was attempted.  91  3.2. Materials and Methods 3.2.1. Strains and growth conditions All strains used in this study are listed in Table 3.1. LIM was prepared as described (Vartivarian et al., 1993) and the pH was adjusted to 7.2, unless specified otherwise. The water used for LIM was treated with Chelex-100 resin (Bio-Rad) to chelate iron. Cells for growth assays were pre-grown overnight at 30°C with shaking in YPD (Fisher). The cells were then washed twice with low iron water, inoculated into LIM at 4 x 106 cells/mL and grown at 30°C for two days to starve the cells for iron.  These cells are designated as pre-starved cells  throughout this chapter. After starvation, the cells were harvested, washed and inoculated in LIM alone or supplemented with heme (porcine hemin; Sigma) or FeCl3 (Sigma) to a final concentration of 5 x 104 cells/mL, unless specified otherwise. Cultures were incubated at 30°C and growth was monitored by measuring the optical density at 600 nm using a DU530 Life Science UV/Visible spectrophotometer (Beckman Instruments).  The pH of the culture  supernatant was measured at specific time points using an Orion 2-star pH meter (Thermo Scientific) and the Orion Ross combination pH electrode (Thermo Scientific). To examine growth on solid media, iron-starved cells were diluted to 1 x 106 cells/mL in low iron water. Ten-fold serial dilutions were made in low iron water and 5 µl of each dilution was plated on LIM alone or supplemented with heme or FeCl3. Agarose (1%, Invitrogen) was added as a solidifying agent. Plates were incubated for three days at 30°C and then photographed.  92  Table 3.1. Strains used in this study Strain Description H99 C. neoformans wild-type strain cig1Δ cig1 disruption mutant cig1Δ +CIG1 CIG1 complemented strain cfo1Δ cfo1 deletion mutant cig1Δ cfo1Δ cig1 cfo1 double deletion mutant cig1Δ cfo1Δ +CIG1 CIG1 complemented in cig1Δ cfo1Δ background rim101Δ rim101 deletion mutant rim101Δ +RIM101 RIM101 complemented strain GAL7p::CIG1in Regulated CIG1 in rim101 deletion rim101Δ mutant background GAL7p::CIG1in H99 Regulated CIG1 in H99 background cas1Δ cas1 deletion mutant cap59Δ cap59 deletion mutant B3501A C. neoformans var. neoformans (serotype D) wild-type strain cqs1Δ cqs1 deletion mutant (serotype D)  Provided by/ Prepared by Dr. Joseph Heitman Carmelo Biondo Tian Lian Dr. Wonhee Jung Brigitte Cadieux Brigitte Cadieux Dr. J. Andrew Alspaugh Dr. J. Andrew Alspaugh Brigitte Cadieux Brigitte Cadieux Dr. Emma Griffiths Dr. Jennifer Lodge Dr. June Kwon-Chung Dr. June Kwon-Chung  3.2.2. Construction of strains Primers used for construction of all strains are listed in Table 3.2.  Table 3.2. Primers used for strain construction Allele Primer Primer sequence constructed identification GAL7p::CIG1 Cig1A-6 ATTCACTGGATCCGTCGGTA Gal7-Cig1-LA-R CTTCCGTGTTAATACAGATAAACCGAGATGAAA ATGTAAAGCTGTCG Gal7-Cig1-RA-F2 GCACTCAATTCTCTCCTGAGAATGATTTTTAATC GTTTCACATTC Gal7-Cig1-RA-R TACAGGAGAAGTCATACAAAGCAT Gal7-Cig1-F CGACAGCTTTACATTTTCATCTCGGTTTATCTGT ATTAACACGGAAG Gal7-Cig1-R GAATGTGAAACGATTAAAAATCATGCACTCAAT TCTCTCCTGAGA Gal7-Cig1-PO-F GCCATTATTGTTTCCCGATG Gal7-Cig1-PO-R TCAGGATCTTCATGGCTCCT  93  3.2.2.1. Construction of the galactose-regulated CIG1 strains The GAL7p::NAT::CIG1 allele was constructed by overlap PCR using the methods described by Davidson et al. (2002) and Yu (2004). The sequence upstream of the 5’ end of the CIG1 gene was amplified from genomic DNA from strain H99 with primers Cig1A-6 and Gal7Cig1-LA-R, and the CIG1 open reading frame was amplified with primers Gal7-Cig1-RA-F2 and Gal7-Cig1-RA-R. The NAT::GAL7p allele was amplified from strain GAL7p::PKA1 kindly provided by Dr. J. Choi (Choi et al., 2012) with primers Gal7-Cig1-F and Gal7-Cig1-R. The primers Cig1A-6 and Gal7-Cig1-RA-R were then used to overlap the three products to yield the 4.5 kb GAL7p::NAT::CIG1 allele.  The construct was introduced into the C. neoformans  serotype A strain H99 and the rim101Δ strain kindly provided by Dr. J. A. Alspaugh (Duke University, Durham, NC, U.S.A.; O’Meara et al., 2011) by biolistic transformation to generate the GAL7p::CIG1 regulated strains (Toffaletti et al., 1993). Transformants were screened by colony PCR using primers Gal7-Cig1-PO-F and Gal7-Cig1-PO-R. Transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic H99 DNA using primers Gal7-Cig1-RA-F2 and Gal7-Cig1-RA-R (Figure 3.1).  94  Figure 3.1 Galactose-regulated CIG1 strains. (A) Diagram of the GAL7p::NAT::CIG1 construct introduced into the wild-type and rim101Δ strains. (B) Southern blot analysis to confirm insertion of the GAL7 promoter. Genomic DNA of the indicated strains was digested with ScaI and hybridized with the downstream arm of the GAL7p::NAT::CIG1 construct, which corresponds to the CIG1 open reading frame.  3.2.3. Quantitative RT-PCR To examine gene expression, the cells were grown in LIM alone or supplemented with heme for 6 hours at 30°C with shaking.  Total RNA was extracted using the RNeasy kit  (Qiagen), treated with DNase (Qiagen) and cDNA was synthesized using the SuperScript First Strand System (Invitrogen) following the manufacturers’ recommendations. PCR reactions were monitored using the 7500 system (Applied Biosystems) as described previously (Tangen et al., 2007), with primers designed using Primer Express software 3.0 (Applied Biosystems) and are listed in Table 3.3. The relative gene expression was quantified using the SDS software 1.3.1 (Applied Biosystems), based on the 2-ΔΔCT method (Livak and Schmittgen, 2001). The average Ct of 18S rRNA was used for normalizing.  Table 3.3. Primers used for quantitative RT-PCR Primer identification Primer sequence CIG1A-RT-F GGTGGTCCGGTTTCCTTCA CIG1A-RT-R GACTCGTGGTCGTGCATAACA 18S-RT-F AACAGGTCTGTGATGCCCTTAGA 18S-RT-R ACTCGCTGGCTCAGTCAGTGT  Target Gene CIG1 CIG1 18S rRNA 18S rRNA 95  3.2.4. Aggregate formation Pre-starved cells were grown in LIM + 10 µM heme and were harvested at the specific time points by centrifugation (13,000 x g for 1 minute). The cells were resuspended in 1/10th volume of LIM, mixed 1:1 in india ink and visualized by negative staining on a Zeiss Axioplan 2 Imaging microscope.  3.2.5. Preparation of conditioned media Pre-starved cells were inoculated in LIM + 10 µM heme as described above and grown at 30°C with shaking until the cells reached stationary phase.  The culture supernatant was  harvested by centrifugation (15,000 x g, 15 minutes, 4°C) and filter-sterilized through a 0.45 µm vacuum filter. The conditioned media (CM) was added 1:1 to fresh LIM + 10 µM heme for a final concentration of 50% CM before inoculation for the start of the growth curves.  3.2.6. Isolation of exopolysaccharides Cells were grown in LIM for seven days at 30°C with shaking.  Extracellular  polysaccharides were isolated as previously described (Wozniak and Levitz, 2008). Briefly, the culture supernatant was collected by centrifugation (15, 000 x g, 2 minutes, 4°C).  The  polysaccharides were precipitated by adding 10% sodium acetate and 2.5 volumes of ethanol, and the solution was incubated overnight at room temperature. The supernatant was decanted and the polysaccharides were air dried and dissolved in 1/10th volume of the original culture in low iron water. The polysaccharide concentration was measured by the phenol sulfuric method (Dubois et al., 1951). A fraction of the isolated polysaccharides was treated with 0.1 mg/mL proteinase K (Roche) for 4 hours at 37°C. Another fraction was dialyzed in SnakeSkin dialysis  96  tubing of 7,000 MWCO (Thermo Scientific) in low iron water at 4°C for 48 hours with two changes of water. All polysaccharide fractions were autoclaved for 10 minutes and 2 mL were added to 20 mL fresh LIM + 10 µM heme prior to inoculating for the start of the growth measurements in liquid cultures.  3.2.7. Absorption Spectrophotometry Titrations of isolated GXM with heme were monitored by absorption spectroscopy in the Soret range as previously described (Gaudin et al., 2011) to detect heme-binding interactions. Briefly, increasing amounts of heme dissolved in 0.1 M NaOH and diluted in 10 mM phosphate buffer (pH 7.0) were added to 1 mL of 0.2 µM polysaccharides in buffer. The preparation was incubated at room temperature for 5 minutes after adding heme to allow binding before measuring the absorbance spectra. Heme added to buffer without polysaccharides served as a reference. Spectra were measured using a Cary 50 Bio UV-visible spectrophotometer (Agilent Technologies) with an optical path length of 1 cm in a quartz cell. The isolated GXM was kindly provided by Dr. D. de Oliveira and was isolated as described by Nimrichter et al., (2007).  3.2.8. Isothermal titration calorimetry experiments ITC was performed using a MicroCal iTC200 (GE Healthcare). Samples were in 10 mM phosphate buffer (pH 7.0). Titrations were performed by injecting consecutive 2 µL aliquots of heme solution (1-5 mM) into the ITC cell (volume = 200 µL) containing isolated GXM (0.1-0.5 mg/mL). All solutions were degassed at room temperature before loading in the calorimeter. The heat of dilution data were obtained by titrating 2 µL injections of heme solution into  97  polysaccharide-free buffer. All titrations were performed at 25 C. ITC experiments were performed by Dr. Louise Creagh.  98  3.3. Results 3.3.1. Prior adaptation of the cig1Δ mutant to growth in LIM supplemented with heme does not alter subsequent growth in presence of this iron source Over the course of the experiments detailed in Chapter 2, it became increasingly clear that a Cig1-independent heme uptake system existed to allow C. neoformans to acquire heme in the absence of Cig1. One possibility is that the cig1Δ cells adapt by accumulating compensating mutations or by shifting their metabolism to allow growth in LIM supplemented with heme. Therefore, growth in LIM supplemented with heme was examined after the cells had previously been grown in LIM supplemented with heme to determine if adaptation was the cause of the eventual growth of the cig1Δ mutant. The strains were grown in LIM supplemented with heme until the cells reached stationary phase. The cells were then harvested, washed and inoculated in fresh LIM with added heme (Figure 3.2). Interestingly, the growth of the strains did not change whether the cells were previously grown on heme or not. Specifically, the lag phase of the wildtype and complemented strains lasted approximately 24 hours and the strains had reached stationary phase by 48 hours for both cells that were pre-starved for iron and cells that were pregrown in LIM supplemented with heme. In contrast, the lag phase of the cig1Δ mutant lasted approximately 48 hours and the stationary phase was reached by 72 hours. Again, this was the same for the cig1Δ cells that had been pre-starved or pre-grown in LIM supplemented with heme. Although these results do not necessarily preclude adaptation of the cells to allow growth in LIM supplemented with heme, they suggest that it is not the major factor involved in heme acquisition via a Cig1-independent pathway.  99  Figure 3.2 Prior adaptation does not rescue the growth defect of the cig1Δ mutant in LIM supplemented with heme. Iron-starved cells were grown in LIM + 10 µM heme for 72 hours before being harvested, washed twice in low iron water, and inoculated at 5 x 104 cells/mL in fresh LIM + 10 µM heme. Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD.  3.3.2. The role of Cig1 in heme uptake is pH dependent In an attempt to characterize the Cig1-independent heme uptake system, the growth of the strains was tested in presence of heme at different pH because pH is known to influence iron availability (Wilkins, 1991; Cotton et al., 1999). Although the cig1Δ mutant exhibited a growth defect at pH 7.2, the cells grew as well as the wild-type and complemented strains at pH 5.6 (Figure 3.3A-B). It is possible that the decreased lag phase of the cig1Δ mutant at pH 5.6 resulted from iron being released from the heme molecule at this low pH which could thereby be taken up by the cells via an alternate iron uptake pathway, such as the high-affinity iron uptake pathway. A strain lacking CFO1, a gene encoding an essential component of the high-affinity iron uptake pathway, was grown in LIM supplemented with heme at both pH levels to test this possibility (Figure 3.3A-B). If iron is released from the heme molecule, one would expect the cfo1Δ and/or the cig1Δ cfo1Δ mutants to show a growth defect. However, all the cells grew as well as the wild-type strain at pH 5.6 indicating that iron is not released from the heme molecule 100  in sufficient amounts to influence growth at that pH. As presented in Chapter 2, the cfo1Δ mutant grew as well as the wild-type strain at pH 7.2, while the cig1Δ cfo1Δ mutant showed a similar growth defect as the cig1Δ mutant. Therefore, these results suggest that Cig1 plays an active role in iron acquisition from heme at physiological pH, and that a Cig1-independent system exists to acquire iron from heme at low pH. To examine this hypothesis further, the expression of CIG1 was tested in cells grown in LIM with and without heme at acidic and physiological pH. The expression of CIG1 was down-regulated more than five-fold at pH 5.6 compared to pH 7.2, independent of the presence of heme in the culture medium (Figure 3.3C). These results further support a role for Cig1 in heme acquisition at physiological pH, and suggest that another heme utilization system exists to acquire iron from heme in a Cig1-independent manner at low pH.  101  Figure 3.3 Cig1 is not required for growth in LIM supplemented with heme at acidic pH. Growth of iron-starved cells in LIM + 10 µM heme, pH 7.2 (A) and pH 5.6 (B). Growth was monitored by measuring the optical density at 600 nm. (C) Expression of CIG1 in wild-type cells grown in LIM ± 10 µM heme at pH 5.6 or 7.2. All experiments were repeated three times and the data are plotted as the average ± SD. The * represents significant differences at P < 0.05 between pH 7.2 relative to pH 5.6. Figure reproduced and modified from Cadieux et al. (2013). The fact that the cig1Δ mutant grew as well as the wild-type strain in LIM supplemented with heme at pH 5.6, suggested that a change in pH might explain the eventual growth of the cig1Δ mutant after the extended lag phase at physiological pH. Therefore, the pH of the culture supernatant during growth in LIM supplemented with heme was measured (Figure 3.4A). All the cultures showed a similar trend, that is an increase in pH (up to 8.2-8.3) during the first 24 hours, followed by a decrease in pH (down to 7.5) once the cells reached the exponential growth phase and another increase in pH (up to 7.9-8.3) when the cells reached stationary phase. The cig1Δ mutant remained at an elevated pH for a longer period, corresponding to the longer lag  102  phase of this strain when grown under these conditions. It is unclear whether the change in pH was a cause or a result of the growth of the cig1Δ mutant cells. Growth of the cells in LIM supplemented with heme was also monitored upon modification of the buffering conditions of the culture medium (Figure 3.4B-D). The cig1Δ mutant exhibited a longer lag phase when no buffer  was  added  to  LIM,  compared  to  when  20  mM  piperazineethanesulfonic acid (HEPES) was added as buffering agent.  4-(2-hydroxyethyl)-1The wild-type and  complemented strains also grew more slowly in the absence of a buffering agent. It is possible that there may be a more dramatic increase in the pH in the first 24 hours of incubation when no buffer is added to the culture medium, and that the high pH results in decreasing the heme availability. Alternatively, the observed spike in pH could also be inhibitory for growth of the cells when it is not buffered. The cig1Δ mutant grew as well as the wild-type cells when the concentration of HEPES was increased five-fold (100 mM) from the original level in the buffer (Figure 3.4D). Increasing the concentration of HEPES in the medium increases its buffering capacity and likely maintains the pH of the culture constant throughout the growth of the cells. Consequently, the pH of the medium would not increase in the first 24 hours, and perhaps that shift in pH contributes to the longer lag phase of the cig1Δ mutant. For example, the increase in pH might be the signal which induces the expression of the Cig1-mediated heme uptake pathway. Another explanation could be that the pH influences local activities of the cell surface that are involved in heme processing (e.g., enzymes like ferric reductases, as discussed in Chapter 2). Together, these results suggest that the environmental pH is extremely important for heme uptake and that a Cig1-mediated heme uptake pathway plays a major role at physiological pH while a Cig1-independent acquisition pathway must exist for acquisition of heme at acidic pH.  103  Figure 3.4 The role of Cig1 in heme uptake is highly dependent on pH. pH of the culture medium for iron starved cells grown in LIM + 10 µM heme (A). Growth of iron-starved cells in LIM + 10 µM heme with no added HEPES (B), 20 mM HEPES (C), or 100 mM HEPES (D). Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD.  3.3.3. Rim101 also contributes to heme acquisition Given that pH influenced both the expression of CIG1 and the ability of Cig1 to contribute to heme utilization, the contribution of the pH-regulatory protein Rim101 to growth in LIM supplemented with heme was examined next. Rim101 is a master regulator of the pH response and is highly conserved in many fungal species (Penalva and Arst, 2002; 2004). In addition, Rim101 was shown to positively regulate the expression of CIG1 and a C. neoformans rim101Δ mutant has a growth defect at alkaline pH (O’Meara et al., 2010). Analysis of growth in LIM supplemented with heme revealed that the rim101Δ mutant showed an extended lag phase (36 hours) and eventual growth to the wild-type cell density, a phenotype similar to that of the cig1Δ mutant (Figure 3.5A). Complementation of the rim101Δ mutation restored the wild-  104  type level of growth in LIM supplemented with heme. These results suggest a role for Rim101 in heme utilization. Next, the expression of CIG1 was measured in the wild-type and rim101Δ strains during growth in LIM supplemented with heme to determine if the regulation of CIG1 by Rim101 observed by O’Meara et al., (2010) was maintained when the cells were grown in LIM supplemented with heme (Figure 3.5B). As expected, the CIG1 transcript was down-regulated in the rim101Δ mutant compared to the wild-type strain, and this regulation was independent of the growth phase.  Figure 3.5 Rim101 also contributes to heme utilization. (A) Growth of iron-starved cells in LIM + 10 µM heme was monitored by measuring the optical density at 600 nm. (B) Expression of CIG1 in the wild-type and rim101Δ strains grown in LIM + 10 µM heme. All experiments were repeated three times and the data are plotted as the average ± SD. The * represents significant differences at P < 0.05 relative to the wild-type at each stage of growth.  In a separate approach to examine the contribution of Rim101 to growth in LIM supplemented with heme (independent of CIG1), the promoter of CIG1 was replaced with that of the GAL7 gene, which is galactose inducible and glucose repressible, in the wild-type and rim101Δ strains. As expected, GAL7p::CIG1 in wild-type exhibited a similar growth defect to cig1Δ mutant in LIM supplemented with heme in media with glucose (Figure 3.6A), but a wildtype growth pattern on galactose (Figure 3.6B). In contrast, the galactose-inducible CIG1 strain 105  in the rim101Δ mutant background remained in lag phase significantly longer than either rim101Δ or cig1Δ when grown in glucose (Figure 3.6A). Although Rim101 regulates the expression of CIG1 (O’Meara et al., 2010) and may therefore play a role in Cig1-dependent heme utilization, the additive growth defect in the GAL7p::CIG1 rim101Δ strain on glucose suggests that Rim101 also plays a role in iron acquisition from heme that is independent of its regulation of CIG1. Surprisingly, growth in galactose extended the lag phase of the wild-type and cig1Δ::CIG1 strains, and particularly exacerbated the growth defect of the cig1Δ mutant (Figure 3.6B). The use of galactose as the carbon source also eliminated the growth defect in LIM supplemented with heme for both the rim101Δ and GAL7p::CIG1 rim101Δ strains relative to wild-type. These results further support the presence of a Cig1-independent mechanism for heme use and suggest an influence of carbon source. That is, galactose may promote the expression of a system to use heme iron that compensates for loss of Rim101. The influence of carbon source on heme acquisition was confirmed by growing the cells in acetate (Figure 3.6C). Utilization of this alternate carbon source resulted in much slower growth for all the strains tested, and the strains that reached stationary phase had a much lower cell density compared to growth of the cells in glucose or galactose. Additionally, the growth defect for the cig1Δ and rim101Δ mutants, and both GAL7p::CIG1 strains was severely exacerbated, and the cig1Δ and both GAL7p::CIG1 strains never entered the exponential phase of growth during the course of the experiment. Quantitative RT-PCR analysis of CIG1 expression confirmed the expected carbon source regulation of the GAL7p::CIG1 construct and also revealed that growth in galactose reduces expression of CIG1 relative to growth in glucose (Figure 3.6D). This may explain the extended lag phase in the wild-type and complemented strains on galactose and heme (compare Figures 3.6A and B).  106  Figure 3.6 Rim101 makes a Cig1-independent contribution to heme uptake. The growth of iron-starved cells in LIM + 10 µM heme is shown for media containing glucose (A), galactose (B), or acetate (C). Growth was monitored by measuring the optical density at 600 nm. (D) Expression of CIG1 in the wild-type and rim101Δ strains and the strains containing a galactoseinducible CIG1 allele grown in LIM containing glucose or galactose. All experiments were repeated three times and the data are plotted as the average ± SD. The * represents significant differences at P < 0.05 in expression of CIG1 in the GAL7p::CIG1 strains relative to the parental strain (WT or rim101Δ). Figure reproduced and modified from Cadieux et al. (2013).  3.3.4. Rim101 plays a role in iron and heme utilization at physiological and alkaline pH The contribution of Rim101 in heme uptake was further investigated by examining growth in LIM supplemented with heme or FeCl3 at different pH (Figure 3.7). The cig1Δ mutant exhibited a growth defect at pH 7.0, but not at lower pH in presence of heme. These results confirm the previous growth assays at pH 5.6 and pH 7.2 (Figure 3.3). In contrast, the cig1Δ 107  mutant grew as well as the wild-type and complemented strains at all pH in LIM supplemented with FeCl3. The rim101Δ mutant exhibited a growth defect at pH 7.5 and above in LIM supplemented with heme, but not at lower pH, while the mutant displayed a growth defect only at pH 8.0 when grown in LIM supplemented with FeCl3. The results were confirmed by growing the cells on solid LIM with added heme or FeCl3 at different pH, with similar growth defects being observed (Figure 3.7B). Together, these data support a role for Cig1 in heme acquisition at physiological pH while Rim101 plays a role in both iron and heme acquisition at physiological and alkaline pH. This conclusion is consistent with the role of Rim101 in regulating Cig1 and the heme uptake pathway, as well as components of the high-affinity iron uptake system (e.g., the iron permease gene CFT1) (O’Meara et al., 2010). However, as indicated above, Rim101 may also regulate components of another heme utilization pathway.  108  Figure 3.7 Rim101 plays a role in heme uptake at physiological pH and in iron uptake at alkaline pH. Growth of iron-starved cells in LIM + 10 µM heme (A) or LIM + 10 µM FeCl3 (B) after 48 hours at 30C. Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD. The * represents significant differences at P < 0.05 relative to the wild-type strain. Ten-fold serial dilutions of iron-starved cells spotted onto LIM + 10 µM heme (C) or LIM + 10 µM FeCl3 (D) at different pH, as indicated. The plates were incubated at 30°C for three days and photographed. The experiment was repeated three times and a representative photograph is shown.  3.3.5. Cells form aggregates in the early stages of growth in LIM supplemented with heme While performing the above experiments, the formation of dark brown/black precipitates in the cultures of cells grown in LIM supplemented with heme was observed (Figure 3.8A). The precipitates were present in cultures of all the strains tested, but were more noticeable in cultures of the cig1Δ mutant. Microscopy was used to visualize the precipitates and to potentially shed light on their nature. Upon examination, a dark granular substance (probably heme) was found  109  to surround some of the cells, especially the larger and possibly older cells, but was not directly in contact with the cells (Figure 3.8B). In fact, a halo where the substance appeared to be excluded could be observed surrounding the cells and this was seen for both the wild-type and cig1Δ mutant strains, albeit at different times during growth. These observations suggest that heme may physically interact specifically with the capsule of C. neoformans.  Further  examination, using India ink to visualize the capsule, revealed that the aggregates were composed of cells aggregated together with heme (Figure 3.8C-D). As time progressed, the aggregates became larger and encompassed more cells associated with heme, but once the cultures reached late exponential or stationary phase, the majority of aggregates dissociated and the cells returned to their initial single-cell form. The cig1Δ cells maintained aggregates for a longer period of time, presumably because the cells required more time to reach late exponential and stationary phases. Also, all cells remained as aggregates for a longer period of time when grown in galactose compared to glucose (Figure 3.8C-D). Again, the slower growth of the cells in galactose may explain this difference, or perhaps changes in capsule composition may occur to influence aggregation. The aggregate formation could possibly be explained by the highly negatively charged nature of the polysaccharide capsule surrounding the cell (Nosanchuk and Casadevall, 1997). Electrostatic interactions between the capsule and the positively charged iron in heme could result in heme binding to the capsular polysaccharides and mediate the formation of aggregates. Alternatively, formation of aggregates could reflect a specific mechanism by which the cells trap heme within the capsular mesh of the aggregates for eventual uptake. Finally, the aggregates could form as a result from the precipitation of heme, forcing the cells to aggregate with the precipitates in order to use the heme. At this point, it is unclear whether the interaction between the capsule and heme or aggregate formation is required for uptake of heme.  110  Further work will be needed to fully understand the heme-capsule interaction and the link between the observed aggregates and heme acquisition.  111  Figure 3.8 The cells and heme form aggregates during growth. Cells were grown in LIM + 10 µM heme in media containing glucose (A-C) or galactose (D). Cells in C and D were harvested and negatively stained with India ink before visualization by differential interference contrast microscopy.  112  3.3.6. Heme uptake via the Cig1-independent pathway is dependent on cell density It is possible that the formation of aggregates described above may be essential in heme acquisition as part of a cell density-dependent heme uptake mechanism sharing similarities with the QS phenomenon. QS is a communication system based on the secretion and diffusion of small molecules by microorganisms and relies on the close proximity of the cells to allow for communication between them. The possibility of a QS-like cell density-dependent heme uptake mechanism was investigated by inoculating cultures of the different strains at different cell densities and monitoring the growth in LIM with added heme (Figure 3.9). At low inoculum (102 cell/mL), all the cells remained in lag phase for a long period of time before reaching exponential phase. Specifically, the wild-type and complemented strains took 48 hours before starting to grow exponentially, while the cig1Δ mutant required 120 hours. Remarkably, the length of the lag phase for all the strains tested decreased as the inoculum size increased. At the highest inoculum (106 cells/mL), the lag phase was not detected for any of the strains, and they had all started to grow exponentially by 12 hours. However, the cig1Δ mutant grew slightly slower than the wild-type or complemented strains. The cell density-dependent growth of the cig1Δ mutant in LIM supplemented with heme was confirmed by plating the iron-starved cells on LIM with added heme. Again, the mutant grew more slowly than the wild-type strain at lower inoculum but grew as well as the wild-type at higher inoculum. These results support a role for cell density as an essential factor involved in the Cig1-independent heme uptake pathway and further suggest a possible link between a QS-like mechanism and heme uptake.  For  example, as more cells are added initially, more QS-like molecules would be secreted within the first few hours of growth which would induce the Cig1-independent heme uptake system. Alternatively, addition of more cells would also potentially increase the activity of any cell  113  surface-bound enzymes that process heme, such as the ferric reductases described previously, which could result in increased uptake of iron from heme to allow more rapid growth. Although growth of the cig1Δ mutant in LIM supplemented with heme at acidic pH did not appear to be dependent on cell density (Figure 3.3), further examination of both pathways was required to determine if the two were linked. In order to do so, the strains were grown in LIM supplemented with heme at acidic pH starting with a low inoculum (102 cell/mL). As expected, even at a low inoculum, all the strains grew as well as when inoculated at a higher inoculum (compare Figures 3.3 and 3.9). Specifically, the cells remained in lag phase for 12 hours and had reached stationary phase by 48 hours. These data suggest that the two Cig1independent heme uptake pathways, the pathway for heme acquisition at acidic pH and the cell density-dependent pathway, are distinct. However, this does not preclude possible overlap between the other components involved in each pathway.  114  Figure 3.9 Cig1-independent heme uptake is dependent on cell density. (A) Growth of ironstarved cells in LIM + 10 µM heme when inoculated at different cell densities, as indicated. Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD. (B) Growth of iron-starved cells on LIM + 10 µM heme when plated at different cell densities. The plates were incubated at 30°C for three days and photographed. The experiment was repeated three times and a representative photograph is shown.  3.3.7. Addition of conditioned media allowed the cells to grow faster QS requires that small signaling molecules be secreted by the cells to coordinate the behaviour of the group, including growth. The concentration of signaling molecules is assumed to be at its peak during stationary phase when the cell density is at its highest. To further investigate the effect of a possible QS-like mechanism on heme acquisition, CM were harvested 115  from different strains that reached stationary phase. The CM was added to fresh LIM plus heme, and growth of the different strains was compared (Figure 3.10). Addition of CM to fresh medium did not decrease the length of the lag phase for the wild-type and complemented strains, but resulted in more robust growth (based on the higher optical density at 36 hours) compared to growth of the same strains without CM (Figure 3.10A,C). The enhanced growth with the added CM for these strains was consistently observed independently of the origin of the CM. However, the effect of adding CM to cultures of the cig1Δ mutant varied depending on the source of the CM. For example, only addition of CM harvested from the cig1Δ mutant strain resulted in a shorter lag phase, possibly because the concentration of the QS-like molecule was higher in CM produced by the cig1Δ mutant strain (Figure 3.10B). Nevertheless, addition of CM from all the strains tested yielded faster growth of the cig1Δ mutant. It is unclear why the cig1Δ mutant strain would produce a higher concentration of the QS-like molecule. One possible explanation could be that in absence of a functional Cig1-dependent heme uptake pathway, more QS-like signaling molecules are produced to induce an alternative pathway for heme uptake which appears to be dependent on cell density.  Overall, these data suggest that some secreted  component found in supernatant of cultures at stationary phase (e.g., polysaccharides, proteins or peptides, lipids, or other small molecules) could act as a QS-like signal that appears to be involved in cell density-dependent heme utilization. Alternatively, the possibility of the CM having more available iron than fresh media because the heme has been processed by cell surface enzymes (e.g., reductases) cannot be discounted and could explain the more robust growth upon addition of CM. In a preliminary attempt to identify the QS-like molecule, addition of CM harvested from cultures of the cqs1Δ mutant were tested to determine if they could allow the cells to grow better  116  in LIM supplemented with heme (Figure 3.10D). The cqs1Δ mutant is defective in secretion of the QS peptide, Qsp1, that is reported to control growth in C. neoformans (Lee et al., 2007). This mutant was prepared in the C. neoformans var. neoformans background, and therefore, the C. neoformans var. neoformans wild-type strain, B3501A, was used for comparison. Addition of CM from both the wild-type and the cqs1Δ mutant resulted in faster growth for all of the strains tested compared to growth without CM implying that the Qsp1 peptide is not the QS-like molecule responsible for the observed phenotype. So far, Qsp1 has been the only identified QSlike molecule produced by C. neoformans, however, one can imagine that an array of QS-like molecules are secreted by this fungus in order to adapt to and grow in the many different environments.  Figure 3.10 Addition of CM allows the cells to grow faster. Growth of iron-starved cells: WT (A), cig1Δ (B), and complement (C) in LIM + 10 µM heme with 50% CM from the WT, cig1Δ, and complemented strains or (D) from C. neoformans var. neoformans WT and cqs1Δ strains. The CM was harvested by centrifugation once the cells grown in LIM + 10 µM heme had reached stationary phase. Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD. 117  3.3.8. Addition of exogenous polysaccharides allowed the cells to grow faster C. neoformans produces and secretes copious amounts of polysaccharides, some of which are used for capsule biosynthesis, while others, the exopolysaccharides, are soluble and found in culture supernatants (Cherniak, 1988).  These exopolysaccharides include GXM, the major  capsular polysaccharide, and other minor polysaccharides, such as GalXM.  To determine  whether exopolysaccharides could act as the QS-like molecule involved in heme acquisition, the CM was fractionated further to isolate the total secreted polysaccharides from the medium. The secreted polysaccharides were then added back to fresh cultures and the growth of the strains in LIM supplemented with heme with and without added exogenous polysaccharides was compared (Figure 3.11A-C). Addition of exogenous polysaccharides did not play a big role in the growth of the wild-type and complemented strains. In contrast, addition of exogenous polysaccharides had a more pronounced effect (although not dramatic) on the growth of cig1Δ mutant, resulting in a shorter lag phase (Figure 3.11B). Specifically, addition of exogenous polysaccharides isolated from the wild-type CM reduced the lag phase of the cig1Δ mutant by approximately 24 hours, compared to approximately 12 hours when adding polysaccharides isolated from the cig1Δ mutant CM. Growth curves obtained after addition of exogenous polysaccharides (Figure 3.11) were very similar to previous experiments with addition of CM described in the previous section (Figure 3.10) suggesting that the QS-like molecule associated with heme utilization is probably also present in the polysaccharide fraction isolated from the CM. During isolation of the secreted polysaccharides, other proteins or small molecules may have been precipitated along with the exopolysaccharides and could be present in that fraction of the CM. Therefore, the nature of the QS-like molecule was assessed further by treating the isolated polysaccharide fraction with proteinase K to determine if the signaling molecule is a  118  protein or peptide. Growth of the cig1Δ mutant in LIM supplemented with heme with added exogenous polysaccharides was similar whether the isolated polysaccharides had been treated with proteinase K or not, suggesting that the QS-like molecule does not have a proteinaceous nature (Figure 3.11D). In a second attempt to identify the nature of the QS-like molecule, the isolated polysaccharide fraction was dialyzed in dialysis tubing of low molecular weight cut-off to remove any small molecules (i.e., smaller than 7 kDa) that could serve as the QS-like signal. Addition of exogenous polysaccharides after dialysis resulted in similar growth of the cig1Δ mutant in LIM supplemented with heme than when non-dialyzed exogenous polysaccharides were added to the culture medium. Together, these results imply that the QS-like molecule is non-proteinaceous and larger than 7 kDa. However, it is important to note that the influence of adding exogenous polysaccharides to the growth medium was not dramatic, and consequently, subtle differences in growth that could have arisen from treating the isolated polysaccharides (i.e., proteinase K, dialysis) may have been difficult to detect. It is possible that the QS-like molecule involved in the Cig1-independent heme uptake pathway is the GXM polysaccharide. To this end, a cap59Δ mutant was used to test the role of GXM in heme acquisition. CAP59 is a gene necessary for capsule synthesis, and its product is presumably involved in the process of GXM export (Chang et al., 1995; Chang and KwonChung, 1999; Garcia-Rivera et al., 2004). The cas1Δ mutant was also used. Cas1 is involved in acetylation of the capsule and the cas1Δ mutant has been shown to have increased shedding of GXM in the supernatant (Janbon et al., 2001; Dr. E. Griffiths, personal communications). Growth of the cap59Δ and cas1Δ mutants in LIM supplemented with heme was compared to that of the wild-type strain and no difference in growth was observed for either strain (Figure 3.11E). Interestingly, growth of the cas1Δ mutant in LIM supplemented with FeCl3 resulted in longer lag  119  phase compared to that of the wild-type strain (Appendix D). Although these observations don’t confirm a role for GXM in heme uptake, they don’t necessarily rule it out either. If GXM is the QS-like molecule, it is possible that no growth defect is observed under the conditions tested because the Cig1-dependent heme uptake pathway overshadows or compensates for the abnormal GXM shedding in the cap59Δ and cas1Δ mutants. Also, as mentioned above, addition of exogenous polysaccharides results in subtle differences in growth, and therefore, the contribution of GXM to heme uptake may have been difficult to detect. Finally, addition of exogenous polysaccharides isolated from the culture supernatant of cap59Δ and cas1Δ mutants also decreased the lag phase of the cig1Δ mutant, similar to what was observed with addition of GXM isolated from the wild-type and cig1Δ strains (Figure 3.11B, F).  Combined, these data  suggest that GXM may not be the QS-like molecule, and instead, other exported components within the isolated exopolysaccharide fraction, including GalXM for example, could be the QSlike signal. Future work will be needed to further assess the role of GXM in heme acquisition and to examine the contribution of GalXM in the cell density-dependent heme uptake pathway. Also, if GXM or GalXM are not the QS-like molecule, the isolated exopolysaccharide fraction might need to be separated further to identify other putative QS-like molecules.  120  Figure 3.11 Addition of exogenous polysaccharides allows the cells to grow faster. Growth of iron-starved cells in LIM + 10 µM heme with added exogenous polysaccharides isolated from CM. Growth was monitored by measuring the optical density at 600 nm. All experiments were repeated three times and the data are plotted as the average ± SD.  3.3.9. Binding of GXM to heme The examination of the aggregates formed during growth in LIM supplemented with heme suggested a possible interaction between the capsule and heme. The capsule is composed mainly of GXM, but also contains GalXM and mannoproteins (Reiss et al., 1985; Murphy et al., 1988; Bose et al., 2003; Zaragoza et al., 2009). Consequently, the interaction between the capsule and heme was further investigated by measuring the absorbance spectra of the isolated  121  GXM after the addition of increasing amounts of heme. As mentioned in Chapter 2, heme groups have characteristic absorbance bands that depend on the ligation and conformational state of the chromophore thus allowing detection of heme binding by spectrophotometry. Titration of GXM with heme did not reveal any binding between GXM and heme (Figure 3.12A). ITC was also used to test the interaction between GXM and heme, however, no binding was detected (Figure 3.12B). These preliminary results suggest that GXM does not bind heme, however, the viscous nature of the GXM solution and the approximate and large size of GXM (1,700-7,000 kDa) could possibly have prevented detection of interactions. For example, the concentration of GXM needed for ITC analysis was too viscous and could not be measured. Therefore, a diluted sample had to be used which could have resulted in a weaker interaction that precluded detection. Also, the approximate molecular weight of the polysaccharide led to approximations of the concentrations of the polysaccharide being used which could have resulted in an ineffective ratio of polysaccharides to heme (leading to poor detection of the potential interaction). Exact ratios are particularly important when measuring interactions by ITC or absorbance spectra. Finally, detection of interaction by absorbance spectra has been extensively used for detection of heme-binding proteins but is not commonly used for measuring the interaction between heme and polysaccharides. The parameters may need to be optimized for detection of the interaction. Future work will be needed to confirm whether GXM interacts with heme, and to examine the possible interaction of GalXM or the capsular mannoproteins with heme.  122  Figure 3.12 GXM does not appear to bind heme. (A) Heme binding curve of GXM isolated from the wild-type strain. The difference between the absorbance of the GXM-heme complex and free heme at 405 nm is plotted against the increasing concentration of heme. (B) Binding of heme to GXM by measuring the isothermal calorimetry of heme alone (black), 0.1 mg/mL GXM plus 1 mM heme (red), or 0.5 mg/mL GXM plus 1 mM heme (green) over the specified period of time. All assays were repeated three times and representative graphs are shown.  123  3.4. Discussion Several lines of evidence indicate that additional Cig1-independent mechanisms exist for iron acquisition from heme in C. neoformans. First, deletion of CIG1 resulted in an extended lag phase in LIM supplemented with heme but eventual growth to a density comparable to the wildtype strain. Although heme likely contains some contaminating inorganic iron that could be acquired by the high-affinity iron uptake system (i.e., the permease Cft1 and the ferroxidase Cfo1) (Jung et al., 2008; 2009), the cig1Δ cfo1Δ mutant behaved similarly to the cig1Δ mutant. This observation rules out a substantial contribution of free iron to the eventual growth of the mutants. An alternate pathway could involve processing of heme by unidentified cell-surface reductases (as discussed in Chapter 2) resulting in free iron to be taken up by a pathway different from the high-affinity iron uptake system. This pathway could possibly mediate heme (or free iron released from heme) uptake via endocytosis. A similar pathway has been described for hemoglobin utilization in C. albicans, and Vps23, an important component of the ESCRT involved in endocytosis has been identified as a key player in iron acquisition from heme in C. neoformans (Weissman et al., 2008; Hu et al., 2013). A second piece of evidence for additional heme uptake mechanisms is the absence of a growth defect of the cig1Δ mutant and down-regulation of CIG1 expression at acidic pH. The solubility and bioavailability of iron is highly dependent on pH (Wilkins, 1991; Cotton et al., 1999). It is possible that the pH effect observed is a consequence of an increased bioavailability of heme for C. neoformans at acidic pH compared to neutral pH. If the bioavailabilty of heme is increased, the uptake of heme could bypass the Cig1-dependent heme uptake pathway and rely on a different heme uptake pathway, for example a low-affinity heme uptake pathway. Alternatively, the iron state is also dependent on pH, being in its ferric form at neutral/alkaline  124  pH and ferrous form at acidic pH. The Cig1-dependent heme uptake system may be specific for transportation of ferric iron heme (heme) inside the cell whereas a different heme uptake system would exist to acquire ferrous iron heme. Another possible explanation could be that the activity of the unidentified cell-surface reductases hypothesized to be involved in heme processing are influenced by the pH. Finally, the endocytic pathway described above may also be involved in uptake of heme (or free iron released upon processing of heme).  This is supported by  unpublished data that indicate a role for the ESCRT components in FeCl3 and heme uptake at acidic pH (Dr. G. Hu, personal communications). The influence of pH on growth in LIM supplemented with heme implicated the fungal transcription factor Rim101 because of its known regulation of pH-responsive genes and genes encoding functions for iron acquisition (Bensen et al., 2004; Nobile et al., 2008; O’Meara et al., 2010). In the latter case, the expression of CIG1 was down-regulated in the rim101Δ mutant of C. neoformans (O’Meara et al., 2010) and deletion of RIM101 resulted in a growth defect in LIM supplemented with heme similar to that of the cig1Δ mutant. Although Rim101 likely influences heme uptake by regulating CIG1, it also makes an independent contribution to growth in LIM supplemented with heme as demonstrated by the exacerbated growth defect of a strain lacking both Cig1 and Rim101.  This independent contribution could potentially be linked to the  ESCRT-mediated endocytic pathway involved in iron acquisition from heme especially since Rim101 is a downstream target of the ESCRT pathway in C. albicans (Xu and Mitchell, 2001; Kullas et al., 2004; Xu et al., 2004). Interestingly, the rim101Δ mutant did not have a growth defect when grown in LIM supplemented with heme in presence of galactose, while it had a more severe growth defect when grown in acetate as a carbon source. Furthermore, the transcript levels of CIG1 were  125  reduced on galactose versus glucose media and the cig1Δ mutant did not grow in LIM supplemented with heme in the presence of acetate. Thus, carbon source appears to influence iron acquisition from heme. A link between carbon source and heme has previously been established in S. cerevisiae. Specifically, the Hap 2/3/4/5 complex, which is a heme-regulated transcription factor involved in activation of genes responsible for respiration has been shown to be repressed by glucose and de-repressed by galactose and non-fermentable carbon sources (McNabb and Pinto, 2005; Lai et al., 2006). Also, the expression of the heme oxygenase, the enzyme responsible for catalyzing heme degradation, is regulated by glucose and is induced upon glucose deprivation (Chang et al., 2002). Further evidence suggests the existence of Cig1-independent heme uptake systems including the correlation between the starting inoculum size and the length of the lag phase of the cig1Δ mutant when grown in LIM supplemented with heme. This heme utilization system could rely on QS-like behaviour as indicated by the formation of cell aggregates during lag phase, the ability of the cig1Δ mutant to grow as well as the wild-type strain in LIM supplemented with heme at higher inoculum, and finally the partial recovery of the growth defect for the cig1Δ mutant when CM or isolated polysaccharides were added to fresh culture. The study of QS in fungi is still in its infancy, and the first evidence of fungal QS was described in C. albicans ten years ago. Farnesol, a QS molecule, was observed to control filamentation which is essential for virulence in this pathogenic fungus (Hornby et al., 2001). Since then, only a few other QS molecules have been identified, including tyrosol necessary for controlling growth, morphogenesis and biofilm formation in C. albicans, and phenylethanol and tryptophol involved in controlling morphogenesis during nitrogen starvation in S. cerevisiae (Chen et al., 2004; Chen and Fink, 2006; Albuquerque and Casadevall, 2012).  126  Cell density-dependent behaviours have also been described in different fungi, including C. neoformans. For example, a cell density-dependent growth phenotype similar to QS was observed in C. neoformans var. neoformans (serotype D) after deletion of the global repressor TUP1 (Lee et al., 2007). A small oligopeptide (11 amino acids), Qsp1, encoded by the CSQ1 genes was identified as an autoregulatory molecule found in CM and was responsible for the cell density-dependent growth phenotype. Another study described the influence of cell density on melanization in both serotypes A and D, in which melanization occurred more rapidly in denser cultures (Eisenman et al., 2011). Finally, growth, secretion of GXM, and biofilm formation were also shown to depend on cell density in C. neoformans, although the QS molecule was never confirmed (Albuquerque, 2011). Additionally, a link between QS and iron uptake has been well documented in many bacteria, notably in P. aeruginosa. For example, expression of QS systems were induced under iron starvation conditions in P. aeruginosa but repressed under high-iron conditions (Kim et al., 2005; Duan and Surette, 2007; Yang et al., 2007; Hazan et al., 2010). Not surprisingly, some QS regulators of P. aeruginosa are also downstream targets of the Fur iron regulator (Ochsner et al., 2002; Oglesby et al., 2008). In contrast, QS regulators also induce many iron responsive genes in P. aeruginosa, including the siderophore, pyochelin (Ochsner et al., 2002; Palma et al., 2003; Schuster et al., 2003; Cornelis and Aendekerk, 2004; Juhas et al., 2004; 2005; Deziel et al., 2005; Hazan et al., 2010). The cell density-dependent heme uptake system is likely distinct from the heme uptake mechanism involved at acidic pH because cell density was not a factor for growth in LIM supplemented with heme at acidic pH. Nevertheless, both pathways may rely on cell-surface reductases involved in heme processing. A higher cell density would result in increased cell-  127  surface reductase activity which could process heme more rapidly. Consequently, it is possible that different components involved in transport of the heme to the inside of the cell are shared among the two pathways. Finally, the capsular polysaccharides could potentially be linked with heme uptake. The figures of the aggregates formed during growth in LIM supplemented with heme imply some sort of physical contact between the capsule and heme.  Furthermore, addition of exogenous  polysaccharides increased the growth of the cig1Δ mutant in LIM supplemented with heme suggesting a possible role for the polysaccharides as the QS-like signaling molecule involved in cell to cell communication during growth in LIM supplemented with heme. The capsule is composed of GXM, GalXM, and mannoproteins. Additionally, chitin-like oligomers are found at the cell surface and appear to be involved in linking the capsule to the cell wall by interacting with GXM (Rodrigues et al., 2008b; Ramos et al., 2012). If any of these components could bind heme, they could potentially help trap the heme molecule in the capsule mesh. In fact, the QS molecule, PQS, characterized in P. aeruginosa was observed to bind iron and acted as an iron trap molecule (Bredenbruch et al., 2006; Diggle et al., 2007).  Although GXM represents  approximately 90-95% of the capsular material, it was not shown to bind heme in the conditions tested (Cherniak et al., 1980). Also, studies involving the cap59Δ strain, a mutant which cannot produce GXM, indicated that GXM does not appear to be the QS-like molecule. Treatment of the isolated polysaccharide fraction with proteinase K did not reduce the ability of the exogenous polysaccharides to rescue the growth defect of the cig1Δ mutant in LIM supplemented with heme suggesting that the QS-like molecule is not a mannoprotein. Other studies will be needed to confirm that GXM does not bind heme but future work should focus on determining whether  128  GalXM, capsular mannoproteins and/or chitin-like oligomers interact with heme, if this interaction is essential for heme uptake, and if it plays a role in QS-like signaling between cells. In summary, C. neoformans appears to have developed multiple heme uptake systems. In this study, two distinct Cig1-independent heme utilization systems have been identified, including a pathway involved in heme utilization at acidic pH, and another cell densitydependent pathway with similarities to QS. The QS-like molecule was not determined, although secreted polysaccharides could be potential candidates. Further work will be needed to elucidate the receptors and mechanisms involved in heme uptake in both these Cig1-independent pathways.  129  Chapter 4. Cig1 plays a role in heme utilization, as well as in secretion and maintenance of cell wall integrity in C. neoformans var. neoformans. 4.1. Introduction In the work presented in Chapter 2, Cig1 was found to play an important role in heme uptake and utilization in C. neoformans var. grubii. Although C. neoformans var. neoformans shares 85-90% similarity with C. neoformans var. grubii at the genomic level, it cannot be assumed that the one protein will play the same role in both varieties (Kavanaugh et al. 2006). In fact, C. neoformans var. grubii and C. neoformans var. neoformans diverged approximately 18 million years ago (Fan et al., 1994; Xu et al., 2000). Over that time, genetic diversity has arisen between the two different C. neoformans varieties leading to important differences in pathogenicity, virulence, host specificity and geographical distribution.  There are multiple  reported cases of the same protein playing an important role in one variety while no role could be determined in the other variety (Cruz et al., 2000; Wang et al., 2002; Hicks et al., 2004; Bahn et al., 2005).  Therefore, this study focuses on further characterizing the role of Cig1 in C.  neoformans var. neoformans to obtain a broader view of the function of the protein. Lian et al. (2005) characterized a cig1Δ mutant in C. neoformans var. neoformans with regard to iron-related phenotypes.  Specifically, the cig1Δ mutant in C. neoformans var.  neoformans was reported to have a growth defect in LIM and a change in the capsule response to iron (Lian et al., 2005). Typically, the wild-type strain elaborated a larger capsule when grown in LIM compared to when it was grown in presence of FeCl3. However, the cig1Δ mutant appeared to be non-responsive to iron levels and produced a larger capsule than the wild-type strain when grown in LIM supplemented with FeCl3. These preliminary results suggested a possible link between Cig1 and the synthesis of the polysaccharide capsule.  130  Secretion is an important component for the pathogenesis of C. neoformans as many of its known virulence factors are secreted to the cell surface or to the extracellular space, including the polysaccharides required for capsule synthesis. Although little is known about secretion in C. neoformans, there is increasing evidence suggesting the existence of more than one secretory pathway, including the conventional and non-conventional pathways (Rodrigues et al., 2007; 2008a; 2008b; 2012; Nosanchuk et al., 2008; Eisenman et al., 2009; Panepinto et al., 2009; Oliveira et al., 2009; 2010; Kmetzsch et al., 2011). Transport through the conventional secretory pathway requires the secreted proteins to have a signal peptide and trafficking of proteins from the endoplasmic reticulum (ER) to the Golgi to subsequently reach the cell periphery in trafficking vesicles. The non-conventional secretory pathway does not require the secreted proteins to have a signal peptide and secretion occurs independently from the ER-Golgi pathway (Rodrigues et al., 2008b; Panepinto et al., 2009; Oliveira et al., 2010; Kmetzsch et al., 2011). Additionally, extracellular vesicles produced by C. neoformans can transport cargo material, including proteins, polysaccharides and lipids, across the cell wall (Rodrigues et al., 2007; 2008a; 2008b; Nosanchuk et al., 2008; Eisenman et al., 2009; Oliveira et al., 2009). These different secretory pathways are involved in the secretion of different components to the cell wall, capsule and extracellular space. However, there also appears to be some redundancy between the pathways. For example, there is evidence that GXM, the major polysaccharide of the capsule, can be transported via all of the described secretory pathways (Yoneda and Doering, 2006; 2009; Rodrigues and Djordjevic, 2012). The link between Cig1 and elaboration of the capsule led to the hypothesis that Cig1 may influence secretion. To test this hypothesis, the role of Cig1 in secretion was assessed by growing the wild-type and cig1Δ strains in presence of secretion inhibitors. The susceptibility to  131  other stresses, including agents that challenge cell wall integrity, as well as osmotic stress and heat stress, were also tested.  Additionally, the activity of secreted proteases was compared  between the different strains and the concentration of total secreted proteins was also measured. Finally, the relative quantity of secreted extracellular vesicles was compared between the different strains.  132  4.2. Materials and Methods 4.2.1. Strains and growth conditions All strains used in this study are listed in Table 4.1. YPD (Fisher), yeast extract peptone galactose (YPG), YNB (Difco), and LIM were used to grow the cells. LIM was prepared as described (Vartivarian et al., 1993) and the pH was adjusted to 7.2. The water used for LIM was treated with Chelex-100 resin (Bio-Rad) to chelate iron. Cells for all assays were pre-grown overnight at 30°C with shaking in YPD, unless specified otherwise.  Table 4.1. Strains used in this study Strain Description B3501A C. neoformans var. neoformans wild-type strain cig1DΔTL cig1 disruption mutant #1 cig1DΔBC cig1 disruption mutant #2 cig1DΔdel cig1 deletion mutant cig1DΔ +CIG1 CIG1 complemented strain in cig1DΔTL cigD1Δstop Strain with a stop codon after the ATG in the disrupted CIG1 open reading frame GAL7p::CIG1D Regulated CIG1 disruption allele in the B3501A background H99 C. neoformans var. grubii wild-type strain cig1AΔdis cig1 disruption mutant cig1AΔdel cig1 partial deletion mutant cig1AΔ +CIG1 CIG1 complemented strain  Provided/ Prepared by Dr. June Kwon-Chung Tian Lian Brigitte Cadieux Brigitte Cadieux Tian Lian Brigitte Cadieux Brigitte Cadieux Dr. Joseph Heitman Brigitte Cadieux Carmelo Biondo Tian Lian  4.2.2. Construction of strains Primers used for construction of all strains are listed in Table 4.2 and a schematic of all of the constructs generated is depicted in Figure 4.1A.  133  Table 4.2. Primers used for strain construction Allele Primer identification Primer sequence constructed cig1DΔdis cig1D:NEO  CIG-drup-F CIG-drup-R CIG1D-1F CIG1D-3R CIG1D-4F  cig1AΔdel cig1DΔstop  CIG1D-6R CIG1D-7F CIG1D-8R CIG1A-1 CIG1A-6 CIG1D-GAL-LA-F CIG1D-STOP-LA-R2 CIG1D-STOP-RA-F2  GAL7p::NAT:: CIG1  CIG1D-STOP-R CIG1D-GAL-LA-R CIG1D-GAL-RA-F Nest-CIG1D-STOP-F  GAL7p::NAT  Nest-CIG1D-STOP-R Gal7-CIG1D-F Gal7-CIG1D-R  NEO Transformant screen  TL2001 TL2002 colPCR-Rev  CAAGGGCTTTTCAACCCTGT AGTACTTCGGCATCCACTTGAC AACAATGTACTGACCTTTCCATACC AATTCTGCAGATATCCATCACACTGGCGGCGGAA TTAATATTGGAAGTTGTCGTG AATTCCAGCACACTGGCGGCCGTTACTAGTCGAT GAGAGTTATTCATTTTCACCT GGGATTGCTATTATACCACAGTGAG CCCAGTGTTCTGTCCCATCT AGGCCAAGGTTCTTGGAAAT TATAGTCATTTTTGAACCAACGACA ATTCACTGGATCCGTCGGTA GTCTACTACGGTCTATGTGCTGGAT TGAATGTGAAGCGATTAAAGATTCACATGGTATT CTGTGTGATCGG TACAACGATCACACAGAATACCATGTGAATCTTT AATCGCTTCACATTC AGAGTCGATTCAGATCACCAACTT ACGGCGAGGCGCCGCCAGGCCCCGCCGATCGGT ATTCTGTGTGATCGTTGTAGGC AGCTTCACGGCTCAGGCTGAACTTGGATTAATGA TCTTTAATCGCTTCACATTCA GTTTCTTGAAGCTTAACTTGAGGTG TATGAACATATGCCTGATAGTCTCG GCCTACAACGATCACACAGAATACCTAGTGAAGT CGTATTGTCTCTGC TGAATGTGAAGCGATTAAAGATGCATTCTCAGGA GAGAATTGAGTGCTG ACTAGTAACGGCCGCCAGTGTGCTGGAATT GCCGCCAGTGTGATGGATATCTGCAGAATT GCCACTCGAATCCTGCATGCTTATG  134  Figure 4.1 Construction and confirmation of mutants. (A) Schematic representation of the constructs generated to create the different mutants used in this study. The coloured boxes represent the indicated open reading frames. NAT and NEO represent the nourseothricin and neomycin resistance cassettes, respectively. The engineered stop codon is represented by the red dot. The galactose-regulated promoter, GAL7p, is in green. Note that the cig1AΔdis strain contains the disrupted allele transferred from the cig1DΔTL mutant as indicated by the CIG1D* segments. (B) Southern blot analyses were performed to confirm disruption and deletion of CIG1, insertion of the engineered stop codon, insertion of the GAL7 promoter in C. neoformans var. neoformans and disruption of CIG1 in C. neoformans var. grubii. In the first four panels, genomic DNA of the indicated strains was digested with HindIII. In the last panel, genomic DNA from the indicated strains was digested with NcoI. In all cases, the digested DNA was hybridized with a probe amplified from the DNA sequence downstream of the CIG1 open reading frame. 4.2.2.1. Construction of the cig1Δ mutant and complemented strains The CIG1 disruption mutant cig1DΔTL and the CIG1 complemented strain cig1DΔ::CIG1 in C. neoformans var. neoformans (serotype D) strain B3501A were described by Lian et al. (2005). The mutation in the cig1DΔTL strain (prepared by Tian Lian) is referred to as a disruption allele because the NAT resistance marker was inserted into the gene without deletion of the open reading frame. The cig1DΔBC strain (prepared by Brigitte Cadieux) was prepared 135  by amplifying the cig1:NAT disruption allele from the cig1DΔTL strain using primer CIG-drupF and CIG-drup-R. The construct was introduced into strain B3501A by biolistic transformation (Toffaletti et al., 1993) and transformants were screened by colony PCR using primers CIG1D7F and colPCR-Rev.  All transformants in which the wild-type allele was replaced were  confirmed by hybridization to genomic DNA blots with a probe amplified using primers CIG1D1F and CIG1D-3R (Figure 4.1B). The transformant cig1DΔBC-12 was used for further studies. To construct the cig1DΔdel strain, a cig1D:NEO deletion allele was prepared by overlap PCR using the methods described by Davidson et al. (2002) and Yu (2004). In the first round of PCR, the 871 bp sequence upstream of the 5’ end of the CIG1 gene was amplified from genomic DNA from strain B3501A with primers CIG1D-4F and CIG1D-6R, and the 941 bp sequence downstream of CIG1 was amplified with primers CIG1D-1F and CIG1D-3R. The 1,970 bp sequence of the NEO gene was amplified from plasmid pJAF1 with primers TL2001 and TL2002. The nested primers CIG1D-7F and CIG1D-8R were then used to overlap the three products to yield the 4,099 bp cig1:NEO knock-out construct. The construct was introduced into strain B3501A by biolistic transformation to generate the cig1DΔBC strain.  The resulting  transformants were screened by colony PCR using primers CIG1D-1F and colPCR-Rev. All transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic B3501A DNA using primers CIG1D1F and CIG1D-3R (Figure 4.1B). The transformant cig1DΔdel-7 was used for further studies. Construction of the cig1AΔdel and complemented strains in the H99 background were described in Chapter 2. Construction of the cig1AΔdis strain was done by amplifying the cig1:NAT disruption allele from the cig1DΔTL strain using primer CIG-drup-F and CIG-drup-R and the construct was introduced into strain H99 by biolistic transformation. Transformants  136  were screened by colony PCR using primers CIG1A-6 and colPCR-Rev. All transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified using primers CIG1A-1 and CIG1D-3R (Figure 4.1B). The transformant cig1AΔdis-10 was used for further studies.  4.2.2.2. Construction of the cig1DΔstop strain The cig1DΔstop allele was prepared by inserting the stop codon, TGA, immediately after the start codon ATG in the CIG1D disruption construct using overlap PCR. Specifically, the 892 bp sequence upstream of the CIG1 gene was amplified from genomic DNA isolated from the cig1DΔTL strain with primers CIG1D-GAL-LA-F and CIG1D-STOP-LA-R2. The CIG1DSTOP-LA-R2 primer was designed to include the sequence of the start codon for the CIG1 gene followed by the stop codon. The 3,378 bp sequence of the cig1:NAT disruption allele, including the sequence downstream of the CIG1 gene, was amplified from genomic DNA from the cig1DΔTL strain using primers CIG1D-STOP-RA-F2 and CIG1D-STOP-R. The nested primers were then used to overlap the two products and yield the 4,037 bp construct. The construct was introduced into the B3501A wild-type strain by biolistic transformation and the transformants were screened by colony PCR using primers CIG1D-STOP-R and colPCR-Rev.  All  transformants in which the wild-type allele was replaced were confirmed by hybridization to genomic DNA blots with a probe amplified from genomic B3501A DNA using primers CIG1D1F and CIG1D-3R (Figure 4.1B). The transformant cig1DΔstop-1 was used for further studies.  137  4.2.2.3. Construction of the galactose-regulated CIG1D strain The GAL7p::NAT::CIG1 allele was constructed by overlap PCR. The sequence upstream of the 5’ end of the CIG1 gene was amplified from genomic DNA from the cig1DΔTL strain using the primers CIG1D-GAL-LA-F and CIG1D-GAL-LA-R, and the cig1D:NAT disruption allele was amplified with primers CIG1D-GAL-RA-F and CIG1D-STOP-R. The NAT::GAL7p allele was amplified from strain GAL7p::PKA1 kindly provided by Dr. J. Choi (Choi et al., 2012) with primers Gal7-CIG1D-F and Gal7-CIG1D-R.  The nested primers Nest-CIG1D-  STOP-F and Nest-CIG1D-STOP-R were then used to overlap the three products to yield the 5.2 kb GAL7p::NAT::CIG1 allele.  The construct was introduced into the C. neoformans var.  neoformans strain B3501A by biolistic transformation to generate the GAL7p::CIG1D regulated strains.  Transformants were screened by colony PCR using primers CIG1D-STOP-R and  colPCR-Rev and confirmed by hybridization to genomic DNA blots with a probe amplified from genomic B3501A DNA using primers CIG1D-1F and CIG1D-3R (Figure 4.1B).  The  transformant GAL7p::CIG1D-1 was used for further studies.  4.2.3. Quantitative RT-PCR To examine gene expression, the cells were grown in LIM (with either glucose or galactose as the carbon source as indicated) for 6 hours at 30°C with shaking. Total RNA was extracted using the RNeasy kit (Qiagen), treated with DNase (Qiagen) and cDNA was synthesized using the SuperScript First Strand System (Invitrogen) following the manufacturers’ recommendations. PCR reactions were monitored using the 7500 system (Applied Biosystems) as described previously (Tangen et al., 2007), while the primers used were designed using  138  Primer Express software 3.0 (Applied Biosystems) and are listed in Table 4.3. The relative gene expression was quantified using the SDS software 1.3.1 (Applied Biosystems), based on the 2-ΔΔCT method (Livak and Schmittgen, 2001). The average Ct of 18S rRNA was used for normalization.  Table 4.3. Primers used for quantitative RT-PCR Primer identification Primer sequence CIG1D-RT-F GCCGTCATCGCTGGCTTA CIG1D-RT-R TGCTTCTTCTTGCCAAAACGA 18S-RT-F AACAGGTCTGTGATGCCCTTAGA 18S-RT-R ACTCGCTGGCTCAGTCAGTGT  Target Gene CIG1D CIG1D 18S rRNA 18S rRNA  4.2.4. Capsule assay Cells pre-grown in YPD were washed twice with low iron water, diluted to 4 x 106 cells/mL in LIM and grown for 24 hours at 30°C. The cells were then resuspended in 1/10th volume of LIM, mixed 1:1 in india ink and visualized by negative staining on a Zeiss Axioplan 2 Imaging microscope.  4.2.5. Growth in LIM supplemented with heme Cells pre-grown in YPD were washed twice with low iron water, inoculated into LIM at 4 x 106 cells/mL and grown at 30°C for two days to starve the cells for iron. After starvation, the cells were harvested, washed and inoculated in LIM supplemented with 10 µM heme (porcine hemin; Sigma) to a final concentration of 5 X 104 cells/mL. Cultures were incubated at 30°C and growth was monitored by measuring the optical density at 600 nm using a DU530 Life Science UV/Visible spectrophotometer (Beckman Instruments).  139  4.2.6. Plate assays Cells pre-grown in YPD were washed once with water and resuspended at 1 x 106 cells/mL in water. Ten-fold serial dilutions were made in water and 5 µL of each dilution were plated on YPD with and without agents that challenge the cell wall integrity: 0.5 mg/mL caffeine (Sigma), 0.0075% sodium dodecyl sulfate (SDS; Sigma) and 0.5% congo red (Sigma), with and without secretion inhibitors: 25 µg/mL brefeldin A (BFA; LC Laboratories), 0.625 µg/µL monensin (Sigma), 625 µM N-ethyl maleimide (NEM; Sigma) and with and without 1.2 M NaCl or 1.2 M sorbitol; 2% agar (Sigma) was added as the solidifying agent. Plates were incubated for three days at 30°C or 37°C as indicated and then photographed.  4.2.7. In silico analysis The predicted CIG1 DNA sequence (CNC01660) was retrieved from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/gene/3256784). Genomic DNA was isolated from C. neoformans strain B3501A and amplified with the primers listed in Table 4.4. The amplified DNA samples were sent for sequencing at the NAPS Unit at the University of British Columbia. The predicted and confirmed DNA sequences were aligned using Clustal W (http://www.ebi.ac.uk/clustalw/).  The Sequence Manipulation Suite was used to predict  translation of the DNA sequence (http://bioinformatics.org/sms2/translate.html).  Table 4.4. Primers used for amplifying and sequencing the DNA Primer identification CIG-Adj-3F CIG-Adj-4R CIG-Adj-5F CIG-Adj-6R CIG-Adj-7F CIG-Adj-8R  Primer sequence ACGTCCTTCGGTTGAGTCAG GCCCCTGACCAAGGTGTT GTACGAGACGACCACGAAGC AGACATGAGGGTTGGTGGAG TTCCGAGCTTGAGATTGCTT CCCAGTGTTCTGTCCCATCT 140  4.2.8. Total protein secretion assay Cells pre-grown in YPD were washed twice with water and inoculated in YNB to a final concentration of 5 x 105 cells/mL. The cultures were grown for four days at 30°C with shaking and the culture supernatant was harvested by centrifugation (13,500 x g, 1 minute, 25°C). The total amount of proteins secreted in the culture supernatant was measured using the Bio-Rad Protein Assay (Bio-Rad) and normalized to the number of cells in the culture.  4.2.9. Protease assay Protease activity was monitored by comparing the size of the clearance zones surrounding cells grown on azoalbumin (Choi et al., 2012; Chen et al., 1996). Briefly, cells pregrown in YPD were harvested by centrifugation (13,500 x g, 1 minute, 25°C), washed once with water and resuspended at 1 x 107 cells/mL in water. Five µL were plated on minimal agar media containing 0.1% azoalbumin (Sigma-Aldrich). The plates were incubated for seven days at 30°C and then photographed.  4.2.10. Secretion of extracellular vesicles Cells pre-grown in YPD were washed in water and inoculated in 500 mL YNB to a final concentration of 5 x 105 cells/mL. Cultures were grown for four days at 30°C with shaking. The extracellular vesicles were isolated from the culture supernatant as described by Rodrigues et al., (2007) with minor modifications.  Briefly, the culture supernatant was harvested by  centrifugation, and the extracellular vesicles were directly isolated by ultracentrifugation (100,000 x g, 1 hour, 4°C). The extracellular vesicles secreted by each strain were compared using thin layer chromatography (TLC; Oliveira et al., 2009). The vesicles were washed once  141  with water and resuspended in a solution of chloroform/ methanol/water (8:4:3) to extract the lipid fraction. The organic phase was dried at room temperature and resuspended in 50 µL of chloroform/methanol (2:1) solution. The lipid extracts were loaded onto a TLC silica plate and separated using a hexane/ether/acetic acid (20:10:0.5) solvent solution. The plate was then sprayed and developed as described (Oliveira et al., 2009). Densitometric analysis using ImageJ (rsbweb.nih.gov/ij) was performed to quantitate the relative amount of ergosterol in each lane.  142  4.3. Results 4.3.1.  Complementation of the cig1DΔ mutation resulted in an enlarged capsule and  overexpression of CIG1 Previously, Lian et al. (2005) had described altered capsule production in response to iron for the cig1DΔTL mutant. Typically, C. neoformans produces a larger capsule in low iron conditions.  Although no significant differences were observed in the capsule size of the  cig1DΔTL mutant compared to the wild-type strain in LIM, Lian et al. (2005) found that the cig1DΔTL mutant produced a larger capsule than the wild-type strain in presence of iron. This experiment was repeated to confirm these observations and similar results were obtained (Figure 4.2A). Interestingly, the inclusion of the complemented strain in the experiment revealed that this strain produced an enlarged capsule compared to the wild-type strain in both low iron and iron-replete conditions. Considering the possible link between the role of Cig1 and regulation of capsule size suggested by the results of Lian et al. (2005), the expression of CIG1 in the wildtype and complemented strains was measured by quantitative RT-PCR (Figure 4.2B). Expression of CIG1 was significantly higher (> 150 times) in the complemented strain compared to the wild-type strain, further supporting a possible link between Cig1 and the control of capsule size. However, upon further investigation, it was found that the transformation event that yielded the complemented strain resulted in ectopic integration of the complementation construct into another gene (CNBE3170) leading to its disruption (Dr. G. Hu, personal communications). Specifically, insertion of the transformation construct resulted in deletion of the last 694 bp of the gene. This region represents approximately half of the sequence encoding the protein and corresponds to the C-terminal portion of the protein.  Although CNBE3170 encodes a  hypothetical protein of unknown function, it is possible that disruption of this gene also  143  contributes to the enlarged capsule phenotype and/or overexpression of CIG1. Therefore, the phenotypes of the complementation strain must be interpreted cautiously.  Figure 4.2 Complementation of the cig1DΔ mutation results in enlarged capsule and overexpression of CIG1. (A) Cells were grown in LIM +/- 100 µM FeCl3, harvested and negatively stained with India ink before visualizing by differential interference contrast microscopy. (B) Expression of CIG1 in the wild-type and complemented strains grown in LIM. All experiments were repeated three times and the data are plotted as the average ± SD. The * represents a significant difference at P < 0.0005. 144  4.3.2. Construction of additional strains with mutated alleles of CIG1 in C. neoformans var. neoformans As described in the previous section, complementation of the cig1DΔ mutant resulted in the unexpected phenotype of an enlarged capsule and, therefore, the strain could not be used as a control to confirm that the phenotypes observed with the mutant resulted from mutation of the CIG1 gene. Although other complemented strains were generated, none of them reverted the phenotypes to those of the wild-type strain. An accepted alternative is to use independent mutants to confirm that loss of a particular gene causes the observed phenotypes. Therefore, two additional independent mutants were constructed. One of these mutants, cig1DΔBC, consisted of a disruption of the CIG1 gene with the identical construct used for the cig1DΔTL mutant, which resulted in partial deletion of the CIG1 gene. The second mutant, cig1DΔdel, was constructed to have a complete deletion of the CIG1 gene. These three mutants (cig1DΔTL, cig1DΔBC and cig1DΔdel) were used in all subsequent studies along with the wild-type and complemented strains.  4.3.3. Cig1 also plays a role in heme acquisition in C. neoformans var. neoformans Given that Cig1 plays an important role in heme utilization in C. neoformans var. grubii, the possibility that Cig1 plays a similar role in C. neoformans var. neoformans was examined. Growth of the cig1DΔ mutants in LIM with added heme as a sole iron source was monitored by measuring the optical density and compared to the wild-type and complemented strains (Figure 4.3). Not surprisingly, all of the cig1DΔ mutants had a growth defect in LIM supplemented with heme similar to that of the cig1Δ mutant in C. neoformans var. grubii. However, the growth defect of the cig1DΔTL and cig1DΔBC mutants was more severe because the cells still remained  145  in lag phase after 96 hours, compared to a lag phase of 60 hours for the cig1DΔdel mutant and the cig1Δ in C. neoformans var. grubii (compare Figures 4.3 and 2.6D). The wild-type strain reached stationary phase by 48 hours of growth while the complemented strain partially allowed the cells to recover, remaining in lag phase for 60 hours before starting to grow.  An unusual  behaviour of the complemented strain was again observed because addition of the wild-type copy of CIG1 to the mutant cig1DΔTL improved growth in LIM supplemented with heme, but growth was not restored to the level of the wild-type strain. Overall, these results indicate that CIG1 also plays a role in heme acquisition in C. neoformans var. neoformans.  Figure 4.3 Cig1 in C. neoformans var. neoformans is required for growth in LIM supplemented with heme. Growth of iron-starved cells in LIM + 10 µM heme was monitored by measuring the optical density at 600 nm. The experiment was repeated three times and the data are plotted as the average ± SD.  4.3.4. Disruption of CIG1 results in increased susceptibility to secretion inhibitors in C. neoformans var. neoformans, but not in var. grubii The overexpression of CIG1 and enlarged capsule phenotype of the complemented strain, as well as the larger capsule on the cig1DΔTL mutant when grown in iron-replete medium led to the hypothesis that Cig1 may play a role in secretion. For example, it is possible that the 146  function of one or more secretory pathways is perturbed when changes occur to Cig1 expression levels. To examine whether Cig1 influences secretion, the wild-type strain, the different cig1DΔ mutants and the complemented strain in C. neoformans var. neoformans were serially diluted and plated on media containing different secretion inhibitors (Figure 4.4).  The cig1DΔTL and  cig1DΔBC mutants were found to be more susceptible than the wild-type strain to BFA, an agent that arrests the anterograde transport between the ER and Golgi (Misumi et al., 1986; Fujiwara et al., 1988; Orci et al., 1991; Betina, 1992). These mutants were also susceptible to monensin, an agent which blocks intracellular transport in both the trans-Golgi and post-Golgi compartments, thereby preventing glycoprotein secretion (Tartakoff and Vassalli, 1977; Uchida et al., 1979; 1980; Griffiths et al., 1983; Tartakoff, 1983).  Finally, the mutants were slightly more  susceptible than the wild-type strain to NEM, which inhibits protein transport between successive compartments of the Golgi stack by inactivating the Golgi membranes (Nelson and Taiz, 1989). It is interesting to note that the cig1DΔBC mutant was susceptible but consistently less so than the cig1DΔTL mutant. Also, the cig1DΔdel mutant did not show susceptibility to any of the secretion inhibitors tested suggesting that a complete loss of Cig1 protein eliminated the influence on secretion. Remarkably, the complemented strain, which has been shown to overexpress the CIG1 transcript, was susceptible to BFA suggesting that overexpression of Cig1 could impact the anterograde transport between the ER and Golgi. However, caution is needed when interpreting the results involving the complemented strain given the possibility of an additional defect in this strain. Overall, these results suggest that perturbations of Cig1 lead to impairment of the secretory pathway as revealed by growth inhibition when the pathway is challenged.  147  The role of Cig1 in secretion was also evaluated in C. neoformans var. grubii. In this background, the cig1AΔdel and cig1AΔdis mutants did not show susceptibility to any of the secretion inhibitors tested suggesting that Cig1 does not influence secretion in this background (Figure 4.4B).  Figure 4.4 Disruption of CIG1 results in susceptibility to secretion inhibitors in C. neoformans var. neoformans but not in var. grubii. Ten-fold serial dilutions of C. neoformans var. neoformans strains (A) and C. neoformans var. grubii strains (B) grown in YPD were spotted onto YPD with and without secretion inhibitors at the concentration indicated. The plates were incubated at 30°C for three days and photographed. The experiments were repeated three times and a representative photograph is shown.  4.3.5. Disruption of CIG1 results in a defect in cell wall integrity in C. neoformans var. neoformans but not in var. grubii Defects in the secretory pathway often lead to changes in cell wall integrity due to the improper delivery of proteins, polysaccharides, or lipids involved in cell wall synthesis. The role of Cig1 in cell wall integrity was therefore tested by plating ten-fold serial dilutions of the different strains on media containing agents that challenge the cell wall or agents that cause osmotic stress.  In C. neoformans var. neoformans, the cig1DΔTL and cig1DΔBC mutants  showed a growth defect at 37°C and had slower growth than the wild-type strain in presence of 148  agents that challenge the cell wall, including caffeine, congo red and SDS (Figure 4.5). Additionally, the mutants grew slower than the wild-type strain in presence of NaCl, which presents a salt and osmotic stress, but not in presence of sorbitol. In a similar manner to growth in presence of secretion inhibitors, the cig1DΔdel mutant did not show susceptibility to any of the stresses tested, while the complemented strain was more sensitive than the wild-type strain to growth in presence of congo red, SDS, and NaCl. In terms of complementation of the cig1DΔTL mutant, the cig1DΔ::CIG1 strain showed reduced susceptibility on caffeine, congo red (slight) and at 37°C, but no difference on SDS or NaCl. Overall, these results point to a role for Cig1 in maintaining cell wall integrity, thereby conferring resistance against various stresses, specifically high temperature and osmotic stress. Further investigation will be required to determine whether Cig1 plays a direct role in maintaining cell wall integrity or if susceptibility to the different stresses arose from an influence on the secretory pathway. The mutants in C. neoformans var. grubii did not show altered susceptibility to any of the stresses tested (Figure 4.5B) indicating that Cig1 does not play a role in maintaining cell wall integrity in this background.  149  Figure 4.5 Disruption of CIG1 results in susceptibility to agents that challenge the cell wall in C. neoformans var. neoformans but not in var. grubii. Ten-fold serial dilutions of C. neoformans var. neoformans strains (A) and C. neoformans var. grubii strains (B) grown in YPD were spotted onto YPD with and without agents that challenge the cell wall at the concentration indicated. The plates were incubated at 30°C or 37°C for three days and photographed. The experiments were repeated three times and a representative photograph is shown.  4.3.6. Evidence that expression of a truncated Cig1 peptide influenced susceptibility to secretion inhibitors, agents that challenge the cell wall, heat and osmotic stress The fact that the cig1DΔTL and cig1DΔBC disruption mutants showed increased susceptibility to secretion inhibitors as well as to agents that challenge the cell wall and different stresses, while the cig1DΔ deletion mutant did not, led to the hypothesis that the disruption allele might represent a gain of function mutation. The construct was therefore amplified from the cig1DΔTL mutant and sequenced to confirm that the predicted sequence was in fact correct and that no mutation had occurred during cloning and transformation. The construct proved to be correct as predicted except for one amino acid substitution in the 5th codon that changed the predicted arginine to a histidine (Figure 4.6A). In addition, the sequence analysis confirmed that the disruption construct could potentially encode a truncated polypeptide (Figure 4.6B-C). This truncated polypeptide would be 150 amino acids in length and encoded by exons 1, 2, 3, and a small portion of exon 4. Thus, it is possible that the expression of a Cig1 truncated polypeptide  150  in the cig1DΔTL and cig1DΔBC disruption mutants, but not in the cig1DΔ deletion strain, could explain the phenotypic differences between the mutants.  predicted confirmed  MIFNRFTFTAAMAASAASAISVQRRAAITDFDTSPVAFAFPEPRGFSASTASEAPCGGFD 60 MIFNHFTFTAAMAASAASAISVQRRAAITDFDTSPVAFAFPEPRGFSASTASEAPCGGFD 60 ****:*******************************************************  predicted confirmed  PVNRTSYPLSGGDVALIQQTDATNVNILWTSESDPTLFHSFSTYSNSILDIAAGHYCQDA 120 PVNRTSYPLSGGDVALIQQTDATNVNILWTSESDPTLFHSFSTYSNSILDIAAGHYCQDA 120 ************************************************************  predicted confirmed  PDFSSLGFAEGDNATLLVIYQLDGADTYYYQCATSNGRQCAGIRP--WFICIN-TEEM-- 175 PDFSSLGFAEGDNATLLVIYQLDGADTYYY------------------------------ 150 ******************************  Figure 4.6 The cig1DΔ disruption mutants express a truncated polypeptide. (A) Multiple sequence alignment comparing the predicted sequence of the wild-type Cig1 and confirmed sequence encoded by the cig1DΔTL construct. The * indicates identical amino acids and : indicates a difference between amino acids. (B) Amino acid sequence of the cig1DΔTL construct. The green M is the start codon, while the red * indicates the stop codons. The sequence in bold letters represents the predicted sequence of the expressed truncated polypeptide. (C) Schematic representation of the cig1DΔTL construct. The coloured boxes represent the indicated open reading frames. The gray lines under the boxes represent the portions of the exons of the CIG1 gene. The red line represents the predicted end of the coding region of the truncated polypeptide.  Two additional strains, GAL7p::CIG1D and cig1DΔstop, were constructed to further investigate the possibility that a truncated polypeptide was expressed and conferred phenotypes on C. neoformans. The first strain was constructed to control expression of the disrupted cig1DΔ  151  construct via regulation by the GAL7 promoter described in Chapter 3. In presence of glucose, the truncated polypeptide should not be expressed and a phenotypic difference might be observed compared with the cig1DΔTL and cig1DΔBC strains. That is, if the truncated polypeptide is the reason for the detected increased susceptibility to secretion inhibitors, agents that challenge the cell wall, and osmotic stress then no difference in susceptibility should be observed on glucose. However, in the presence of galactose, the truncated polypeptide should be expressed resulting in increased susceptibility to those stresses. Quantitative RT-PCR analysis of CIG1 expression confirmed the expected carbon source regulation of the GAL7p::CIG1 construct in the GAL7p::CIG1D strain (Figure 4.7A). The second strain, cig1DΔstop, was engineered to have a stop codon exactly after the start codon in the disrupted cig1DΔ construct thus eliminating translation of the truncated Cig1 polypeptide. This strain would be expected to behave exactly like the cig1DΔdel mutant and not have increased susceptibility to any of the chemicals tested. As in sections 4.3.4. and 4.3.5., all of the strains pre-grown in YPD were serially diluted and plated on media containing different secretion inhibitors, agents that challenge the cell wall, as well as osmotic stressors. As expected, the GAL7p::CIG1D strain did not show susceptibility to any of the stresses tested when grown in glucose (Figure 4.7B). Similarly, the cig1DΔstop strain did not show susceptibility to any of the agents. Together, these results support the conclusion that expression of the Cig1 truncated polypeptide resulted in the increased susceptibility to secretion inhibitors, agents that challenged cell wall integrity, heat and osmotic stress. Interestingly, the results were markedly different when the cells were grown in galactose (Figure 4.7C). In these conditions, the cig1DΔ disruption mutants (cig1DΔTL and cig1DΔBC)  152  were generally less susceptible to the agents tested, compared to when the cells were grown in glucose, and all strains grew to a similar level as the wild-type strain.  However, the  complemented strain was still susceptible to BFA and more resistant to SDS. Quantitative RTPCR analysis of CIG1 expression in the wild-type strain reveals that growth in galactose reduces expression of CIG1 relative to growth in glucose (Figure 4.7A), as was seen in C. neoformans var. grubii described in Chapter 3. Hence, the change in CIG1 expression in galactose could explain the observed differences in phenotypes.  153  Figure 4.7 Expression of a Cig1 truncated polypeptide results in increased susceptibility to secretion inhibitors, agents that challenge the cell wall, heat and osmotic stress. (A) Expression of CIG1 in the wild-type and GAL7p::CIG1D strains grown in LIM with glucose or galactose as indicated. The experiment was repeated three times and the data are plotted as average ± SD. The * represents significant differences at P < 0.005. (B-C) Ten-fold serial dilutions of C. neoformans var. neoformans strains grown in YPD with glucose (B) or galactose (C) were spotted onto YPD or YPG, respectively with and without secretion inhibitors and agents that challenge the cell wall at the concentration indicated. The plates were incubated at 30°C or 37°C for three days and photographed. The experiments were repeated three times and a representative photograph is shown.  4.3.7. Cig1 may influence the export of total proteins, proteases and extracellular vesicles The role of Cig1, and the truncated polypeptide, in secretion in C. neoformans var. neoformans was examined further. Initially, the total amount of protein secreted by the mutants was measured and compared to the amount of protein secreted by the wild-type strain (Figure  154  4.8A).  Approximately 1.5 times more protein was measured in the supernatants of the  cig1DΔTL, cig1DΔBC, cig1DΔdel, GAL7p::CIG1D and cig1DΔstop mutants compared to the wild-type and complemented strains, possibly suggesting increased secretion by the mutants, however the difference is not statistically significant. The protease activity of the different strains was also evaluated by plating the cells on azoalbumin medium. Once proteases are secreted, they degrade the albumin to create a halo of clearing surrounding the colonies. Therefore, the size of the cleared zone is indicative of the amount of secreted protease activity. A clearing zone was observed around all of the strains tested and appeared to be of similar size, except the one around the cig1DΔTL mutant was larger (Figure 4.8B). It is not clear why the cig1DΔTL strain would secrete more protease activity than the wild-type strain, and these results indicated that additional, more quantitative assays are needed to examine differences between the strains. Finally, the relative quantity of extracellular vesicles that accumulated in the culture supernatant of each strain was examined and compared by TLC as another method to evaluate the influence of Cig1 on export (Figure 4.8C). Ergosterol has been shown to be present in the membranes of extracellular vesicles and therefore serves as a marker for determining the relative quantity of vesicles. Similar amounts of ergosterol were detected in the wild-type strain and most of the cig1DΔ mutants indicating that similar numbers of extracellular vesicles were secreted by the different strains. However, significantly more ergosterol was detected in the lipid extract isolated from the culture supernatant of the complemented strain suggesting an increased number of extracellular vesicles secreted by this strain.  155  Overall, these first experiments suggest that Cig1 may influence the export of proteins and lipids to the extracellular environment. However, more quantitative work will be needed to better understand the contribution of Cig1 in secretion and the mechanisms involved.  Figure 4.8 Cig1 influences secretion of proteins and extracellular vesicles. (A) Total secreted proteins were measured in the culture supernatant of cells grown for 4 days in YNB. Protein concentrations were determined by Bradford assay and normalized to the total number of cells. The experiment was repeated three times and the data in the graph are plotted as the average ± SD. (B) Protease activity of cells grown in YPD overnight. Cells were spotted onto azoalbumin media and the plates were incubated at 30°C for 7 days and photographed. The experiment was repeated three times and a representative photograph is shown. (C) Relative quantity of extracellular vesicles based on detection of ergosterol by TLC. The densitometry values serve as an index to compare the relative quantity of extracellular vesicles produced by the different strains. The extracellular vesicles were extracted from the culture supernatant of cells grown in YNB for 4 days. The experiment was repeated two times and the data are plotted as the average ± SD and a representative photograph of the TLC plate is shown. The * represents a significant difference at P < 0.005.  156  Additional data showing the capsule response to iron of the cig1DΔBC and cig1DΔdel strains (absent from Section 4.3.1) are presented in Appendix E, while growth of the GAL7p::CIG1D and cig1DΔstop mutants in LIM supplemented with heme (absent from Section 4.3.3) can be found in Appendix F. Briefly, the cig1DΔBC mutant responded to iron similarly than the cig1DΔTL mutant, that is, both mutants produced a capsule larger than the wild-type strain when grown in presence of iron. In contrast, the cig1DΔdel mutant behaved like the wildtype strain (i.e., smaller capsule in presence of iron compared to LIM). When growth of the mutants was compared in presence of heme, both the GAL7p::CIG1D and cig1DΔstop mutants remained in lag phase for 60 hours, but then initiated exponential phase and reached stationary phase at a similar cell density as the wild-type strain. These results closely resemble the data obtained with the cig1DΔdel mutant. A summary of all the phenotypes observed throughout this study is presented in Table 4.5.  157  Table 4.5 Summary of phenotypes observed with the different strains of C. neoformans var. neoformans Assay Capsule  WT + in LIM  cig1DΔTL Enlarged on FeCl3  cig1DΔBC Enlarged on FeCl3*  cig1DΔdel + in LIM*  GAL7p::CIG1D n/t  cig1DΔstop n/t  cig1DΔ::CIG1 Enlarged in LIM  Growth in LIM with heme  +  No  No  slow  slow*  slow*  slow  Secretion inhibitors  R  S  S  R  R  R  S to BFA, R to others  Agents that challenge the cell wall  R  S  S  R  R  R  S  NaCl  R  S  S  R  R  R  S  37°C  R  S  S  R  R  R  +/- S  Secretion of total proteins  +  ++  ++  ++  +/-  ++  +  Secretion of proteases  +  ++  +  +  +  +  +  Secretion of extracellular vesicles  +  +  n/t  +  +  +  ++  +: present; R: resistant; S: susceptible; +/- S: slightly more than wild-type; ++: more than wildtype; n/t: not tested; -: less than wild-type; *: data presented in Appendices E-F  158  4.4. Discussion Over the course of this study, evidence was accumulated which suggests a role for Cig1 and/or a Cig1 truncated polypeptide in secretion. For example, the strains expressing a Cig1 truncated polypeptide displayed an increased susceptibility to secretion inhibitors and were more susceptible to agents that challenge the cell wall and to different stresses (i.e., osmotic stress and temperature stress) compared to the wild-type strain. It is possible that Cig1 may also play a role in maintaining cell wall integrity and thereby render the cells resistant to osmotic and heat stress. Alternatively, the cell wall defect observed could be a consequence of compromised secretion of the components necessary for cell wall synthesis and repair. Future studies will be needed to examine the contribution of Cig1 in maintaining cell wall integrity. The link between the Cig1 truncated polypeptide and secretion was reinforced by the observation that the strain expressing the truncated polypeptide had increased protease activity. Considering that these strains were susceptible to BFA, an agent that arrests the anterograde transport between the ER and Golgi, it would imply that the truncated polypeptide plays a role in the conventional secretory pathway (Misumi et al., 1986; Fujiwara et al., 1988; Orci et al., 1991; Betina, 1992). This would also indicate that the proteases measured in this study are secreted via this pathway. Furthermore, the extracellular vesicles are known to transport proteins, enzymes and lipids and have been proposed to be involved in a secretory pathway separate from the conventional pathway (Rodrigues and Djordjevic, 2012). Indeed, the strains expressing the Cig1 truncated polypeptide secreted the same amount of extracellular vesicles than the wild-type strain suggesting that the non-conventional secretory pathway is not affected by the truncated polypeptide. Consequently, it is possible that the Cig1 truncated polypeptide may be poisoning or blocking the conventional secretory pathway. According to the sequence information, the  159  predicted transmembrane domain would be absent from the Cig1 truncated polypeptide. Without this domain, the protein may not reach its proper destination and may result in accumulation of the protein within the secretory pathway leading to inhibition of secretion. Also, the truncated polypeptide would probably be misfolded which could further interfere with the secretory pathway. For example, altered secretion of proteins involved in heme uptake could explain the exacerbated delay in growth in LIM with added heme of the strains expressing the Cig1 truncated polypeptide compared to the cig1DΔdel mutant.  Further work involving the  examination of the localization of the Cig1 truncated polypeptide could potentially help explain the role and mechanism of the truncated polypeptide in secretion. It is important to consider that an alternative explanation for the increased secretion of proteases by the strains expressing the Cig1 truncated polypeptide could be the result of protein leakage from the damaged cell wall rather than actual secretion according to the observation that the mutants were susceptible to agents that challenge the cell wall and different stresses. Finally, the phenotypes observed with the complemented strain, which overexpresses the CIG1 transcript, further support a role for Cig1 in secretion. This strain, which overexpresses CIG1, produced a larger capsule than the wild-type strain.  Additionally, that same strain  appeared to export more extracellular vesicles in the culture supernatant, as judged by ergosterol analysis. This is not surprising as the extracellular vesicles have been linked with capsule synthesis and shown to transport the polysaccharides necessary for capsule formation (Yoneda and Doering, 2006; Rodrigues et al., 2007). In some instances, the complemented strain showed phenotypes similar to that obtained with the strain expressing the Cig1 truncated polypeptide. The combined results of expression of CIG1 in the complemented strain and the growth of the strain in presence of secretion inhibitors suggest that it could produce both wild-type CIG1 and  160  the Cig1 truncated polypeptide. Hence, expression of the truncated peptide in the complemented strain may alter the secretory pathway, as evidenced by the impaired growth of the complemented strain in presence of BFA.  Furthermore, the altered secretion of proteins  involved in heme uptake could explain delay in growth in LIM supplemented with heme of the complemented strain. It is important to note that the complemented strain also contains a disruption of a different gene, CNBE3170, encoding a hypothetical protein of unknown function. The phenotypes associated with this strain could be a consequence of overexpressing CIG1 alone, or of disrupting the CNBE3170 gene, either alone or in combination with overexpression of CIG1, or of disrupting both CIG1 and the CNBE3170 gene in the same strain. Future work, including deletion of CNBE3170 and/or construction of a new CIG1 overexpressing strain in the wild-type background, will be needed to confirm that the phenotypes observed with the complemented strain are indeed related to the overexpression of CIG1. Interestingly, susceptibility to secretion inhibitors and agents that challenge the cell wall integrity was observed for the cig1DΔBC strain, but it was not as severe as for the cig1DΔTL mutant. The two mutants were prepared by different people, with approximately five years in between their construction. It is therefore possible that differences accumulated in the wild-type strain during that time could account for differences in severity of the phenotypes. That is, it is possible that a different stock of the B3501A parental strain was employed for the construction of each mutant. C. neoformans is well known for phenotypic plasticity and there are anecdotal and published accounts of changes in laboratory stocks over time (Hu et al., 2008; 2011; Ngamskulrungroj et al., 2012; Sionov et al., 2010; Fries et al., 1996; Okagaki et al., 2010; Zaragoza et al., 2010).  161  No susceptibility to the secretion inhibitors and agents that challenge the cell wall was observed for the cig1AΔ mutants in C. neoformans var. grubii. The difference in phenotypes between the two Cryptococcus varieties is not surprising. Although Cig1 from C. neoformans var. neoformans shares 97.1% similarity and 87.5% identity at the amino acid level with Cig1 from C. neoformans var. grubii, there are many cases of the same protein having altered functions in different varieties of C. neoformans. For example, the catalytic subunit of protein kinase A, Pka1, is involved in mating, haploid fruiting, melanin synthesis and elaboration of the capsule in C. neoformans var. grubii, but plays no role in these activities in C. neoformans var. neoformans (Hicks et al., 2004). However, the second catalytic subunit, Pka2, is required for mating, haploid fruiting, melanin and capsule production in C. neoformans var. neoformans but not in C. neoformans var. grubii (Hicks et al., 2004). Nevertheless, it is interesting that the cig1AΔdis mutant in C. neoformans var. grubii did not show any susceptibility to the secretion inhibitors and agents that challenge the cell wall. The cig1AΔdis mutant was prepared by transforming the C. neoformans var. grubii wild-type strain with the cig1DΔTL construct amplified from C. neoformans var. neoformans.  Therefore, similar phenotypes would be  expected considering that the exact same construct was used. It is possible that the regulation of Cig1 might be different between the two varieties or that differences in other genes modify the influence of Cig1. In summary, Cig1 plays a role in heme utilization in C. neoformans var. neoformans as was observed in C. neoformans var. grubii. A link between Cig1 and secretion was identified, although more work will be needed to fully understand the contribution of Cig1 in secretion as well as the mechanisms involved. Future studies will also be needed to determine whether Cig1 plays a role in maintaining cell wall integrity.  162  Chapter 5. Discussion 5.1. Heme uptake The research presented in this thesis makes an important contribution to understanding mechanisms of iron acquisition from heme in C. neoformans.  Iron is essential for the  proliferation of C. neoformans and, therefore, iron uptake is an important component of its pathogenesis. The level of free iron in the mammalian host is kept to an extremely low level by sequestration with iron-binding proteins. The control of free iron has the dual purpose of protecting the host cells against the toxicity of iron (i.e., by preventing free radical formation) and also protecting the host against infection by pathogenic microorganisms. C. neoformans has developed multiple iron uptake mechanisms to circumvent the problem of iron unavailability (Jacobson et al., 1997; 1998; Tangen et al., 2007; Jung and Kronstad, 2008; Jung et al., 2008; 2009).  For example, the reductive high-affinity iron uptake pathway, comprised of the  ferroxidase Cfo1 and the iron permease Cft1, is the best characterized system and it appears to be a key iron uptake pathway in C. neoformans (Jung et al., 2008; 2009; Jung and Kronstad, 2008). Specifically, deletion of either CFO1 or CFT1 results in attenuated virulence and poor accumulation in the central nervous system in a mouse model of infection (Jung et al., 2008; 2009). One of the most abundant iron sources in the mammalian host is hemoglobin, a tetrameric protein containing one heme molecule per protein subunit. It is unclear whether C. neoformans can utilize hemoglobin because the fungus lacks the hemolytic activity required to rupture erythrocytes, the major source of hemoglobin (Drs. W. Jung and G. Hu, personal communications).  However, it is possible that the fungal pathogen acquires heme and/or  hemoglobin from erythrocytes that have already lysed.  Indeed, free hemoglobin has been  163  detected in various fluids in the human host (Pynnonen et al., 2011; Kipnis et al., 2008). Although C. neoformans has been shown to utilize heme as a sole iron source, the mechanism of heme uptake was unknown prior to the work in this thesis (Jung et al., 2008). In fact, when the studies presented in this thesis were started, no proteins involved in heme utilization had yet been identified in C. neoformans, and very little was known about heme uptake in other fungi. In C. albicans, a family of cell surface proteins (Rbt5 and Rbt51) involved in heme and hemoglobin acquisition had been identified, however the details of the mechanism of heme/hemoglobin uptake were still unknown (Weissman and Kornitzer, 2004). Since then, endocytosis has been proposed as a pathway for hemoglobin acquisition and this pathway depends on the activity of the ESCRT system (Weissman et al., 2008).  Recently, Vps23, a protein in the ESCRT-I  complex, has been identified as a key player in iron acquisition from heme in C. neoformans, suggesting that a similar pathway may be present in this organism (Hu et al., 2013). The research presented in this thesis identified the first protein, Cig1, involved in heme uptake and use in C. neoformans. Cig1 may play a role as a hemophore to deliver heme for endocytosis via receptors at the cell surface or to deliver heme to the cell surface for uptake via some unidentified cell surface transporter. Heme uptake has been well characterized in bacteria and most heme uptake pathways generally consist of specific cell surface heme receptors and transporters to traffic heme molecules from the extracellular environment to the intracellular space (Tong and Guo, 2009; Nobles and Maresso, 2011). Some bacteria have a relay of heme receptors across the cell wall to accommodate the transport of heme to the cell membrane (Hammer and Skaar, 2011; Nobles and Maresso, 2011).  Alternatively, other bacteria secrete heme-binding proteins, termed  hemophores, in order to sequester heme from the environment and deliver it to the cell surface  164  receptors (Hanson et al., 1992; Letoffe et al., 1994; 1998; 1999; 2000; Jarosik et al., 1995; Cope et al., 1998; Arnoux et al., 1999; 2000; Wandersman and Stojiljkovic, 2000; Rossi et al., 2001; Maresso et al., 2008; Tong and Guo, 2009; Honsa and Maresso, 2011; Klebba et al., 2012; Wandersman and Delepelaire, 2012). As described below, the characterization of Cig1 suggests that a fungal pathogen of humans also possesses components of a heme uptake system with similar features to the bacterial uptake systems.  165  5.2. A novel Cig1-dependent heme uptake pathway in C. neoformans In C. neoformans, the gene encoding Cig1, a secreted mannoprotein, represented the most abundant transcript in cells grown in LIM and was strongly regulated by iron suggesting an important role for the protein in iron homeostasis (Lian et al., 2005). The study presented in Chapter 2 revealed that Cig1 is a component of a novel heme uptake pathway in C. neoformans. A key finding was that growth of a mutant in which CIG1 had been deleted displayed an extended lag phase compared to the wild-type strain in the presence of heme as a sole iron source. Additionally, the mutant was less sensitive than the wild-type strain to GaPPIX, a toxic heme analog, indicating that Cig1 played a role in heme uptake. A recombinant Cig1 protein was shown to bind to heme, although a Kd could not be determined, possibly because the binding was weak and/or transient. Cig1 was found to be localized at the cell surface and was also secreted to the extracellular space in association with extracellular vesicles produced by C. neoformans. Given its extracellular localization, its ability to bind heme, and its role in heme utilization and uptake, it was hypothesized that Cig1 could act as a heme receptor, as illustrated in Figure 5.1A. However, the fact that Cig1 is also secreted to the extracellular space could indicate that it is a fungal hemophore. If this were the case, it would be the first fungal hemophore ever reported. In either scenario, heme uptake could be mediated via unidentified membrane-localized transporters or via endocytosis. Cig1 displayed low affinity for heme in the conditions tested but this finding does not discount its possible role as a heme receptor or hemophore. It is important to keep in mind that the biochemical experiments used to test heme binding were performed with a recombinant Cig1 protein expressed in E. coli, a prokaryotic host. Secreted eukaryotic proteins, including Cig1, are subject to post-translational modifications (i.e., glycosylation) that do not generally take place in prokaryotic cells. It is possible that the  166  glycosylation of Cig1 is important for its interaction with heme. Post-translational modifications of fungal proteins have been shown to influence their stability, their interactions with other proteins, as well as their potential for a contribution to virulence (Leach and Brown, 2012). Additionally, the recombinant Cig1 protein was tagged with GST which could interfere with the proper folding of the protein and result in decreased affinity for heme. Interestingly, the study presented in Chapter 2 showed that deletion of CIG1 in the absence of the reductive high-affinity iron uptake pathway resulted in attenuated virulence in a mouse model of infection for C. neoformans.  This observation confirms that the high-affinity iron uptake pathway is an  important iron uptake pathway in the host, but it also indicates that heme uptake is important in the pathogenesis of C. neoformans, thus warranting further studies of this pathway. Cig1 also appears to play a role in heme acquisition in C. neoformans var. neoformans as presented in Chapter 4. The MS analysis of secreted cryptococcal proteins capable of binding to heme-agarose beads identified multiple proteins, many of which are involved in carbohydrate metabolism as presented in Chapter 2 (Table 2.4). Cig1 was identified as one of those proteins suggesting that endogenously produced Cig1 binds to heme-agarose and further confirming the extracellular location of the protein. Additionally, a predicted transmembrane receptor protein was also identified as a putative heme-binding protein and was only detected in cells grown in irondepleted media, just like Cig1. This could be a very interesting candidate for future studies in identifying other key players in the novel heme uptake pathway in C. neoformans. Future work will be needed to determine if the transmembrane receptor protein and the other proteins that were detected are involved in heme utilization.  167  5.3. Multiple heme uptake pathways in C. neoformans The strain in which CIG1 was deleted had a much longer lag phase than the wild-type strain when grown in LIM supplemented with heme added as a sole iron source. However, the mutant did eventually start to grow and it reached the same final cell density as the wild-type strain. Additionally, prior adaptation of the cells to growth on heme did not shorten the lag phase of the mutant thus suggesting that accumulation of mutations or changes in metabolism could not explain the eventual growth of the cig1Δ mutant. Therefore, the eventual growth of the mutant suggested that other heme uptake pathways must exist in C. neoformans to allow the cells to utilize iron from heme. Consequently, the cig1Δ mutant proved to be an excellent tool to investigate the other heme uptake pathways, as presented in Chapter 3. Upon further investigation, the requirement of Cig1 for heme uptake appeared to be limited to the condition of physiological pH because the cig1Δ mutant grew as well as the wildtype strain in LIM supplemented with heme at lower pH. Additionally, the expression of CIG1 was down-regulated at acidic pH. Hence, some other mechanism of heme uptake, independent of Cig1, probably plays a more important role at acidic pH. One possible scenario could involve uptake of heme via the ESCRT-mediated endocytic pathway (Hu et al., 2013) as illustrated in Figure 5.1B. This scenario is supported by the defect in growth on heme observed for the mutants of the ESCRT system at acidic pH (Dr. G. Hu, personal communications). Alternatively, proteins (e.g., cell surface reductases) could extract iron from the heme molecule resulting in free iron which could also be transported into the cell via the ESCRT-mediated endocytic pathway, for example, or by another transporter which has not yet been identified (as illustrated in Figure 5.1C). A role for the endocytic pathway in this scenario is also possible as evidenced by the defect in growth in LIM supplemented with FeCl3 observed for the mutants of  168  the ESCRT system at acidic pH (Dr. G. Hu, personal communications). The iron is probably not taken up by the high-affinity iron uptake pathway because deletion of CFO1, the gene encoding an essential component of the pathway, did not result in delayed growth in the presence of heme at low pH. Finally, another pathway for uptake of the entire heme molecule could possibly exist, such as a low-affinity heme transporter for example (Figure 5.1D).  The solubility and  bioavailability of iron from heme would be increased at lower pH which could support the idea of a functional low-affinity iron or heme uptake pathway at acidic pH (Wilkins, 1991; Cotton et al., 1999).  169  Figure 5.1. Proposed model for different heme uptake pathways in C. neoformans. (A) Cig1 heme uptake pathway. Cig1 could be a hemophore which sequesters heme for eventual uptake by a cell-surface receptor. Transport inside the cell could be achieved via endocytosis or via an unidentified transporter. (B) ESCRT-mediated endocytosis for heme. (C) Cell surface reductases may process heme, thereby releasing iron which can be transported to the intracellular space via ESCRT-mediated endocytosis or a predicted iron transporter. (D) Low-affinity heme uptake via an unidentified transporter.  Additional evidence suggests the presence of a third heme uptake pathway which relies on cell density in C. neoformans. This heme uptake pathway shares similarities with QS behaviour, including cell aggregation, dependence on cell density and a decreased lag phase for cells grown in LIM supplemented with heme with added CM. QS is a means of cell-cell communication among populations that is common in microorganisms. Signaling molecules are secreted in the environment and when the concentration of signal is high enough, indicating a 170  sufficient number of cells, the population mounts a coordinated response. This phenomenon is extremely beneficial in order to increase the population’s survival by allowing the cells to adapt to low nutrient availability, to organize an attack on the host, or to escape the immune response of the host. QS has been widely studied in bacteria and, in the last decade or so, has also been reported in different fungi including C. neoformans (Bassler, 2002; Joint et al., 2007; Lee et al., 2007; Williams, 2007; Albuquerque, 2011; Eisenman et al., 2011; Albuquerque and Casadevall, 2012). It is possible that C. neoformans requires QS to induce heme uptake in this specific pathway. Alternatively, QS and heme uptake in C. neoformans could share common regulators or the regulators of QS could be downstream of the regulators of heme uptake. A link between iron and QS has previously been established in P. aeruginosa (Ochsner et al., 2002; Palma et al., 2003; Schuster et al., 2003; Cornelis and Aendekerk, 2004; Juhas et al., 2004; 2005; Deziel et al., 2005; Kim et al., 2005; Duan and Surette, 2007; Yang et al., 2007; Oglesby et al., 2008; Hazan et al., 2010). It is important to note that the different heme uptake pathways described above (i.e., Cig1, acidic pH, cell density) may not be three completely independent heme uptake pathways and they may share some key players and/or machinery for transportation of heme inside the cell. For example, the cell surface reductases for heme processing (Figure 5.1C) could be involved in both the pathway contributing at acidic pH and the cell density-dependent pathway and could be regulated differently depending on the conditions. That is, at acidic pH, the cell surface reductases could be up-regulated, while the regulation could be dependent on cell density at physiological pH. Alternatively, ESCRT-mediated endocytosis could be involved in all three pathways for heme uptake (Figure 5.1A-C). Further work will be needed to elucidate the receptors, transporters and mechanisms involved in all the heme uptake pathways identified in the studies presented in this thesis.  171  As mentioned earlier, the CIG1 transcript is highly abundant in cells grown in low iron medium. The transcription factor Rim101 plays a role in regulating heme uptake along with other transcription factors (Fig. 5.1), and part of this role is most likely due to the regulation of CIG1 transcript levels (O’Meara et al., 2010). However, part of the role of Rim101 in heme uptake is independent of Cig1 based on the observation that a rim101Δ mutant in which the expression of CIG1 was repressed by a regulated promoter, displayed a longer lag phase than both the rim101Δ and the cig1Δ mutants when grown in LIM supplemented with heme. Given the role of Rim101 as the master regulator of the pH response, it is possible that Rim101 is linked to the heme uptake pathway that makes a major contribution at acidic pH as illustrated in Figure 5.1 (Tilburn et al., 1995; MacCabe et al., 1996; Ramon et al., 1999; Lamb and Mitchell, 2003). Specifically, Rim101 could be involved in the ESCRT-mediated endocytic pathway given that Rim101 is a downstream target of the ESCRT system. Although the Cig1-dependent heme uptake pathway may be a larger contributor to heme uptake at physiological pH, the proposed ESCRT-mediated endocytic pathway may not be restricted to heme uptake at acidic pH but may also be involved in heme uptake at physiological pH. A growth defect of the mutants of the ESCRT system on heme at physiological pH supports this hypothesis (Hu et al., 2013). Additionally, it is possible that Rim101 regulates the two pathways (i.e., Cig1 and endocytosis) in response to pH. Future work is needed to fully understand the role of Rim101 and the full range of targets that function in heme utilization.  172  5.4. Expression of the Cig1 truncated polypeptide alters secretion and leads to loss of cell wall integrity A link between Cig1 and secretion was identified in C. neoformans var. neoformans but not in C. neoformans var. grubii.  In fact, the expression of a predicted Cig1 truncated  polypeptide may alter the function of the conventional secretory pathway as evidenced by the increased susceptibility of the strains to secretion inhibitors, especially BFA. BFA is known to specifically inhibit conventional secretion, which is typically driven by an N-terminal signal sequence and requires traffic between the ER and Golgi, passage through the Golgi apparatus and transport to the cell membrane via intracellular vesicles (Doms et al., 1989; Rodrigues and Djordjevic, 2012). The integrity of the cell wall of the cells expressing the Cig1 truncated polypeptide was affected, possibly as a result of altered secretion, and rendered the cells more sensitive to osmotic and heat stress. Alternatively, Cig1 could play a role in maintaining cell wall integrity and, therefore, loss of CIG1 would result in loss of cell wall integrity. Furthermore, expression of the Cig1 truncated polypeptide resulted in increased secretion of proteases, although this could be due to loss of cell wall integrity rather than a specific influence on the secretory pathway. The link between Cig1 and secretion was further reinforced by the observation that a strain overexpressing Cig1 elaborated a larger capsule and secreted more extracellular vesicles than the wild-type strain. The extracellular vesicles have previously been shown to contain the capsular polysaccharides and have been linked with capsule biosynthesis (Yoneda and Doering, 2006; Rodrigues et al., 2007). A current working hypothesis is that expression of the Cig1 truncated polypeptide somehow blocks the secretory pathway. It is possible that the expression of the Cig1 polypeptide without the transmembrane domain leads to the protein not reaching its proper destination. Instead, the Cig1 truncated polypeptide could  173  potentially accumulate somewhere within the secretory pathway and inhibit secretion. More work will be needed to fully understand the contribution of Cig1 to secretion and to determine the mechanism involved.  174  5.5. Key areas for future work 5.5.1. Cig1 binding to heme In this study, the Kd of recombinant Cig1 to heme could not be determined.  As  previously discussed, it is possible that the weak binding observed was due to issues arising because of expression of an eukaryotic protein from a recombinant vector in a prokaryotic system. Attempts were made to express Cig1 in C. neoformans but the expression levels achieved were too low to allow further studies. It would be interesting to fuse a stronger constitutive promoter, for example the TEF1 promoter, to the CIG1 gene in order to increase the level of expression of Cig1. Additionally, the GST tag used to label the recombinant protein may have interfered with proper folding. A HIS tag, which is comprised of 6 histidine residues is significantly smaller and could potentially reduce misfolding of the tagged protein. Tagged and endogenously produced Cig1 could be used to further study the interaction with heme and potentially determine the Kd.  5.5.2. Identifying the mechanisms involved in Cig1-dependent heme uptake pathway Cig1 has been shown to play a role in heme uptake but additional work is needed to define the mechanism. Identifying the structure of the protein by crystallography could provide invaluable information about the role of Cig1 in heme binding. The Cig1 protein expressed in and purified from C. neoformans (as described above) could be used to help ensure that the properly folded protein was evaluated. Also, identifying the heme binding domain of Cig1 would be important to understand the mechanism involved in heme uptake. One approach would be to delete different parts of the CIG1 gene to determine which region of the protein is essential for heme binding activity. The predicted chitin-binding domain would be a good place to start.  175  Eventually, deletion or substitution of single amino acids by site directed mutagenesis could also be performed to determine the residues needed for Cig1-mediated heme utilization. A better understanding of the biochemical details of heme uptake could be useful. The work done with the non-iron MP suggested that Cig1 is involved in heme uptake but this method provides an indirect measure of heme uptake. A more direct approach may further confirm the role of Cig1 in uptake of heme. Future work could include examining uptake of radioactive Fe55-heme. The use of Fe55-heme has previously been used in studying heme transport in different organisms (Protchenko et al., 2008; Le Blanc et al., 2012). However, preliminary results using Fe55-heme yielded variable data and no difference was observed between uptake of Fe55-heme by the wild-type strain and the cig1Δ mutant. It is possible that the Fe55-heme was contaminated with free iron which could be taken up via the high-affinity iron uptake pathway. A different source of Fe55-heme will be needed to further study heme uptake. Identifying the heme receptor and/or transporter involved in the Cig1-dependent heme uptake pathway would also be very useful in defining the mechanism involved in heme uptake. Examining the predicted transmembrane receptor identified as a putative heme-binding protein during the MS analysis presented in Chapter 2 would be the easiest approach and could potentially provide a key receptor involved in heme uptake. Another approach would be to perform a pull-down assay of cell membrane/cell wall proteins using heme-agarose beads followed by MS analysis to potentially identify the heme transporters involved in the Cig1dependent and/or Cig1-independent heme uptake pathways. Following the MS analysis, deletion mutants of the genes encoding the proteins that were identified could be constructed and characterized to assess heme transport function. It is possible that the interaction between some of the proteins identified by MS and the heme-agarose beads may not be specific. Additionally,  176  some of the identified proteins may be the result of secondary interactions and may not be true direct heme-binding proteins. Nonetheless, this approach would be relatively simple and could yield the missing transporters and/or other cell surface heme receptors. The method for triple peptide formaldehyde labeling could be used to quantify the relative abundance of proteins in each sample but this approach will need to be optimized in order to allow detection of the majority of proteins, as Cig1 was not detected in previous attempts.  5.5.3. Identifying other proteins involved in the Cig1-independent heme uptake pathway(s) The key players involved in the Cig1-independent heme uptake pathways also need to be identified to fully understand heme uptake in C. neoformans. Construction of deletion mutants for the genes encoding the proteins identified as secreted heme-binding proteins in the MS analysis presented in Chapter 2 (specifically the predicted transmembrane receptor), followed by characterization of these mutants would be a logical place to start. Upon construction of the mutants, their role in heme utilization could be confirmed. The interaction between heme and the proteins involved in heme utilization could also be examined. potentially identify other heme receptors and/or hemophores.  Such analyses could  Alternatively, comparative  transcriptome analysis of the wild-type strain and the cig1Δ mutant in the presence of heme could also be used to identify candidate functions for subsequent genetic analysis. Different technologies, such as RNA-seq and microarrays, have been extensively used to compare the transcriptomes of C. neoformans cells from different growth conditions, and this approach has also been used in many other organisms. It is likely that in the absence of Cig1, the key players involved in the other heme uptake pathways would be more highly expressed to compensate, and this property might facilitate their identification. Again, deletion of the candidate genes and  177  characterization of the mutants could provide the important additional information about heme uptake. Additionally, further characterization of the role of the other C. neoformans putative surface reductases, as predicted by protein homology, in heme utilization are already underway in the laboratory (Dr. S. Saikia, personal communications).  5.5.4. Identifying the QS-like molecule involved in cell density-dependent heme uptake The cell density-dependent heme uptake pathway described in Chapter 3 has similarities to QS behaviour.  Identification of the QS-like molecule is essential in order to further  characterize this pathway. Although some crude fractionation of the components found in the culture supernatant has already been done, as presented in Chapter 3, it would be useful to separate the different components further using liquid chromatography.  After separation,  samples with activity could be analyzed by MS or nuclear magnetic resonance (NMR) to determine the molecules found in these fractions. These molecules in pure form (if available) could be again tested for their ability to decrease the lag phase of the strain in which CIG1 was deleted, and this approach would hopefully confirm the identity the QS-like molecule. Preliminary data presented in Chapter 3 indicate that the QS-like molecule could potentially be a polysaccharide (e.g., GXM, GalXM, chitin-like oligomers) secreted into the culture supernatant of C. neoformans.  It would be interesting to determine if these  polysaccharides can bind to heme. Binding of heme to proteins or polysaccharides can be visualized using different chemicals, including 3,3’-dimethoxybenzidine (Mazoy and Lemos, 1996). Additionally, imaging MS of cells grown on agar in the presence of heme could provide invaluable information based on the spatial localization of the QS-like molecule (Watrous and Dorrestein, 2011). For example, this approach could indicate whether more QS-like molecules  178  are distributed at the surface or within the capsule of the cells. Also, some genes have been found to be essential for elaboration of the capsule, including CAP10, CAP59, CAP60, and CAP64, while the CAS genes are involved in modification of the capsule (Chang and KwonChung, 1994; 1998; 1999; Chang et al., 1996; Moyrand et al., 2002; 2004). Mutants could be constructed in which both CIG1 and genes involved in capsule synthesis and/or modification are deleted. Alternatively, mutants expressing only GXM or GalXM could also be constructed in the cig1Δ mutant background. Characterization of these mutants could provide insights into the role of capsular polysaccharides in heme uptake.  5.5.5. Further characterization of the link between Cig1 and secretion Finally, Cig1 has also been linked to secretion through the unusual behaviours of a mutant carrying a disruption allele and of a complemented strain. A key finding was that the complemented strain appeared to overexpresses CIG1 and produced a larger capsule than the wild-type strain. It was later found that the integration of the complemented construct resulted in disruption of the CNBE3170 gene. This gene encodes a predicted protein without sequence similarity to other proteins and it is therefore difficult to predict its function. Mutants in which the CNBE3170 gene was deleted should be constructed to confirm that the phenotypes associated with the complemented strain are in fact related to overexpression of CIG1 and not to disruption of CNBE3170. If the phenotypes are not a result of overexpression of CIG1 but of disruption of CNBE3170, characterization of the mutant would still provide useful information about the secretory pathway involved in capsule production. Alternatively, a strain overexpressing CIG1 at the endogenous locus could be constructed. For example, the TEF1 promoter could be inserted upstream of the CIG1 gene to promote elevated and constitutive expression of CIG1.  179  Based on the data presented in Chapter 4, it is possible that the expression of the Cig1 truncated polypeptide could somehow block the secretory pathway. Creating a GFP-tagged construct for the Cig1 truncated polypeptide would be very useful for future studies. For example, proof for expression of the truncated peptide could be provided by Western blot analysis of the lysates of cells expressing the tagged truncated polypeptide.  Also, cells  expressing the GFP-tagged truncated polypeptide could allow visualization of the localization of the protein by fluorescence microscopy which could give insight into the mechanism involved in altering secretion.  Using dyes (e.g., ER-Tracker, Golgi-Tracker) which stain the different  compartments of the secretory pathway and looking for co-localization of the Cig1 truncated polypeptide could help determine whether accumulation of the truncated polypeptide occurs at a specific step within the pathway (as a possible reason for altered secretion). Alternatively, proteins which are known to be found in the specific components of the secretory pathway could be also be used as markers.  180  5.6. Conclusion In conclusion, the studies presented in this thesis identified a major role for Cig1 in heme uptake at physiological pH in the fungal pathogens C. neoformans var. grubii and C. neoformans var. neoformans. Additionally, it was determined that C. neoformans has evolved multiple heme uptake pathways, including a Cig1-independent pathway required for heme uptake at acidic pH and a cell density-dependent heme uptake pathway. The work presented here is novel and important because heme uptake has never been described in C. neoformans and very little is generally known about the process in fungi. Although the key players and the mechanism involved in these pathways were not characterized, the data provide the ground work for eventually understanding all of the mechanisms of heme uptake in C. neoformans and this could potentially lead to further elucidating heme uptake in other fungi too. Iron is essential for pathogenesis of C. neoformans and the majority of iron in the mammalian host is found in heme. Therefore, identifying the key players and the different steps involved in heme uptake could provide new targets for novel drug development in order to offer better treatment to patients with cryptococcosis. There is a pressing need for development of novel treatments as the current antifungal drugs that are available suppress but do not cure cryptococcosis (van der Horst et al., 1997; Bicanic et al., 2005; McCarthy et al., 2006). Finally, the expression of a Cig1 truncated polypeptide in C. neoformans var. neoformans was linked to secretion and maintenance of cell wall integrity. More work will be needed to fully understand the underlying mechanism of the altered secretion phenomenon and the relationship with cell wall integrity. 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FEMS Yeast Res. 5, 1-10. 210  Appendices Appendix A  Figure A.1. The HA::Cig1 strain has a growth defect when grown in LIM supplemented with heme. Growth of iron-starved cells in LIM + 10 µM heme was monitored by measuring the optical density at 600 nm.  211  Appendix B Polysaccharide binding assays A 100 µg/ml solution of Cig1::GST protein in 50 mM sodium phosphate, pH 7 was mixed one to one with a 1 mg/ml polysaccharides solution, including different forms of cellulose and chitin, in the same buffer. The mixture was incubated at room temperature with shaking for 4 hours. The insoluble polysaccharides were precipitated by centrifugation and the residual unbound protein in the supernatant was measured by absorbance spectrophotometry at 280 nm. Cig1::GST protein in buffer without insoluble polysaccharides served as a reference. Binding to polysaccharides was also monitored by ITC, as described in Chapter 2, using a solution of 50 µM Cig1::GST in 50 mM sodium phosphate, pH 7 and injection of consecutive 2 µL aliquots of 500 µM glucose, mannose, galactose, xylose, or glucosamine prepared in the same buffer. ITC experiments were performed by Dr. Louise Creagh. Although not conclusive, these preliminary results suggest that Cig1 does not bind sugars or the tested polysaccharides.  212  Figure B.1. Cig1 does not appear to bind polysaccharides. (A) Cig::GST was mixed with cellulose, chitin, α-cellulose and microgranules of cellulose. Binding was determined by comparing the spectral absorbance at 280 nm of the supernatant of Cig1::GST protein in buffer alone or in presence of the insoluble polysaccharide after 4 hours of incubation at room temperature. ITC of glucose (B), galactose (C), mannose (D), xylose (E), and glucosamine (F) alone (red) or Cig1::GST plus sugars (black) over the specified period of time. All assays were repeated three times and representative graphs are shown.  213  Appendix C Table C.1. Potential heme-binding proteins identified in the culture supernatants of cells grown in LI-YNB Accession  # AAs  Description  Coverage  # PSMs  # Peptides  Score  CNAG_01239  412  chitin deacetylase  10.44  29  3  1004.92  Times Detected (n = 2) 2  CNAG_02030  677  glyoxal oxidase  26.14  47  11  773.78  2  CNAG_00407  664  glyoxal oxidase  14.16  21  6  575.23  2  CNAG_01854  835  heparinase II/III family protein  12.57  16  6  574.18  2  CNAG_06501  564  1,3-beta-glucanosyltransferase  7.62  17  3  510.53  2  CNAG_03465  624  laccase  15.87  22  7  497.78  2  CNAG_02860  452  endo-1,3(4)-beta-glucanase  7.30  32  2  379.03  2  CNAG_06081  867  glucose oxidase  8.77  12  5  315.21  2  CNAG_04373  499  alginate lyase  15.63  15  6  305.52  2  CNAG_06291  249  deacetylase  12.45  19  2  302.52  2  CNAG_01653  281  cytokine inducing-glycoprotein  18.15  8  3  298.55  2  CNAG_00588  171  conserved hypothetical protein  30.99  12  2  285.88  1  CNAG_06835  588  glucosidase  8.16  6  3  243.33  2  CNAG_02966  395  carboxypeptidase D  11.90  9  4  224.07  2  CNAG_05138  785  exo-beta-1,3-glucanase  8.92  10  5  212.26  2  CNAG_03525  728  trehalase  3.57  7  2  199.52  2  CNAG_00799  789  cellulase  3.42  5  3  181.56  1  CNAG_00919  548  carboxypeptidase D  6.39  8  3  179.72  1  CNAG_00150  931  peptidase  7.52  5  4  178.36  2  CNAG_05799  470  chitin deacetylase  7.87  6  3  177.47  2  CNAG_02189  532  alpha-amylase  5.64  5  3  171.95  2  CNAG_02225  431  cellulase  9.98  5  4  151.97  2  CNAG_06085  637  secreted phospholipase B  4.55  5  3  120.49  2  CNAG_04524  862  zinc metalloprotease  2.67  2  2  120.28  2  CNAG_00250  422  conserved hypothetical protein  7.58  2  2  119.81  1  CNAG_01405  700  conserved hypothetical protein  6.00  3  3  108.08  1  CNAG_02850  896  2.46  2  2  107.75  2  CNAG_03120  2430  glucan endo-1,3-alpha-glucosidase agn1 alpha-1,3-glucan synthase  2.55  5  5  106.72  2  CNAG_05471  972  alpha-glucosidase  5.14  3  3  98.04  1  CNAG_00581  438  endopeptidase  5.25  4  2  91.59  1  CNAG_03524  1717  transmembrane receptor  1.51  2  2  90.30  2  CNAG_01562  299  conserved hypothetical protein  10.37  3  2  89.51  1  CNAG_01019  154  Cu/Zn superoxide dismutase  22.08  4  3  85.96  2  CNAG_05918  547  F0F1 ATP synthase subunit beta  8.78  3  3  85.07  1  CNAG_01984  323  transaldolase  14.24  3  3  83.53  1  CNAG_04869  620  carboxylesterases  7.58  4  3  79.03  1  214  Accession  # AAs  Description  Coverage  # PSMs  # Peptides  Score  CNAG_07445  687  transketolase  7.28  4  4  75.18  Times Detected (n = 2) 1  CNAG_04601  499  glycine hydroxymethyltransferase  6.21  2  2  71.14  1  CNAG_05731  631  glyoxal oxidase  4.44  3  3  67.96  1  CNAG_04753  467  lactonohydrolase  5.14  2  2  67.49  2  CNAG_02944  551  acid phosphatase  5.08  2  2  65.07  2  CNAG_01577  451  glutamate dehydrogenase  6.21  2  2  63.67  2  CNAG_01040  543  carboxypeptidase D  6.08  4  3  62.85  1  CNAG_07736  715  2.94  2  2  59.11  1  CNAG_03072  444  glucan endo-1,3-alpha-glucosidase agn1 phosphopyruvate hydratase  4.95  2  2  56.47  1  CNAG_04944  422  conserved hypothetical protein  5.92  2  2  46.68  1  CNAG_01890  763  5methyltetrahydropteroyltriglutamatehomocysteine S-methyltransferase  2.75  2  2  45.89  1  AAs: amino acids, PSMs: peptide spectrum match, n: number of replicates.  Table C.2. Proteins binding to agarose beads identified in the culture supernatants of cells grown in LI-YNB Accession  # AAs  Description  Coverage  # PSMs  # Peptides  Score  CNAG_00588  171  conserved hypothetical protein  33.33  7  2  97.52  Times detected (n = 2) 1  AAs: amino acids, PSMs: peptide spectrum match, n: number of replicates.  215  Table C.3. Potential heme-binding proteins identified in the culture supernatants of cells grown in LI-YNB + FeCl3 Accession  # AAs  Description  Coverage  # PSMs  # Peptides  Score  CNAG_04373  499  alginate lyase  11.62  13  4  365.71  Times detected (n = 2) 1  CNAG_02030  677  glyoxal oxidase  9.16  12  4  353.30  1  CNAG_02860  452  endo-1,3(4)-beta-glucanase  8.41  18  2  297.55  1  CNAG_00799  789  cellulase  4.56  10  4  295.58  1  CNAG_00407  664  glyoxal oxidase  10.39  12  5  265.02  1  CNAG_06501  564  1,3-beta-glucanosyltransferase  6.03  7  2  264.51  2  CNAG_06081  867  glucose oxidase  5.07  6  3  200.99  1  CNAG_02850  896  3.13  3  2  166.85  1  CNAG_00587  185  glucan endo-1,3-alpha-glucosidase agn1 conserved hypothetical protein  15.14  5  2  152.94  1  CNAG_01984  323  transaldolase  8.98  3  2  143.51  1  CNAG_02225  431  cellulase  4.64  6  2  131.57  1  CNAG_05799  470  chitin deacetylase  3.62  3  2  124.55  1  CNAG_02189  532  alpha-amylase  6.77  5  3  118.76  1  CNAG_01890  763  5.37  5  3  110.50  1  CNAG_06085  637  5methyltetrahydropteroyltriglutamatehomocysteine S-methyltransferase secreted phospholipase B  4.24  3  2  102.60  1  CNAG_01854  835  heparinase II/III family protein  3.83  5  2  88.01  1  CNAG_03525  728  trehalase  3.57  2  2  86.19  1  CNAG_00581  438  endopeptidase  5.25  2  2  48.88  1  AAs: amino acids, PSMs: peptide spectrum match, n: number of replicates.  Table C.4. Proteins binding to agarose beads identified in the culture supernatants of cells grown in LI-YNB + FeCl3 Accession  # AAs  Description  Coverage  # PSMs  # Peptides  Score  CNAG_05918  547  F0F1 ATP synthase subunit beta  10.60  4  4  175.90  Times detected (n = 2) 1  CNAG_01984  323  transaldolase  18.27  4  4  118.34  1  CNAG_03072  444  phosphopyruvate hydratase  7.88  2  2  78.43  1  CNAG_00588  171  conserved hypothetical protein  33.33  9  2  74.01  1  CNAG_06699  339  6.49  2  2  71.15  1  CNAG_03225  338  glyceraldehyde-3-phosphate dehydrogenase malate dehydrogenase  7.99  2  2  66.82  1  CNAG_01890  763  3.01  2  2  44.03  1  CNAG_02225  431  5methyltetrahydropteroyltriglutamatehomocysteine S-methyltransferase cellulase  4.64  2  2  41.62  2  AAs: amino acids, PSMs: peptide spectrum match, n: number of replicates. 216  Appendix D 3  Absorbance 600 nm  2.5 2 WT  1.5  cas1Δ  1  cap59Δ  0.5 0 0  20  28  44  53  68  76  92  100 164  Time (hours)  Figure D.1. Cas1 may be involved in iron acquisition. Growth of iron-starved cells in LIM + 100 µM FeCl3 was monitored by measuring the optical density at 600 nm. The experiment was repeated three times and the data are plotted as the average ± SD.  217  Appendix E  Figure E.1. Elaboration of the capsule by additional cig1DΔ mutants in C. neoformans var. neoformans. Cells were grown in LIM +/- 100 µM FeCl3, harvested and negatively stained with India ink before visualizing by differential interference contrast microscopy.  218  Appendix F 3  Absorance 600 nm  2.5 WT  2  cig1DΔTL cig1DΔBC  1.5  cig1DΔdel 1  cig1DΔ::CIG1 GAL7p::CIG1  0.5  cig1DΔstop 0 0  12  24  36 48 60 Time (Hours)  72  84  96  Figure F.1. All cig1DΔ mutants in C. neoformans var. neoformans grow more slowly than wild-type in LIM supplemented with heme. Growth of iron-starved cells in LIM + 10 µM heme was monitored by measuring the optical density at 600 nm. The experiment was repeated three times and the data are plotted as the average ± SD.  219  

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