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Genomic approaches to explore virulence in the fungal pathogen Cryptococcus neoformans Tangen, Kristin Lynne 2006

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GENOMIC APPROACHES TO EXPLORE VIRULENCE IN THE FUNGAL PATHOGEN CRYPTOCOCCUSNEOFORMANS  by KRISTIN LYNNE TANGEN B.Sc. (Microbiology), The University of Victoria, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (MICROBIOLOGY AND IMMUNOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA March 2006 © Kristin Lynne Tangen, 2006  ABSTRACT The fungal pathogen Cryptococcus neoformans is the leading cause of encephalitis in people with the acquired immunodeficiency syndrome (AIDS). The range of drugs available to treat C. neoformans is limited in number and efficacy; therefore, there is a dire need for new, more powerful therapeutics. To achieve this we need to better understand how the fungus can cause disease, which will lead to the identification of factors relating to virulence and ultimately may provide new drug targets. The plethora of emerging genomic resources has allowed for targeted biology to focus on key genes in important cellular processes that relate to virulence. The work described in this thesis contributes to the development of genomic resources for biological investigations in Cryptococcus neoformans. This work has three specific components: 1) physical mapping of the genomes for strains serotypes A and D; 2) analysis of the low iron transcriptome in a serotype A strain; and 3) characterization of a putative siderophore (iron) transporter gene, SIT], that was identified by transcriptome analysis. The first component involved the hybridization of 125 markers to a set of 9,216 BAC clones from the strain JEC21 (serotype D) and to 6,528 clones from the strain H99 (serotype A). These data provided the first genome-wide comparison of gene synteny between two strains of the fungus, and linked contigs to specific karyotype bands. The second component of the work involved the analysis of the low iron transcriptome for the serotype A strain, H99 using serial analysis of gene expression (SAGE). A number of interesting genes were identified in the low iron transcriptome including those involved with the response to stress and mechanisms of iron uptake. A key finding was that the low iron transcriptome was remarkably similar to the in vivo library from cells grown in rabbit cerebral spinal fluid (CSF) and significantly distinct from the libraries grown in yeast nutrient broth (YNB). The third component of the work focused on the gene SIT], which encodes a putative siderophore transporter. backgrounds of varying virulence.  The gene was characterized in three strain  This work showed that SIT] was important for iron  utilization in conditions of low iron or when a siderophore was provided as the sole iron source. Further, there were pleiotrophic phenotypes for a number of virulence-related attributes including melanization, cAMP signaling and cell wall integrity. Finally, throughout the entire body of work, multiple differences were identified between strains of the same or different serotypes on a genomic and biological level, and this variation may lend insight into differences in virulence between strains.  11  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF TABLES  viii  LIST OF FIGURES  x  LIST OF ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xiv  CHAPTER ONE: INTRODUCTION A. Cryptococcus neoformans and Cryptococcosis a. C. neoformans varieties, serotypes and molecular types b. C. neoformans infections c. Environmental sources of Cryptococcus d. C. neoformans life cycle e. Transmission of C. neoformans and etiology of cryptococcosis in humans f. Epidemiology of cryptococcosis g. The immune response to C. neoformans h. Treatment of cryptococcosis B. Genomic Analyses of C. neoformans a. Karyotype studies b. Physical mapping projects c. EST sequencing projects d. Genome sequencing efforts e. Serial analysis of gene expression (SAGE) C. C. neoformans Signaling and Virulence Factors a. The cAMP and PKC]/MAP kinase pathways b. Structure and biology of the polysaccharide capsule: contribution to virulence c. Melanin and its effects on virulence d. Mating type specificity: prevalence in clinical strains e. Superoxide dismutase (SOD) and effects f. Resistance to oxidative stress g. Mannose production and virulence h. The ability to grow at 37°C i. Other virulence factors D. Iron and Siderophore Transport a. Importance of iron transport and iron responsive genes b. Iron acquisition  111  1 1 1 2 2 3 3 3 4 5 6 6 7 8 8 9 11 11 13 14 15 15 16 17 18 18 20 20 20  c. Reductive fungal iron transport systems in fungi d. Reductive fungal iron transport systems in C. neoformans e. Siderophores and siderophore transport E. Melanin Production a. Melanin synthesis and placement b. Redox interactions of iron and melanin F. Rationale and Aims of this Study  20 21 22 23 23 24 24  CHAPTER TWO: Physical Mapping of the Genomes of Serotype A and D Strains of C. neoformans 27 INTRODUCTION  27  MATERIALS AND METHODS A. Filter Preparation and Layout B. Probe Design C. Hybridization Protocol D. Data Collection and Analysis E. Data Integration F. Southern Analysis of HindIII Digested DNA and CHEF Gel Separated Chromosomes  31 31 31 31 32 32 32  RESULTS 33 A. Summary of Hybridization Experiments 33 B. Integration of the Markers into the BAC Fingerprint Maps 38 C. Comparison of the H99 and JEC21 Fingerprint Maps 43 D. Relationship of Specific Contigs to Chromosome-Sized Bands From the C. neoformans Electrophoretic Karyotype 48 SUMMARY  54  CHAPTER THREE: Serial Analysis of Gene Expression (SAGE) of C. neoformans under Iron Limited Conditions 55 INTRODUCTION  55  MATERIALS AND METHODS A. Determination of Growth Conditions a. Fungal strains b. Media and growth conditions c. Capsule microscopy B. RNA Extraction C. Construction of a Serial Analysis of Gene Expression (SAGE) Library D. Sequencing and Data Processing E. Tag Identification  57 57 57 57 57 57 58 59 59  iv  RESULTS .61 A. Determination of Growth Conditions for SAGE Library Construction 61 B. Summary of SAGE Library Construction 62 C. SAGE Tag Annotation 64 D. Analysis of the Low Iron Transcriptome 65 Pairwise and Multiple E. Library Comparisons 70 a. Pairwise comparison of 37°C low iron vs. 25°C libraries 70 b. Pairwise comparison of 37°C low iron vs. 37°C libraries 77 c. Pairwise comparison of 37°C low iron vs. in vivo libraries 84 d. Iron regulated tags: Three-way overall comparison of the libraries 37°C low iron, 25°C and 37°C in YNB 89 e. Comparison of multiple libraries including: 25°C, 37°C low iron, 37°C and in vivo libraries 91 SUMMARY AND DISCUSSION  93  CHAPTER FOUR: Analysis of the Siderophore Transporter Gene, SIT1  97  INTRODUCTION  97  MATERIALS AND METHODS A. in silico Analysis of SIT1p and SIT] a. Determination of the coding sequence and exon-intron boundaries of CnSitlp b. Comparison of SIT] and Sitlp in serotypes A, B and D c. Identification of siderophore-related genes in C. neoformans d. Comparison of Sitip to fungal homologs B. Strains and Growth Conditions C. RNA Isolation and Northern Analysis D. Construction of sit]:: URA5 (serotype D) and sit]::NEO (serotype A) Alleles E. Plate Assays a. Siderophore utilization b. Melanin production c. Temperature sensitivity d. Cell wall integrity F. Microscopy a.DIC b. Transmission electron microscopy G. Antibiotic Susceptibility Assays H. Virulence Assays in the Murine Model  98 98  101 102 102 102 103 103 104 104 104 104 105  RESULTS A. Characterization of SIT] a. Comparison of SIT] and Sitip in serotypes A, B and D  106 106 106  v  98 98 98 99 99 100  B.  C.  D. E.  F.  G. H. I.  b. Identification of siderophore-related genes in C. neoformans c. Comparison of Sitip to fungal homologs d. Expression of SIT] in low iron and iron replete conditions Construction of Siderophore Transporter Mutants a. SIT] gene and Sitlp protein structure b. Construction and confirmation of null mutants The Role of SIT] in Iron Acquisition a. Growth in various iron conditions b. Siderophore utilization Involvement of the cAMP Pathway in Siderophore Utilization SIT] and Melanization a. Production of DOPA melanin b. Presence of extracellular melanin-like granules c. Effects of other factors on melanization Influences of SIT] on Cell Wall Integrity a. Temperature sensitivity and cell wall integrity b. Alteration of cell wall structure Summary of Additional Phenotypic Analyses of sit] Mutants Drug Susceptibility Virulence in the Murine Model a. SerotypeD b. SerotypeA  SUMMARY AND DISCUSSION  110 113 118 119 119 121 125 125 133 135 137 137 137 140 143 143 145 148 149 150 150 151 153  CHAPTER FIVE: GENERAL DISCUSSION 155 A. Phenotypic and Genomic Differences Between Serotypes A and D Strains. 155 B. Iron-regulated Genes in C. neoformans 157 C. The Role of SIT] in Iron Acquisition 159 D. The Role of SIT] in the Structure of the Cell Wall 161 E. The Possible Role of SIT] in Endosomal Trafficking 162 F. The Potential Influences of the cAMP and PKC] Signal Transduction Pathways on SIT] 163 G. Susceptibility of sit] Mutants to Antifungal Compounds 165 H. SIT] is Not Required for Virulence in Serotype A 166 I. Models of Possible Relationships Between SIT] and Cellular Processes in C. neoformans 166 J. Future Directions 170 REFERENCES  173  APPENDIX I: Websites Used In These Studies  193  APPENDIX II: Supplementary Physical Mapping Data (CHAPTER TWO) A. Probes sequences for hybridization experiments B. Hybridization strategy for pooled probes  194 194 199  vi  C. D.  Directions to interpret ResGen High density filters Raw hybridization experiment results i. JEC21: integrated markers ii. JEC21: pooled probes iii. H99: integrated markers iv. H99: pooled probes  200 201 201 203 212 214  APPENDIX III: Supplementary SAGE Data (CHAPTER THREE) 225 Construction of the SAGE library from H99 cells grown in low iron media A. at 37°C 225 i. Figure lIla. agarose gel of RNA extraction 225 ii. Figure IlIb. polyacrylamide gel of PCR Optimization 225 iii. Figure IlIc. polyacrylamide gel of PCR for 102 bp band 226 iv. Figure hId. polyacrylamide gel of digest of 26 bp band 226 v. Figure hlIe. polyacrylamide gel of ligation to form concatemers 227 vi. Figure IlIf. agarose gel of colony PCR results 227 vii. Tag Identification for SAGE Analysis 228 B. Raw SAGE data for H99 low iron media library 228 APPENDIX IV: Supplementary Data Analysis of SIT] (CHAPTER FOUR)  229  A. SIT] Sequence information 229 a. SIT] nucleotide and amino acid sequence from serotype D (SGTC) and A (DUMRU) and B (MSGSC) 229 b. Sequences of SIT] homologs in other fungi 232 c. Sequence of knock out cassettes with primers 235 d. Sequence of the reconstituted fragment with primers 237 B. SIT] Mutant Construction 239 a. Agarose gel showing PCR products of individual overlap knock out fragments 239 b. Agarose gel showing amplification of knock out cassette 239 C. Confirmation of homologous recombination of iXsit] allele 241 a. Agarose gel showing positive colony PCR results 241 D. Reconstitution of sit] with SIT] 243 a. PCR: primers SIT1A and SIT1F to differentiate wild-type and mutant bands 243 E. Raw Data and Calculations 244 a. Raw growth curve data 244 b. Sample calculation for cell count assays 247 APPENDIX V: Construction of Yeast Two Hybrid Libraries  248  APPENDIX VI: Author’s Contributions to Research and Development  249  vii  LIST OF TABLES Table 1: Summary of the hybridization of selected markers to BAC clones of JEC21andH99  33  Table 2: Summary of hybridization experiments for individual probes  35  Table 3: Summary of the contigs in the fingerprint map of JEC2 1  39  Table 4: Summary of the contigs in the fingerprint map of H99  40  Table 5: Most abundant SAGE tags for H99 cells grown at 37°C in low iron  67  Table 6: Comparison of SAGE data for strain H99 cells grown at (3 7°C low iron) vs. YNB (25°C) media  71  Table 7: Differentially abundant SAGE tags from H99 cells grown at (3 7°C low iron) vs. YNB (25°C) media 73 Table 8: Differentially abundant SAGE tags from H99 cells grown in YNB (25°C) vs. (3 7°C low iron)  75  Table 9: Comparison of SAGE data for strain H99 cells grown at (3 7°C low iron) vs. YNB (37°C) media 78 Table 10: Differentially abundant SAGE tags from H99 cells grown in at (37°C low iron) vs. YNB (37°C) media  80  Table 11: Differentially abundant SAGE tags from H99 cells grown in YNB (37°C) vs. (3 7°C low iron)  82  Table 12: Comparison of SAGE data for strain H99 cells grown at (3 7°C low iron) vs. cells isolated from rabbit cerebral spinal fluid (in vivo)  85  Table 13: Differentially abundant SAGE tags from H99 cells grown at (3 7°C low iron) vs. cells isolated from rabbit cerebral spinal fluid (in vivo) 87 Table 14: Strains used and constructed in these studies  100  Table 15: Siderophore-related genes in C. neoformans  112  Table 16: Summary of melanin production for sit] mutants in serotype D strains  142  Table 17: Summary of cell wall defects for sit] mutants noted by TEM  146  viii  Table 18: Minimum inhibitory concentrations of antifungal agents for sit] mutants.... 150 Table 19: Summary of DBA1 mouse weight loss with wt and the sit] mutant of the strain B3501A (serotype D)  150  Table 20: Summary of AJJcr mouse survival with SIT] wild-type , sit] mutants and sit] + SIT] reconstituted strains of the strain H99 (serotype A) 5 x 1 0 cell innoculum  151  Table 21: Summary of AJJcr mouse survival with SIT] wild-type , sit] mutants and sit] + SIT] reconstituted strains of the strain 1199 (serotype A) 5 x 1 cell innoculum  .  151 Table 22: Summary of pleiotrophic phenotypes for sit] mutants of JEC21, B3501A and H99 backgrounds  ix  154  LIST OF FIGURES Figure 1: Example of an autoradiograph showing the results of a row pooi of 12 overgo probes hybridized to a high-density BAC clone filter  34  Figure 2: FPC display from the map of strain JEC21  41  Figure 3: Conservation of gene synteny between the genomes of JEC21 and H99  45  Figure 4: Determination of Hindlil fragment sizes for 26S rDNA  47  Figure 5: Relationship between electrophoretically separated chromosomes and the contigs of the JEC21 and H99 maps  52  Figure 6: Genomic location of 26S rDNA and HIS3  53  Figure 7: Elaboration of the polysaccharide capsule in low iron or iron replete Medium  63  Figure 8: Expression profiling comparing relative transcript levels of SAGE tags from strain H99 cells grown in YNB (25°C) and at (3 7°C low iron) media 72 Figure 9: Expression profiling comparing relative transcript levels of SAGE tags from strain H99 cells grown in YNB (3 7°C) and at (3 7°C low iron) media 79 Figure 10: Expression profiling comparing relative transcript levels of SAGE tags from strain H99 cells isolated from rabbit cerebral spinal fluid (in vivo) or grown at (37°C low iron) media 86 Figure 11: Comparison of SAGE tag occurrences for cells grown in low iron (37°C), YNB (25°C) or YNB (3 7°C) medium 90 Figure 12: Comparison of SAGE tag occurrences for cells grown in YNB medium (25°C), isolated from rabbit cerebral spinal fluid (in vivo), low iron (3 7°C) medium or YNB (37°C) medium 92 Figure 13: Amino acid alignment of the Sitip protein from serotypes A, B and D strains of C. neoformans  107  Figure 14: Similarity tree of the Sitlp protein from serotypes A, B and D strains of C. neoformans 108 Figure 15: Comparison of exon and intron nucleotide sequence similarity for the SIT] gene in serotypes A, B and D strains of C. neoformans  x  109  Figure 16: Amino acid alignment of the Sitlp protein from the serotype D strain B3501A of C. neoformans with fungal homologs  114  Figure 17: Similarity tree of the Sitip protein from the serotype D strain B3501A of C. neoformans with fungal homologs 117 Figure 18: Transcript levels for the Sitlp gene in culture media with low or high iron levels for strains JEC2 1, B3 501 A and H99 118 Figure 19: Structure of the SIT] gene and Sitip protein  120  Figure 20: Structure of the Asit] knock out constructs  122  Figure 21: Confirmation of the iXsitl knock out in the serotype D strains B3501A and JEC21 123 Figure 22: Confirmation of the isit] knock out in the serotype A strain H99  124  Figure 23: Disruption of the SIT] gene significantly affects growth in LIM and LIM+BPDA+Deferoxamine in B3501A and H99 but not JEC21  126  Figure 24: sit] mutants are unable to use a siderophore to acquire iron in all strains as shown by siderophore utilization assays  134  Figure 25: The cAMP pathway is involved in siderophore utilization in serotype A but not serotype D strains  136  Figure 26: Melanin production on DOPA medium  138  Figure 27: DIC microscopy of melanized cells at four days growth on DOPA Medium  139  Figure 28: Temperature sensitivity and cell wall integrity assays  144  Figure 29: Transmission electron microscopy (TEM) of melanized cells for wild type and sit] mutant strains  147  Figure 30: Virulence assays in the murine model for serotype A wild type and sit] mutant strains  152  Figure 31: Model of possible relationships between SIT] and cellular processes in C. neoformans  169  xi  LIST OF ABBREVIATIONS ABC  ATP Binding Cassette  AIDS  Acquired ImmunoDeficiency Syndrome  ATP  adenosine triphosphate  BAC  Bacterial Artificial Chromosome  BPDA  Bathophenanthroline disulfonic acid  BLAST  Basic Local Alignment Search Tool  cAMP  cyclic adenosine mono-phosphate  CFU  colony forming units  CHR  chromosome  contig  contiguous piece of DNA  CR  congo red  CSF  cerebral spinal fluid  CW  calcofluor white  DIC  differential interference contrast  DUMRU  Duke University Mycology Research Unit  EtBr  ethidium bromide  JR  iron replete medium  Ga1XM  galactoxylomannan  GFP  green fluorescent protein  GTP  guanine triphosphate  GXM  glucuronoxylomannan  LGA  low glucose asparagine medium  LiCl  lithium chloride  LIM  low iron medium  MAPK  mitogen-activated protein kinase  MIC  minimum inhibitory concentration  MP  mannoprotein  MSGSC  Michael Smith Genome Sciences Center  NAT  nourseothricin  xii  NCCLS  National Committee for Clinical Laboratory Standards  NEO  neomycin  NCBI  National Center for Biotechnology Information  ORF  open reading frame  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PKC  protein kinase C  poly-A  poly-adenylated  SAGE  Serial Analysis of Gene Expression  SDS  sodium dodecyl sulphate  SSC  saline sodium citrate  SGTC  Stanford Genome Technology Center  TEM  transmission electron microscopy  TIGR  The Institute for Genomic Research  YNB  yeast nitrogen broth  YPD  yeast extract peptone dextrose medium  xlii  ACKNOWLEDGEMENTS I would first like to thank Dr. James W. Kronstad for his exceptional leadership, resources, advice, expertise and patience. His laboratory and mentorship provided a progressive, supportive and positive environment that allowed me to develop skills both personal and professional that will continue to provide me with strong guidance and knowledge in my future career. To my committee: Dr. B.B. Finlay, Dr. P. Hieter and Dr. S.J.M Jones, I thank you for your input, encouragement and dedication to my studies. I also would like to acknowledge the Michael Smith Genome Sciences Center, a world-class institution that was an integral partner in much of my thesis work. Specifically, I would like to thank the mapping and SAGE groups including J. Schein, S.J.M. Jones, M. Marra and S. Zuyderduyn for a successful collaboration and constructive experience. I thank Dr. J. Heitman from Duke University for many strains and vectors as well as expert advice on C. neoformans. Thank you to the UBC Bio-Imaging Facility: E. Humphrey, K. Rensing, G. Martens, D. Home for technical assistance and expertise and to L. Samuels for helpful discussion on TEM. I would like to note institutions whose Cryptococcus databases without which I could not have completed these studies: these include DUMRU, SGTC, the Broad Institute of Harvard and MIT, the University of Oklahoma and TIGR. To my family: Mom and Dad (Joy and Kenneth Tangen), Brother (Darren) and his family: Nicole and Gabrielle Riley, Grandma Mary Tangen, Sage, all the other Tangens and Sinclairs. I appreciate every day how blessed I am to have such a wonderful family with an incredible belief in me. Mom and Dad, your love and support have made my life beautiful and have taught me to follow my dreams and that with hard work, passion and commitment anything is possible. To my friend in celebration of life Matt Galloway, thank you for being by my side and sharing in dreams and ideas for the present and future. To my friend and classmate Dr. M.D. Brazas your friendship was exemplary in support and offering a sharing of experience both professional and in life. Thank you to members of the Kronstad laboratory past and present for an encouraging and collaborative environment. To Dr. B. R. Steen who provided friendship and mentorship, J. Klose (technical expertise and friendship), Dr. S, Kidd (gene genealogy, PCR fingerprinting, AFLP, RFLP), A.P. Sham (mouse work), Dr. T. Lian (Northerns and technical expertise), Dr. G. Hu (technical expertise), Dr. W. Jung (information on iron uptake and regulation genes) and Dr. C. D’Souza (technical expertise and comments). I would also like to thank many friends professional and personal who have enhanced my life in numerous ways: Dr. P.R. Hardwidge, Dr. H. Ledebur, Dr. D.M. Martin, M and M. Shepard, Dr. C. Maxwell, M. Smith, LL.B., E. Thomsen, J. Hagel, S. Duck, A. Harbott, J. Keefer, D. and J. Madsen, C and B. Gannon, J. Prenter and The Vancouver Spurs. This work was funded through grants to J.W.K. from The Natural Sciences and Engineering Research Council of Canada (NSERC): mapping and the Canadian Institutes of Health Research (CIHR): SAGE and SIT].  xiv  CHAPTER ONE: INTRODUCTION A. a.  Cryptococcus neoformans and Cryptococcosis. C. neoformans varieties, serotypes, molecular types and phylogeny. Cryptococcus neoformans is a saprophytic basidiomycete fungus that has adapted to survival in the mammalian host. Strains of the fungus are separated into five serotypes (A, B, C, D and AD) based on antigenic differences in their polysaecharide capsule and three varieties are also recognized: neoformans (serotype D), grubii (serotype A) and gattii (serotypes B and C). Recently, the variety gattli has been designated as the separate species C. gattii. Cryptococcus has been further classified into molecular types by PCR-fingerprinting, URA5-RFLP (restriction fragment length polymorphism) analysis and amplified fragment length polymorphism (AFLP) analysis (Meyer et a?., 1999; 2003; Kidd et a?., 2004). PCR-fingerprinting involves using a single minisatellite specific primer specific to M13 phage core sequence, producing an electrophoretic profile that differentiates strains within varieties and serotypes (Meyer et al., 1999; 2003). URA5-RFLP analysis separated molecular types based on polymorphisms in the gene for orotidine monophosphate pyrophosphorylase (URA5) resulting from a HhaI and Sau961 double digest (Meyer et al., 2003). AFLP analysis is a multilocus genotyping method and has been used to type Cryptococcus (Boekhout et al., 2001).  These molecular types  correspond to the same groupings found in PCR-fingerprinting and the URA5-RFLP method, and the authors that performed these analyses were the first to recommend the two species divisions of C. neoformans (serotype A and D) and C. basillisporus (serotypes B and C) rather than the now accepted C. neoformans (serotype A and D) and C. gattii (serotypes B and C). Each of these methods group strains into eight succinct molecular types (VN or VG designations for PCR-fingerprinting and the URA5-RFLP method; and corresponding AFLP types). The accepted designations that have appeared are: C. neoformans serotype A var. grubii (VNI/AFLP1 and VNII/AFLP1A), serotype AD (VNIII/AFLP3), serotype D var. neoformans (VNIV/AFLP2), serotype B  C.  gattii (VGI/AFLP4), (VGII/AFLP6), (VGIII/AFLP5) and serotype C  (VGIV!AFLP7). Molecular phylogenetic work revealed that the grubii and neoformans varieties are separated by ‘—4 8.5 million years of evolution, and these diverged from the gattii variety —‘-37 million years ago (Xu et a?., 2000). This divergence was based on the gene genealogies of four genes: the mitochondrial large ribosomal subunit RNA, the internal transcribed spacer region of  1  the nuclear rRNA, including ITS 1, 5.8S rRNA subunit and ITS2, orotidine monophosphate pyrophosphorylase (URA5) and diphenol oxidase (LA Cl) (Xu et al., 2000).  b.  C. neoformans infections. C. neoformans primarily affects people with a compromised immune system, although multiple isolated infections have been documented in normal individuals (Christianson et al., 2003; D’Souza et al., 2004). C. neoformans is especially detrimental to those people with AIDS, or Diabetes mellitus, and in patients undergoing long-term corticosteroid therapy, organ transplantation or cancer treatment. Since the onset of the AIDS era, there has been a marked increase in the prevalence of cryptococcal infections. Previously, the fungus was rarely encountered as a human pathogen. Presently, approximately 10-3 0 % of AIDS patients contract C. neoformans and it is the leading mycological cause of death in these people (Forche et al., 2000). The average survival rate for an AIDS patient diagnosed with cryptococcosis is less than 6 months (Casadevall and Perfect, 1998). C. gattii has been associated with a wider host range including immunocompetent humans, as well as wild and domestic animals (Krockenberger et al., 2002; Kidd et a!., 2004)  c.  Environmental sources of Cryptococcus. Cryptococcus neoformans is found in a wide range of environmental locations including water, soil, air, trees and pigeon feces (Casadevall and Perfect, 1998). The prevalence of the fungus in urban areas, specifically in pigeon guano, increases the risk of transmission to humans. C. neoformans was initially hypothesized to exist in nature as a wood rotting fungus, however, extensive genomic and biological investigation has shown no evidence for wood degradation enzymes such as cellulases or lignin degradation enzymes (Jacobson et a!., 2003). C. gattii has been more extensively studied with respect to environmental sources. To that effect, C. gattii was historically isolated from eucalypts (Sorrell et al., 1996; Krockenberger et a!., 2002), however now many tree species have tested positive for C. gattii even in temperate climates (Davel et al., 2003; Kidd et a!., 2004; Granados et a!., 2005). Environmental isolates of C. neoformans are largely found in soil and pigeon excreta (Baro et a!., 1999; Trilles et al., 2003: Granados et aL, 2005), but have also been associated with trees (Trilles et a!., 2003; Kidd et a!., 2004: Granados et a!., 2005).  2  d.  C. neoformans life cycle. C. neoformans exists in the anamorphic yeast-like state as a or  ,  mating type haploid  strains. The fungus can undergo sexual reproduction: conjugation occurs following signaling via G-protein-coupled receptors and pheromones. A dikaryon with clamp connections forms as a result of mating partner fusion.  Fusion is followed by karyogamy and the development of  basidia. Following meiosis, the fungus produces haploid basidiospores, which then germinate to form the yeast-like state. The fungus is most commonly found in the yeast form and this is the infectious state of the organism. C. neoformans can also undergo sporulation in the anamorphic state when nitrogen is limited through a process called haploid fruiting. The sexual stage has not been conclusively identified in nature and the bias for the a mating type in the environment suggests that mating is not a common event (Casadevall and Perfect, 1998). The vast majority of clinical isolates are also of the a mating type, and it has been suggested that the a mating type is a virulence-associated factor.  e.  Transmission and etiology of cryptococcosis in humans. C. neoformans is commonly contracted by inhalation of the desiccated yeast cells, poorly encapsulated yeast forms, or basidiospores (Casadevall and Perfect, 1998). In individuals with a normal immune system, C. neoformans usually only produces an asymptomatic  •  respiratory infection. In immunocompromised individuals the infection may disseminate to cause cryptococcosis. Once disseminated, the fungus may cross the blood brain barrier where it causes encephalitis in the form of gelatinous cysts with a high fungal load. Disseminated C. neoformans may also cause lesions in the lung, heart, skin, and bone (Casadevall and Perfect, 1998).  f.  Epidemiology of Cryptococcus. C. neoformans serotypes A, D and AD are pandemic, the majority of clinical isolates in North America are serotype A strains; serotype D strains are more prevalent in specific European countries, for example, Denmark, France and Italy (Bennett et al., 1984; Dromer et al., 1996; Franzot et al., 1998). Serotypes B and C were previously considered to be mainly restricted to tropical and subtropical regions, although isolates have been obtained from temperate regions (Mitchell and Perfect, 1995; Sorrell, 2001). More recently, serotype B C. gattii has emerged as  3  the cause of cryptococcosis in humans and animals on the East Coast of Vancouver Island. This outbreak (the fungus is now considered endemic to the region) is the largest of its kind in history. The emergence of C. gattii on Vancouver Island is unique for two reasons: 1) this variety had previously mainly been identified in tropical regions, whereas Vancouver Island is a temperate region; 2) the fungus was responsible for infections in a wide range of hosts including marine, wild and companion animals, in addition to immunocompetent humans. In the past C. neoformans was largely considered to be an opportunistic pathogen. Since the onset and identification of this outbreak in 1999, significant strides have been made in the molecular, genetic, evolutionary and biological characterization of these strains including the novel discovery that these strains are a distinct molecular type to those found in tropical regions (Kidd et al., 2004).  g.  The immune response to C. neoformans. The immune response to C. neoformans involves both physical and nonspecific immunity, as well as a specific acquired response. The initial physical factors encountered by the fungus include: high temperature, slightly alkaline pH and nutrient depletion. The cells also undergo an attack by host effector cells of the innate immune system (Casadevall and Perfect, 1998). If the fungus can overcome the physical barriers and initial immune response to survive and persist in the human host, a specific immune response ensues. This response includes both humoral and cellular immunity. In humoral immunity, anticryptococcal antibodies are formed in response to fungal antigens.  In the cellular response, T-lymphocytes are generated that  recognize cryptococcal antigens (Casadevall and Perfect, 1998). Specific cellular immune responses are dependent on nonspecific immune mechanisms and opsonization by professional phagocytes and complement.  Previously, cell-mediated immune mechanisms were regarded as  the primary immune response to C. neoformans. Now it is accepted that a combination of antibody- and cell-mediated responses are required for the control of C. neoformans (Perfect, 2005; Casadevall and Pirofski, 2005).  A strong cellular response results from cryptococcal  infection through Th- 1 and requires the cytokines tumor necrosis factor, interferon gamma and IL-2 in addition to the chemokines MCP- 1 and MIP- 1 a for recruitment of inflammatory cells (Aguirre et al., 1995; Kawakami et al., 1996; Huffnagle, 1997; Huffnagle, 2000). Effector cells involved in the control of C. neoformans include CD4+ and CD8+ lymphocytes in addition to  4  activated macrophages (Hill, 1992; Levitz, 1994).  The capsular polysaccharides and  cryptococcal proteins can elicit an antibody response. The majority of research has focused on the response to capsular polysaccharides (Casadevall and Perfect, 1998; Vecchiarelli, 2005). The major component of the polysaccharide capsule is glucuronoxylomannan (GXM). This polysaccharide is highly immunogenic when opsonized and elicits innate and adaptive immune responses (Vecchiarelli, 2005). Minimal macrophage binding occurs in the absence of opsonins, however, C. neoformans is a potent activator of complement, which results in the deposition of the final component C3, an opsonin, on the fungal cell. Macrophages recognize opsonized C. neoformans by three major complement receptors designated CD35 (CR1), CD11b/CD18 (CR3), and CD11c/CD18 (CR4) (Levitz, 2002). In the cellular response, GXM binding involves the following receptors: toll-like receptor (TLR)4, CD14, TLR2, CD18 and Fc gamma receptor II (FcgammaRPi). This combination of receptor binding can actually lead to a suppressive effect. Interestingly, this can be reversed by the presence of GXM protective antibodies (Vecchiarelli, 2005).  Shed polysaccharide and mannoproteins represent a problem for the human immune  system in that they use up many of the circulating opsonins in the serum and cerebral spinal fluid, thereby reducing the ability of the immune system to identify the whole pathogen vs. shed polysaccharide.  h.  Treatment of cryptococcosis. The present therapy for cryptococcosis includes treatment with the polyene drug amphotericin B and azole drugs such as fluconazole and 5-flucytocine. Amphotericin B is often used in cases of rapidly progressing, life-threatening illness and fluconazole is used in chronic suppression or prophylaxis in immunosuppressed individuals. Unfortunately, there is a high occurrence of relapse even after apparent successful therapy (Casadevall and Perfect, 1998) as well as a significant problem with increasing drug resistance worldwide (Pfaller et aL, 2005). In addition, antifungals such as amphotericin B can be highly toxic to the host. There is a great need for new anticryptoccocal compounds with higher efficacy and lower toxicity. Understanding pathogenesis in C. neoformans may allow researchers to identify new targets for development of more effective drug therapies for treating and controlling cryptococcosis.  5  B.  Genomic Analyses of C. neoformans. There are notable differences between serotypes A (grubii) and D (neoformans) in virulence, epidemiology and clinical prevalence, with serotype A generally being the more virulent serotype and the most common among clinical isolates. Due to these differences, many studies have been initiated to compare the serotypes on a genomic level.  a.  Karyotype studies. Several studies have attempted to characterize the differences between serotype A and D strains.  Thus, isolates have been characterized with respect to 26S rRNA sequences, PCR  fingerprints, enzyme electrophoretic profiles and electrophoretic karyotypes (Perfect et al., 1989, 1993; Guehó et a!., 1993; Meyer et a!., 1993; Brandt eta!., 1993; Wickes, eta!., 1994; Boekhout and van Belkum, 1997; Bertout et a!., 1999). Strains of serotype A and D were distinguished by the RFLP patterns obtained upon hybridization with a repeated element (CNRE-1) and by the nucleotide sequence analysis of specific genes (e.g. URA5) (Franzot et al., 1998; Xu et a!., 2000). Xu et al. (2000) performed a more detailed phylogenetic analysis of strains from the different serotypes using sequence analysis of the mitochondrial large ribosomal subunit, the internal transcribed spacer region of nuclear rRNA, and the genes encoding orotidine monophosphate pyrophosphorylase (URA5) and diphenol oxidase (CnLACJ).  This work  supports the current separation of strains into the three varieties and two species and provides a phylogenetic framework for understanding the evolution and geographic distribution of C. neoformans. The population structure of C. neoformans has also been examined in detail using AFLP genotyping (Boekhout et at., 2001) and PCR fingerprinting (Meyer et at., 1999; Ellis et at., 2000). Finally, although strains of serotype A and D have been shown to be genetically distinct, it is possible to obtain mating between isolates from the two different serotypes (Kwon Chung, 1975). Several groups have performed electrophoretic karyotyping to determine the size and number of chromosomes in strains representing the different varieties of C. neoformans (Perfect et a!., 1989; Polachek and Lebens, 1989; Perfect et a!., 1993; Wickes et a?., 1994; Boekhout and van Belkum, 1997; Boekhout eta!., 1997; Forche et at., 2000). In addition, Wickes eta?. (1994) and Spitzer and Spitzer (1997) assigned several markers to electrophoretically separated  6  chromosomes by hybridization with known genes or ESTs. At the time of these studies, the view of the karyotype in C. neoformans indicated a genome size in the range of 15 to 27 Mb with an average chromosome number of 12 for variety neoformans and 13 for variety gattii. Forche et at. (2000) described the construction of a meiotic linkage map for C. neoformans serotype D. A mapping population of 100 progeny was employed with a total of 181 AFLP, RAPD and gene markers to identify 14 major linkage groups. Six of the linkage groups were assigned to specific chromosomes. A more recent genetic linkage map has been reported for serotype D (Marra et a?., 2004).  These studies further refined estimates of genome size to 20.2 Mb, with 14  chromosomes ranging in size from 0.8-2.3 Mb.  b.  Physical mapping projects. The Kronstad laboratory in collaboration with the Michael Smith Genome Sciences Center (MSGSC) initiated a physical characterization of the C. neoformans genome as part of an international effort (Heitman et a?., 1999) to obtain the complete genomic sequence of two strains representing the A and D serotypes. Physical maps for one strain of each serotype were reported in 2002 (Schein et al., 2002), with 82 markers placed on the serotype D (strain JEC21) genome and 102 markers placed on the serotype A (strain H99) genome. A portion of the map construction is detailed in Chapter Two of this thesis and has been published (Schein et a?., 2002). Conservation of gene synteny was generally good between the two genomes but there were notable examples of chromosomal rearrangements such as inversions and translocations. The BAC fingerprinting technology first described by Marra et a?., (1997) was used to generate large contigs that formed the framework for assembly and finishing of the genomic sequences for the serotype A and D strains (Loftus et a?., 2005). The ends of the fingerprinted BAC clones were also sequenced and the traces contributed to the shotgun sequence databases for both strains. The mapping approach was employed previously for whole-genome, random BAC clone fingerprinting projects that supported sequencing of the Arabidopsis thaliana (Marra et al., 1999; Mozo et a?., 1999) and human (McPherson et a?., 2001) genomes. Finally, the contributions described in Chapter Two placed markers on the BAC maps and these markers were employed both to compare the conservation of synteny between the serotype A and D strains and to attempt to correlate BAC clone contigs with specific chromosomes. Five additional mapping projects for different Cryptococcus genomes have been initiated and completed since the successful initial  7  maps. This resource allows the comparison of multiple strains and provides the framework for additional sequencing efforts and genomic studies (detailed below).  c.  EST sequencing projects. Sequencing of expressed sequence tags (ESTs) assists in the annotation of genomes and in gene expression studies. ESTs are a single pass sequence read from cDNAs, thus representing gene transcripts from an organism. They are particularly useful because they represent the coding regions of the genes and provide key information to assist in the annotation of features such as intron and exon locations. They also provide transcript identification for expression-based data derived from techniques such as serial analysis of gene expression (SAGE). EST sequencing projects were completed for the serotype D strain B3501A and the serotype A strain 1199 at the University of Oklahoma (http://www.genome.ou.edu/cneo.html). For B3501A, 4000 clones were sequenced from both ends (directionally cloned inserts) resulting in 8000 ESTs with an additional 1700 clones sequenced directionally (‘-3300 EST5) from cells grown in low iron medium. For the serotype A strain H99, 3750 ESTs were obtained from both ends of clones resulting in  7500 reads. Additional ESTs were sequenced for H99 at the Duke University  Mycology Research Unit (DUMRU) (http://cneo.genetics.duke.edu!).  These databases were  utilized in the total genome assembly projects, in addition to providing a crucial resource for transcript identification in SAGE projects (Steen et at., 2002; 2003, Lian et at., 2005). Finally, the largest effort to collect EST sequences was performed more recently at The Institute for Genomic Research (TIGR) for the serotype D strain JEC2 1 (Loftus et at., 2005).  d.  Genome Sequencing Efforts. A number of genome sequencing efforts are now complete or near completion including the publication of two serotype D genomes JEC21 and B3501A (Loftus et at., 2005). JEC21 was sequenced, assembled and annotated at The Institute for Genomic Research (TIGR) (http://www.tigr.org/tdb/edb2/ctypt1htmls/) and B3501A was completed at the Stanford Genome Technology Center (SGTC) (http ://sequence-www. stanford.edulgroup/C. neoformansl).  In  addition to variation in virulence and genome structure between serotypes, major differences in the genome structure of the closely related strains JEC2 1 and B3 501 A were reported, and this is interesting because B3501A is a more virulent strain than JEC21.  8  Specifically, chromosomal  rearrangements between these two strains were documented with a chromosomal translocation and segmental duplication identified in the strain JEC2 1 (Fraser et al., 2005).  Other genome  sequencing projects near completion are for the serotype A strain, 1199 at the Broad Institute of Harvard and MIT (http ://www.broad.mit.edu/annotationlfungi/cryptococcus_neoformans/), an environmental isolate, W1v1276 (serotype B strain), at the University of British Columbia (http://www.bcgsc.ca!about/news/crypto_public/view) and a Vancouver Island clinical isolate, R265  (serotype  B  strain),  at  the  Broad  Institute  of  Harvard  (http ://www.broad.mit.edu/annotation/fungi/cryptococcus_neoforrnansb/).  and  MIT  Information  from mapping and sequencing projects has identified the number of chromosomes in C. neoformans for strains JEC21, B3501, H99 and WM276 to be 14. The availability and comparative analysis of these genomes, in addition to many transcriptional studies in progress, will be powerful resources to provide further insight into key virulence factors in the fungus.  e.  Serial analysis of gene expression (SAGE). Serial analysis of gene expression (SAGE) is an innovative molecular-biology based technique to analyze expressed genes in an organism. The SAGE technique, initially developed by Velculescu et a!. in 1995, provides a snapshot of the mRNA transcript abundance in a given cell state. The set of mRNA sequence “tags” in a SAGE library reflect the content and relative abundance of mRNA in the cells isolated for that library. This profile of transcripts is known collectively as the transcriptome. A transcriptome is dynamic in contrast to the genome, which is static.  The technique involves isolation of mRNA, capture of the poly-A 3’ ends and  conversion to cDNA. The DNA is cut with a restriction enzyme that has a 4 bp recognition site. This is the “anchoring” enzyme NlaIII (5-CATG-3’).  A linker is added that contains the  recognition site for a type ITS enzyme BsmJI (5’-GGGAC-3’), which cleaves the cDNA 15 bp downstream of the recognition site. The fragments are blunt end ligated, linkers are removed and tags are concatenated, and then cloned into vectors for sequencing. Data output from a SAGE library is in the form of 10 base pair tags, each of which is 3’ adjacent to the 3’ most NlaIII (CATG) site in a eDNA. The abundance of unique SAGE tags may be compared from cells grown in different conditions. The corresponding transcript for each unique tag gives an indication of expression for that transcript in that cell state. The technique allows for tags from multiple transcripts to be sequenced in a single read. This in turn increases the efficiency and  9  lowers the cost of transcriptome analysis.  The tags are extracted and quantified through  automated programs, they can then be annotated using EST and genomic sequence databases. Some of the advantages of SAGE include the real quantification of transcripts and the unbiased nature of the technique with respect to the identification of novel or unknown genes. Many variations of the technique have been developed since the original SAGE protocol such as MicroSAGE (Datson et al., 1999), a modified procedure to allow for smaller sample sizes, and LongSAGE (Saha et al., 2002) that produces longer tags (20 bp) using the TypellS enzyme MmeI (which has a recognition site further from the cut site than the original enzyme Bsmfl). This technique was used to confirm annotations from the original SAGE technique. Recently, an even more robust SAGE technique has been developed. SuperSAGE (Matsumura, 2005) utilizes the Typelli enzyme EcoP 151, which produces 26 bp tags and allows for more definitive transcript annotation. SAGE techniques have been successfully utilized in many transcriptional studies of eukaryotic organisms thus far, including C. neoformans (Steen et al., 2002 and 2003; Lian et al., 2005). SAGE was initially developed to differentiate gene expression in cancer vs. normal cells of humans (reviews by Porter and Polyak, 2003; Liu et al., 2004; Tuteja and Tuteja, 2004). SAGE has also been adopted for use in a wide variety of applications in other organisms. SAGE has been extensively used in plant studies, for example, to investigate root nodule formation in the legume Lotus japonicus (Asarnizu et al., 2005), virally infected Cassava plants (Manihot esceulenta) (Fregen et al., 2004) and tomato transcriptional regulation factors in Arabidopsis thaliana (Chakravarthy et al., 2003). SAGE has also been used to investigate gene expression in parasitic pathogens (Knox et al., 2005). After its development, SAGE was very quickly applied to the model yeast S. cerevisiae and the SAGE transcriptome was reported in 1997 (Velculescu et al., 1997).  This study was quickly followed by SAGE analysis that was adapted to ask  specific questions for example, about the variation of transcriptome size (Holland, 2002), vinification (Varela et aL, 2005) or identification of transcriptional start sites (Zhang and Dietrich, 2005). Many other fungi have been studied by SAGE for industrial processes e.g. Monascus aurantiacus to determine the efficient fermentation for citrinin production (Xiong et al., 2005) as well as other fungal pathogens e.g. Magnaporthe grisea for appressorium formation (Inc et a!., 2003) and barley mildew (Blumeria graminis) (Thomas et al., 2002). Multiple SAGE projects and analyses are ongoing in the Kronstad laboratory and are detailed in Chapter Three.  10  There are advantages of SAGE over microarray technology. First, SAGE is unbiased in its ability to identify novel genes of unknown functions, this is not possible with microarrays where DNA sequence must be determined prior to array printing. Second, SAGE can also be used to effectively quantify fold differences in transcription by comparison of SAGE tag abundance between conditions. The abundance of SAGE “tags” corresponds directly with the mRNA copy number in the cell. Until recently, a complete genomic sequence was not available for C. neoformans, therefore the construction of comprehensive microarrays was not possible. Since the onset of this project, total genome sequence has become available and has been followed by the advent of microarray technology for C. neoformans (Fan et al., 2005; Pukkila Worley et al., 2005).  C.  C. neoformans Signaling and Virulence Factors. The remainder of this introductory chapter presents background information on the biology and pathogenesis of C. neoformans. This information sets the stage to appreciate the application of genomic and genetic approaches to understand virulence in this important pathogen.  a.  The cAMP and PKC1/MAP kinase pathway. Cell signaling plays an important role in the regulation of many cellular processes. Many virulence factors in C. neoformans are known to be regulated through the cAMP/PKA and FKC1/IVIAP kinase pathways (Alspaugh et al.,  1998; Kronstad et al.,  1998). Various  environmental signals such as nitrogen, glucose or iron deprivation activate the Ga protein cAMP-PKA pathway, which regulates differential transcription of genes involved with melanin formation, mating and capsule formation. Protein kinase A contains two regulatory and two catalytic subunits. The kinase is activated when four molecules of cAMP bind to the two regulatory subunits (Pkrlp); this causes a conformational change in the regulatory subunits that consequently releases the catalytic subunits (Pkalp). The catalytic subunits of protein kinase A are then active to phosphorylate downstream targets that include factors related to expression of a number of virulence genes (D’Souza et al., 2001).  11  The Heitman laboratory at Duke University has performed extensive studies characterizing components of the cAMP pathway. Important components include products of the GPA1, CAC1, PKA1 and PKR] genes. GPA1 codes for the alpha subunit of a heterotrimeric GTP-binding protein (Aispaugh et a!., 1997) and CAC] codes for adenylyl cyclase (Aispaugh et a!., 2002). The Heitman laboratory has characterized the PKA] gene in the serotype A strain H99 that encodes the major cAMP-dependent protein kinase catalytic subunit in addition to the FKR] gene that encodes the protein kinase A (PKA) regulatory subunit (D’Souza et al., 2001). In the serotype D strain, the major catalytic subunit is PKA2 (Hicks et al., 2004). PKA2 is present in H99 and FKA 1 is present in JEC2 1, however it has been shown that the subunits have divergent roles between serotypes A and D (Hicks et a!., 2004). In the serotype A strain H99, pkal mutants were sterile, failed to produce melanin or capsule and were avirulent. p/cr] mutants overproduced capsule and were hypervirulent in animal models. The pkrlpkal double mutant had a similar phenotype to the pkal mutant indicating that the pkal mutation is epistatic to the pkrl mutation. Protein kinase A was also shown to function downstream of Gc protein and adenylyl cyclase and to regulate cAMP production by feedback inhibition (Alspaugh et a!., 2002). In serotype D, PKA2 not PKAJ is involved in the regulation of mating, haploid fruiting and virulence factor production, but is not a key regulator of virulence as in serotype A (Hicks et al., 2004). Recently a study of the transcriptional effects dependent on the G-alpha protein, encoded by GPA], was undertaken (Pukkila-Worley et a!., 2005). This study further identified and confirmed multiple genes that were controlled by the cAMP pathway, specifically focusing on those involved in capsule production and melanin formation. Two laccase genes (LA Cl and LAC2) encoding laccase enzymes were controlled by the cAMP pathway through GPA]. LAC] had been previously characterized in C. neoformans and was required for melanin formation (Williamson, 1997). This work showed that similar to LAC1, LAC2 was induced by low glucose, however deletion of the gene only resulted in a minor delay in melanization. Overexpression of LAC2 was able to restore melanization to compensate for the loss of LAC] or GPAJ in addition to partially restoring virulence to lad and gpal mutants (Pukkila-Worley et al., 2005). The PKC] pathway is responsible for maintaining cell wall integrity in C. neoformans (Kraus et al., 2003; Gerik et a!., 2005). The terminal kinase in the pathway is Mpklp/Slt2p and deletion mutants of mpk] are sensitive to temperature and cell wall damaging compounds and are attenuated for virulence in the murine model (Kraus et a!., 2003). More recent studies have  12  characterized other components of the PKC1 pathways including kinases and regulatory proteins (Gerik et al., 2005). These studies showed that the kinases Bcklp and Mkk2p are critical for maintaining cell wall integrity, in addition to the regulatory protein Lrglp and phosphatase Ppglp. The PKC]/MAP kinase pathway has also been implicated in melanogenesis, where the loss of the Cl-domain (for diacyiglycerol) of PKC] led to loss of laccase activity and melanin production (Heung et aL, 2005). These changes were proposed to be mediated by a change in cell wall integrity due to the displacement of laccase from the cell wall. There is also involvement of the C. neoformans PKC] pathway in cell wall integrity. It is known that the cell wall integrity pathway is activated in C. albicans when Pkclp phosphorylates the MAP kinase Mkclp (Bates et a!., 2005). A homolog of Mkclp is present in the C. neoformans serotype D strain JEC21 (TIGR Identifier 162.m02645 ICNIOO41OI). The MAP kinase Mkc 1 p has already been implicated in regulation of cell wall integrity in response to antifungal drugs and loss of calcineurin function in C. neoformans (Kraus et a!., 2003). As described in Chapter Four, the work in this thesis documents a possible relationship between a gene (SIT]) involved in iron uptake to melanogenesis and cell wall integrity, possibly through PKCJ.  b.  Structure and biology of the polysaccharide capsule: contributions to virulence. The polysaccharide capsule of C. neoformans is generally regarded as the most significant virulence factor in the fungus. The capsule is a distinguishing feature in comparison to other medically important yeasts, and is often used diagnostically for the identification of C. neoformans using India ink stain. All acapsular strains produced thus far have reduced virulence or are avirulent (Perfect, 2005). Extensive studies have been performed and summarized on the structure, antigenicity, biology, synthesis and regulation of the capsule (Casadevall and Perfect, 1998; McFadden and Casadevall, 2001; Bose, 2003; Janbon, 2004; Perfect, 2005). Three major polysaccharide  components  are  described  for  C.  neoformans  exopolysaccharide:  glucuronoxylomannan (GXM), galactoxylomannan (Ga1XM) and mannoprotein (MP). GXM accounts for approximately 90% of the capsule, and it is differences in GXM structure leading to antigenic variation that led to the classical serotype designations A, B, C, D and AD (Casadevall and Perfect, 1998). The capsule contributes to virulence in a number of respects. Components  13  of the capsule do elicit a response from the immune system but can also be immunosuppressive. Mannoproteins are immunogenic and elicit a cell-mediated response in a delayed-type hypersensitivity reaction (Murphy et al., 1988). GXM and Ga1XM are poor antigens in the immune response and immunization of mice with large amounts of these polysaccharides resulted in a reduced ability to produce an antibody response (Kozel et at., 1977). In contrast, opsonized GXM is highly immunogenic and produces an antibody response characteristic of T cell dependent antigens, with predominantly IgG isotypes (Casadevall et a!., 1992). immunosuppressive effect of the capsule includes: antiphagocytosis  The  (Kozel and Gotschlich,  1982; Kozel et a!., 1988; Vecchiarelli et a?., 2003; Ellerbroek et al., 2004; Gates et al., 2004), complement depletion (Macher et al., 1978; Kozel, 1993), antibody unresponsiveness (Kozel et al., 1977), inhibition of leukocyte migration (Dong and Murphy, 1995; Lipovsky et a!., 1998; Murphy, 1999; Vecchiarelli, 2000; Ellerbroek et at., 2004), dysregulation of cytokine secretion (Retini et at., 1996; Vecchiarelli et at., 1995; 1996; 1998; Shoham et a?., 2001), brain enhancement of HIV infection (Pettoello-Mantovani et al., 1992; 1994), L-selectin and tumor necrosis factor receptor loss (Dong and Murphy, 1996; Murphy, 1999; Ellerbroek et a?., 2004), and negative charge (Nosanchuk and Casadevall, 1997).  c.  Melanin and its effects on virulence. Melanin production and the laccase enzyme represent another significant virulence factor in C. neoformans. Melanin and the production of laccase is necessary for survival within alveolar macrophages (Liu et a?., 1999) and for extrapulmonary dissemination to the brain (Noverr et at., 2004). DOPA type melanin is synthesized from diphenolic catecholate compounds by the phenol oxidase enzyme, laccase (Lacip).  Disruption of LAC1 leads to an “albino” phenotype on  selective DOPA medium the mutant is and is attenuated for virulence in animal models (Salas et at., 1996). Melanin has multiple affects on cell stability and protection from the host including its ability to act as an antioxidant, a contribution to cell wall integrity, reduction of drug susceptibility by antifungal binding, interference with antibody-mediated phagocytosis and protection from high temperatures (Casadevall and Perfect, 1998). The enzyme laccase has also been implicated in protection from alveolar macrophages even in the absence of the DOPA substrate (Liu et at., 1999). This protection is proposed to be a result of the iron oxidase activity  14  of the enzyme, which allows for the oxidation of Fe2+ to Fe3+ thereby reducing the ability of macrophages to produce damaging free radicals.  d.  Mating type specificity: prevalence in clinical strains. The predominant mating type in environmental and clinical isolates is a, at an a:a ratio of 40:1 (Casadevall and Perfect, 1998). This led scientists to propose that asexual reproduction is the primary mode of reproduction supporting the idea that the a mating type is a virulenceassociated factor (Kwon-Chung et a!., 1992). This hypothesis was reached because there would be few The  mating type available to mate if the predominant mating type in the population was a.  mating type is even more infrequent among serotype A strains. The majority of clinical  isolates in North America are serotype A and a study of environmental isolates in New York City indicated a single pattern for CNRE- 1 repetitive DNA RFLP pattern which suggests a clonal origin (Casadevall and Perfect, 1998; Currie et at., 1994). The haploid a mating type is also plausible as the infectious agent due to its ability to undergo haploid fruiting under conditions of dryness and low ammonia concentration where many strains formed hyphae and basidiospores (Wickes et at., 1996).  Haploid fruiting allows for the generation of particles that are small  enough to account for route of infection through the alveoli of the lung, without the need for sexual reproduction (Wickes et al., 1996). A very interesting recent finding was that mating can occur between strains of the same mating type, a. (Fraser et al., 2005; Lin et a!., 2005). This unique biological phenomenon may further explain the prevalence of the alpha mating type in the environment, in clinical prevalence and further would allow for genetic variation within the population in the absence of the mating type.  e.  Superoxide dismutase (SOD) and effects. Superoxide dismutase (SOD) converts superoxide radicals into hydrogen peroxide and oxygen. This enzyme is widely believed to be key in the survival of bacterial and fungal pathogens in the host. Cox et al. (2003) showed that a null mutant of the Cu,Zn SOD gene SOD] was more susceptible to reactive oxygen species in vitro, had significantly reduced virulence in the murine inhalation model and had attenuated growth compared to wild-type in macrophages that produced reduced amounts of nitric oxide. These findings indicated that superoxide dismutase contributes to virulence but is not absolutely required for pathogenesis in C. neoformans. The  15  authors hypothesize that the reduction in virulence may be due to an increased susceptibility to oxygen radicals within macrophages and may not be required if other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo. Another gene, SOD2, encoding a mitochondrial superoxide dismutase is involved in resistance to oxidative stress, growth at high temperature and is required for virulence in the murine inhalation model (Giles et al., 2004). Similar work has been done in C. gattii, where the sod2 mutant was also found to be sensitive to growth at high temperature and avirulent in the intranasal model in mice (Narasipura et al., 2005). A SOD] homolog is also present in C. gattii and has been implicated in virulence (Narasipura et a!., 2003). The sod] mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The mutant was attenuated for virulence in the murine model, and it was significantly more susceptible to in vitro killing by human neutrophils (PIVINs) than wild type. The deletion of SOD] also resulted in defects in the expression of a number of virulence factors, e.g. laccase, urease and phospholipase. Overall, these results suggested that the SOD] was required for virulence but not saprophytic survival. The virulence defect of sod2 was not believed to be a result of increased killing by phagocytes. In addition, the sod2 mutant was also highly susceptible to redox-cycling agents, high salt and nutrient limitations (Narisipura et a!., 2005). This group also constructed double mutants of sodlsod2. The sod]sod2 double mutants were also avirulent in mice (not surprisingly). The sodlsod2 double mutants also showed a marked reduction in the activities of other known virulence factors and they were more susceptible to killing by macrophages than was the sod2 single mutant. The group had previously shown that sod] mutants of C. gattii were attenuated for virulence, likely due to an increase in killing by phagocytes, and had a reduction of activities of other virulence factors. Therefore this group hypothesizes that Sodlp and Sod2p play distinct roles in the biology and virulence of C. gattii via independent modes of action. f.  Resistance to oxidative stress. A number of genes have been identified that are implicated in the resistance to oxidative stress. Superoxide dismutases SOD] and SOD2 were discussed previously. Glutathione peroxidases are responsible for the reduction of peroxides, which leads to resistance to oxidative  16  stress. Two glutathione peroxidases GPX1 and GFX2 have been characterized in C. neoformans (Missall et al., 2005a). Both genes were induced by t-butylhydroperoxide or cumene hydroperoxide stress and repressed during nitric oxide stress. Further, GPX2 was induced by hydrogen peroxide.  The gpxl and gpx2 mutants were sensitive to peroxide stress but not  superoxide or nitric oxide stressors. The mutants did show a slight sensitivity to killing by macrophages but were dispensable for virulence in the murine model. The thioredoxin system is also involved in resistance to oxidative stress in C. neoformans (Missall et al., 2005 b and c). This antioxidant system consists of thioredoxin, thioredoxin reductase and NADPH. Missall et al., showed that the thioredoxin proteins Trxlp and Trx2p were important for resistance to stress and for virulence. The trxl mutant was sensitive to multiple stresses and the trx2 mutant to nitric oxide stress. It was shown that TRXJ was necessary for virulence in mice and survival in macrophages. They also showed that two putative transcription factors differentially regulate the thioredoxin system. Aftip is involved in oxidative stress induction and Yap4p for nitrosative stress induction of the thioredoxin genes. Two thiol peroxidases were identified and characterized in C. neoformans (Missall et al., 2004; 2005d). Tsalp and Tsa3p were identified in proteomic studies as more highly expressed at 37°C. Missall et al. showed that the TSA 1 and TSA3 were transcriptionally induced by hydrogen peroxide. The group also identified a third thiol peroxidase gene, TSA4 from genomic sequence. Through the construction of single, double and triple mutants they show that the tsal mutant is sensitive to hydrogen peroxide, t butylhydroperoxide, nitric oxide, has reduced growth at 25 and 3 8.5°C and has reduced virulence in two mouse models.  Finally, an alternative oxidase gene, AOX1 that showed increased  transcription at 37°C was characterized with respective to virulence (Akhter et al., 2003). Alternative oxidases are involved with the cytochrome oxidative pathway to produce ATP and may be involved with the response to oxidative stress in the mitochondria. The aoxl mutant was more sensitive to the oxidative stressor tert-butyl hydroperoxide, had reduced virulence in the murine inhalation model and had impaired growth in a macrophage-like cell line.  g.  Maunose production and virulence. Mannose production has been implicated in virulence in C. neoformans.  The GDP  mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose  isomerase  (PMI),  phosphomannomutase  17  (PMM)  and  GDP-mannose  pyrophosphorylase (GMP). Hills et al. (2001) produced a null mutant of the MAN] gene, encoding for the PMI enzyme, to block mannose synthesis. They showed that the gene was required for virulence in both the rabbit and mouse model, resulting in the elimination of C. neoformans from the hosts.  The mutants were also poorly encapsulated, had reduced  polysaccharide secretion and showed morphological abnormalities. These results indicated that the production of mannose is important for pathogenesis in C. neoformans.  h.  The ability to grow at 37°C. The ability to grow at 37°C is an inherent virulence factor of any human pathogen and this trait can be attributed to many genes. A number of genes have been implicated in the survival of C. neoformans at 37°C thus far, including genes involved with stress resistance pathways, signaling networks and basic metabolism (Perfect, 2005).  Some of these include:  manganese superoxide dismutase, SOD2 (Giles et al., 2005), calcineurin, CNA] (Rhodes et al., 1986; Odom et al., 1997), a vacuolar ATPase, VPH] (Erickson et al., 2001) and a gene involved in RNA/DNA splicing, CCNJ (Chung et al., 2003). The signaling genes RAS] (Alspaugh et al., 2000), CNA] (Odom et al., 1997), CNB] (Odom et al., 1997), MPK] (Kraus et al., 2003) and CTSJ (Fox et al., 2003) have been implicated in high temperature growth. Amino acid metabolism genes ILV2 (Kingsbury et al., 2004a) and SPE3/LYS9 (Kingsbury et al., 2004b), and trehalose synthesis genes TPS] (trehalose-6-phosphate synthase) and TPS2 (trehalose-6phosphate phosphatase) (Wills et aL, 2003) are also required. Many other genes have been identified in transcriptional studies that are regulated by temperature but are not necessarily required for high temperature survival (Steen et aL, 2002; Perfect et al., 2005). In S. cerevisiae over 70 genes have been identified that are essential for growth at 3 7°C, indicating that there are likely many more genes required for survival at 37°C in C. neoformans.  Other virulence factors. A number of other virulence factors have been characterized in C. neoformans. These include the production of urease, phospholipase secretion, vacuolar acidification, and mannitol production, and an RNA helicase. Urease is a nickel metalloenzyme that catalyzes the hydrolysis of urea to ammonia and carbamate. C. neoformans is a prolific producer of urease and this characteristic is often used to diagnose the fungus (Casadevall and Perfect, 1998). Urease acts as  18  a scavenger enzyme and is important in conditions of low nitrogen. It is likely important during infection due to its ability to change the local pH in the host, for example when C. neoformans is in the phagolysosome of the macrophage (Casadevall and Perfect, 1998).  Urease has been  implicated in virulence during experimental cryptococcosis in the inhalation and intravenous murine model but not the intercisternal rabbit model (Cox et al., 2000). Phospholipase secretion has been linked to virulence because strains that produced higher levels of phospholipase caused more significant infections than lower producers (Chen et a!., 1997). More recently, the gene PLB1 encoding an enzyme that has phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities has been implicated in virulence (Cox et al., 2001). The pib] mutants did not show defects for any other cryptococcal virulence factors in vitro (e.g. capsule formation, growth at 37°C, laccase activity or urease activity). However, they did show reduced virulence in the murine inhalation and rabbit meningitis model and exhibited a growth defect in a macrophage—like cell line. Many virulence factors have been shown to be dependent on the vesicular proton pump (ATPase) VPH] (Erickson et a!., 2001). The vphl mutant was defective in capsule production, laccase and urease expression, growth at 37°C and was required for virulence in the murine meningoencephalitis model.  Mannitol has been implicated in  virulence as a possible osmoticum or antioxidant during infection. Extracellular mannitol may contribute to the elevation of intercranial pressure found in heavily infected patients because a high concentration of yeast has been implicated in this syndrome (Denning et al., 1991). A serotype A, low-mannitol producing mutant, was isolated by UV mutagenesis from the serotype A strain H99. The mutant was more susceptible to heat and NaCl and had reduced virulence in mice (Chaturvedi et a!., 1996). The DEAD-box RNA helicase-encoding gene (VAD]) has been implicated in virulence in C. neoformans (Panepinto et al., 2005), where the vad] mutant had significantly reduced virulence in the murine model.  The loss of VAD] resulted in the  upregulation of NOT] which is a global repressor of transcription. Of particular interest, NOT] was found to be an intermediate repressor of laccase. The group further found that Vadip was located within macromolecular complexes that formed cytoplasmic granular bodies in mature cells and during infection of mouse brain. In addition, VAD] was shown by in situ hybridization to be expressed in the brain of an AIDS patient coinfected with C. neoformans.  19  D.  Iron and Siderophore Transport. a. Importance of iron transport and iron responsive genes. The low iron environment found in human serum, brain and cerebral spinal fluid may provide a nutritional cue for the induction of key virulence factors (e.g. capsule formation) in C. neoformans. Low iron conditions are not only important in relation to the elaboration of the polysaccharide capsule (Casadevall and Perfect, 1998), but iron is also an essential cofactor for the survival of virtually all organisms. Therefore, pathogens possess specific mechanisms that allow survival in the mammalian host environment including the ability to acquire iron that is tightly bound by host proteins.  b. Iron Acquisition. The quest for iron by organisms is a complex relationship involving complicated iron chemistry, limited bioavailability, iron cytotoxicity and fierce competition for this valuable nutrient. Iron by its chemical nature is more predominant as the ferric (Fe3+) form. That is, in air, the ferrous (Fe2+) form will spontaneously oxidize to the Fe3+ form. In aqueous solution Fe3+ is essentially insoluble at neutral pH and thus not bioavailable. Fe2+ on the other hand is relatively soluble at neutral pH.  Fe3+ binds to its ligands more tightly than Fe2+, partly due to  charge difference, which makes it more difficult to exchange with an exogenous ligand because it has a larger kinetic barrier to overcome (Wilkins, 1991).  This makes Fe3+ relatively  inaccessible to organisms in the absence of sophisticated iron acquisition mechanisms. The other consideration is that ferrous (Fe2+) iron is highly cytotoxic, and can actively participate in the formation of damaging superoxide and hydroxyl radicals. So, although iron is essential for the survival of virtually all organisms, iron acquisition and storage must be tightly regulated. Organisms have developed strategies to reduce Fe3+ to Fe2+ with plasma membrane reductases, but also must limit the level of Fe2+, sequester ferric iron from competitors through chelation systems, and regulate iron levels by utilization of high and low affinity uptake systems (Kosman, 2003).  c. Reductive fungal iron transport systems. In fungi, the most developed understanding of fungal iron transport and accumulation is in the model organism S. cerevisiae. In this yeast, reductive iron acquisition occurs through the  20  cooperation of iron permeases, reductases and oxidases in both high and low affinity uptake systems as well as non-reductive uptake through siderophore facilitators. Genes involved in iron uptake in S. cerevisiae are summarized in a review by Kosman in 2003. The ferric to ferrous uptake system is dependent on extracellular reduction of Fe3+ by ferric reductases and there are seven reductases in S. cerevisiae (FREJ- 7).  Internalization of the iron can occur by direct  ferrous uptake through divalent metal iron transporters Smfl -3p (Pinner et a!., 1997; Chen et al., 1999; Cohen et al., 2000). Another direct uptake system is through Fet4p, a low affinity process. Indirect ferrous uptake can occur though a collaboration of Ftrlp, a high affinity iron permease and Fet3p, a multicopper oxidase. Thus, this system is dependent of ferric to ferrous to ferric redox cycling prior to uptake, a process that is not well understood (Kosman, 2003).  The  advantage of the latter system is its high affinity, the KM value for Fet3p/Ftrlp uptake is 0.2 1 iM (Dancis et a!., 1992) whereas the KM for Smflp and Fet4p transport of ferrous iron is 2 tM (Chen et a!., 1999) and 35 tM (Dix et a!., 1994:1997) respectively.  In conditions where  bioavailable iron is abundant, the high affinity uptake system can be down regulated and iron will be transported by the low affinity systems (Dancis, 1998; Leong and Winkelmann, 1998; Nelson, 1999; Radisky and Kaplan, 1999).  d. Reductive iron transport mechanisms in C. neoformans. A number of iron transport systems have been identified in C. neoformans. Similar to S. cerevisiae, these include both high and low affinity uptake systems (Nyhus and Jacobson, 1999, Lian et a!., 2005). The high affinity uptake system is comprised of a high affinity iron permease, Ftrlp and the multicopper oxidase, Fet3p and is found on Chromosome 12 in the serotype D strain JEC2 1. Jacobson et a!., (1998), showed biochemically that there is a low-affinity iron uptake system in C. neoformans. Wonhee Jung in the Kronstad Laboratory has found a second cluster of genes for a putative iron permease and an oxidase on chromosome three of the strain JEC2 1 and these are strong candidates for a low affinity uptake system. He is presently characterizing this uptake system along with a putative iron responsive regulator protein, CIR] (personal communication). He has found by Northern analysis that the high affinity uptake system on chromosome 12 is iron dependent and a target of Cirip, but the putative low affinity permease is independent of iron and not a direct target of Cir ip. He also identified three putative homologs of ferric reductases in C. neoformans, denoted by the TIGR identifiers (163.m03777,  21  163.m06361 and 177.m02865) Unlike S. cerevisiae, C. neoformans does not appear to have the ability to undergo reductive iron uptake from siderophores, as it does not possess the FIT genes necessary to reduce the tightly bound Fe3+ from siderophores (Protchenko et al., 2001).  e. Siderophores and siderophore transport. Recently, the transcript for a homolog of a siderophore transporter (SIT]) was identified by SAGE (Lian et a!., 2005) as upregulated in low iron conditions. Siderophores are small molecules that bind ferric iron with high affinity and the resulting complexes can then be transported into cells. SIT] therefore may be necessary for the utilization of siderophore bound Fe3+. The use of siderophores for the acquisition of iron is common for both fungi and bacteria (Barasch and Mori, 2004) and siderophores allow pathogens to steal tightly bound iron from human proteins such as transferrin or ferritin or from nutritional competitors in their environment. Enzymes necessary for siderophore synthesis but not uptake include an ornithine -monooxygenase and a non-ribosomal peptide synthase (Haas, 2003). Specific transporters 5 N are required for non-reductive uptake of ferric-siderophore complexes. Interestingly, some organisms such as C. neoformans, Candida albicans and S. cerevisiae have homologs for siderophore transporters even though they do not appear to possess the enzymes necessary for the synthesis of these molecules (Hans, 2003).  This may allow utilization of siderophores  produced by competitors for efficient iron acquisition. In S. cerevisiae there are four siderophore transporters ARN], ARN2/TAF], ARN3/SIT] and ARN4/ENB] (Lesuisse et al., 1998 and 2001; Haas, 2003). C. neoformans has many putative siderophore transporter homologs (strain dependent) including SIT], which is a homolog of S. cerevisiae ARN3/SIT] and C. albicans ARN1/SITA. Siderophore transporters are part of the major facilitator superfamily (MFS) of secondary transporters (Lesuisse et al., 1998; Kim et a!., 2005).  The CnSitlp homolog  ScArn3p/ Sitip has a high affinity for the hydroxamate siderophore ferrioxamine B (FOB; feroxamine) and lesser affmity for ferrichromes (FCH5) and ferricrocin (FC) (Lesuisse et a!., 2001).  22  E.  Melanin Production. a. Melanin synthesis and placement. There are two major types of melanin in fungi: Dihydroxynapthalene (DHN) melanin is synthesized through the polyketide synthase pathway and DOPA melanin is synthesized through phenol oxidase enzymes such as laccases and tyrosinases (Langfelder et a!., 2003).  C.  neoformans produces DOPA melanin through laccase enzymes. As mentioned above, two such  enzymes are present in the fungus encoded by LAC] and LAC2 with the former being the dominant enzyme responsible for the synthesis of melanin under glucose-starved conditions (Zhu and Williamson, 2003). Laccase can oxidize a wide range of substrates including ortho- and para diphenols, aminophenols, diaminobenzenes and catecholamines, notably 3-4, dihydroxyphenyl alanine (DOPA) and dopamine (Williamson, 1997). The reaction catalyzed by laccase involves the oxidation of the diphenolic compound e.g. DOPA to a highly reactive intermediate, dopaquinone (DQ).  Spontaneous autooxidation reactions then take place to form  leucodopachrome, then dopachrome (DC). This intermediate non—enzymatically decarboxylates to form 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole carboxylic acid (DHCI), which then undergoes first a two electron oxidation to indole-5,6-quinones. The final step of synthesis is slow and involves the polymerization to melanochrome and then to melanin (Williamson, 1997). Melanin has been implicated in virulence in C. neoformans due to its antioxidant capabilities that likely protect the fungus against reactive nitrogen and oxygen species produced by host effector cells. Since laccase can oxidize neurologic catecholamines such as DOPA, dopamine, norepinephrine and epinephrine, this has been suggested to partly explain its predilection for colonization of the brain (Casadevall and Perfect, 1998). Melanization is in fact required for extrapulmonary dissemination to the brain (Noverr et al., 2004).  The microstructure of the  cryptococcal cell wall was recently reported (Eisenman et al., 2005) where it was shown that melanin is an integral part of the cell wall structure. The report suggests that melanin is placed in the cell walls in concentric layers of spherical granular particles of about 40-130 nm in diameter. The incorporation of melanin into the cell walls lends a contribution to rigidity and may further contribute to the survival of the fungus in the human host.  23  b. Redox interactions of iron and melanin. Aside from laccase oxidizing a wide range of diphenolic compounds, it also carries iron oxidase activity (Williamson, 1997).  Melanin has the ability to reduce or oxidize iron. These  characteristics set the stage for possible redox interactions between melanin, laccase and iron and may have implications in the already complex mechanisms involved in iron acquisition. Melanin and its redox buffering capability is responsible for neutralizing antioxidants released by host effector cells. Studies have suggested that electrons exported by the yeast to form extracellular Fe(II) maintain the reducing capacity of the extracellular redox buffer (Jacobson and Hong, 1997).  F.  Rationale and Aims of this Study. The onset of the genomic era and the advent of computer technology have exponentially increased the efficiency and scope possible in genetic research. The work described in this thesis involved the identification and characterization of iron regulated genes in C. neoformans, through the development of genomic resources and functional biological studies. The first objective was to contribute to the construction of physical maps for a serotype A and a serotype D strain. The first portion of this work involved the hybridization of 125 markers to set of 9,216 BACs from the JEC21 library and 6,528 BACs from the 1199 library on a highdensity filter.  First, a set of 96 overgo (Ross et al., 1999; Materials and Methods) probes  (4Omers) was employed in a pooled format to rapidly match genes and ESTs with contigs. Second, overgo probes to BAC end sequences were employed to fill in missing data in the crossreference analysis of the contigs.  Finally, overgo and plasmid-derived probes for specific  markers linked to the electrophoretic karyotype (Spitzer and Spitzer, 1997). This data was integrated into the fingerprint maps and was key in the determination of many joins in the maps, provided the first genome-wide comparison of gene synteny between two strains of the fungus, and linked contigs to specific karyotype bands. The physical maps for two serotypes, A and D also provided structural genomic information that was key in the assembly of the total genomic sequence for serotype D at the Stanford Genome Technology Center (SGTC) and The Institute for Genomic Research (TIGR) and serotype A at the Broad Institute of Harvard and Massachusetts Institute of Technology (MIT). The robust assemblies provided the resources necessary to annotate SAGE data.  24  The second objective was to analyze the low iron transcriptome of the serotype A strain, H99. This portion of the work involved the analysis of the low iron transcriptome for the serotype A strain, H99 using SAGE. Low iron conditions are important in relation to the elaboration of the polysaccharide capsule. Iron is also an essential cofactor for the survival of virtually all organisms and pathogens must possess mechanisms to acquire iron when low free iron is available as is the case in the mammalian host environment. This work was specifically focused on iron regulated genes and mechanisms of iron regulation and uptake. A SAGE library was constructed from mRNA isolated from cells grown in low iron medium (LIM) using the MicroSAGE protocol (Datson et al., 1999). 19,278 high quality tags were obtained. The 100 most abundant tags were annotated, in addition to the 100 more and less abundant tags in comparison to three other libraries (700 tags total): 1) from cells grown at 25°C on yeast nutrient broth (YNB); 2) from cells grown at 37°C in YNB (Steen et al., 2002); 3) and from cells isolated from rabbit cerebral spinal fluid (CSF) (Steen et a!., 2003), A number of interesting genes were identified in the transcriptome analysis including those involved with the response to stress and iron uptake. A key finding was that the low iron transcriptome was remarkably similar to the in vivo library from cells grown in rabbit CSF and significantly distinct from the libraries grown in YNB. The functional characterization of the low iron transcriptome in the strain B3 501 A led to the identification of a key gene necessary for iron uptake. The third objective was to characterize the gene SIT] with respect to iron uptake and cellular function in C. neoformans. Thus, this work focused on the gene SIT], which encodes a putative siderophore transporter. The gene was characterized through the construction of null mutants in three strain backgrounds. Serotype D strains B3501A and JEC21 and the serotype A strain, H99.  The SIT] transcript was found to be upregulated in low iron vs. iron replete  conditions. These data were concordant with findings from previous SAGE analyses (Lian et a!., 2005). Deletion of SIT] led to pleiotrophic phenotypes involving melanization and cell wall structure in the serotype D strains and defects in iron uptake for all strains. This work showed that SIT] is required for the use of siderophore bound iron in all strains, for growth in the low iron environment (for strain B35O1A and H99), for melanization in both serotype D strains (JEC2 1 and B3 501 A), and for proper cell wall organization in all strains. It was shown that the cAMP pathway affects siderophore utilization in serotype A but not D.  The SIT] mutants have  increased tolerance to the iron-dependent drug phleomycin and to amphotericin B in the JEC21  25  background. Finally, this work showed that SIT] is not required for virulence in the murine model in serotype A. In summary, the work described in this thesis contributed to the development of robust genomic resources, provided information about gene transcription under an important nutritional cue and further characterized a key gene involved in iron utilization. In addition, significant differences were noted in the phenotypes between strains of two different serotypes, A and D as well as two strains within the same serotype, D. The accumulated information provides lasting resources for the Cryptococcus scientific community, reinforces the importance of the low iron nutritional cue, furthers our understanding of iron regulated genes and iron acquisition and provides new insights about genomic and biological differences between strains of this important human pathogen.  26  CHAPTER TWO: Physical Mapping of the Genomes of Serotype A and D Strains of C. neoformans INTRODUCTION The construction of physical maps was initiated for two C. neoformans genomes as part of an international effort to obtain the complete genomic sequences for strains representing the A and D serotypes. In this chapter, hybridization experiments are presented that place markers on the maps for both serotypes. These experiments contributed significant value to the maps and provided the first genome-wide comparison of gene synteny for two strains of the fungus. Further, hybridization experiments allowed a correlation of specific contigs to electrophoretic karyotypes of C. neoformans. Molecular typing has documented numerous variations between the serotype A and serotype D genomes (Meyer et al., 1993; 1999; 2003; Kidd et al., 2005). The C. neoformans physical maps further investigated the variation by comparison of the two serotypes to identify regions of polymorphism. Polymorphic regions are of interest because serotype A strains are more prevalent clinically and generally more virulent than the serotype D strains. Regions of difference in the genomes may contain virulence factors. The construction of the maps was performed at the MSGSC. The map construction and analysis was comprised of multiple steps including: 1) BAC clone fingerprinting; 2) contig size estimation; 3) BAC end sequencing; 4) BAC end sequence alignment to TIGR shotgun sequence; 5) hybridization of probes to BAC clones and integration of markers to maps through the FPC program; 6) comparison of gene synteny; and 7) analysis of the relationship between electrophoretically separated chromosomes and the contigs of the JEC2 1 and H99 maps. The first four steps were not directly part of this thesis work but will be summarized as they relate to the final three steps (5-7) that will be detailed in these results. The maps were published in Genome Research in 2002 (Schein et al., 2002). Note that J. Schein and K. Tangen contributed equally to this publication and were listed as co-first authors. C. neoformans BAC libraries were prepared at ResGen (Huntsville, AL) in the BAC vector pBe1oBAC11 (Wang et al., 1997).  For the BAC clones that were fingerprinted, the  average insert size was reported to be 114.54 kb for the H99 library and 110.74 kb for the JEC21 library (based on a sample of clones). High throughput, agarose gel-based BAC fingerprinting, fingerprint map assembly and manual editing was performed as described previously (Marra et  27  al., 1997; Marra et al., 1999; McPherson et al., 2001; Schein et al., 2004) with the exception that restriction fragment identification, fragment mobility and size determination was performed using recently developed automated analysis software (Furhmann et al., 2003). For each library, a total of 3,072 bacterial clone glycerol stocks arrayed randomly into eight 384-well plates were processed for fingerprint map construction. Each BAC clone was fingerprinted to determine the number and size of Hindill restriction fragments contained in the insert.  Fingerprints were  successfully obtained for 2,642 JEC21 clones and 2,612 H99 clones. The average insert size for fingerprinted clones in the JEC21 library was 108,560 bp and 107,648 bp for the H99 library, as determined by the fingerprint analysis. A fingerprint database for each library was created and analyzed using the program FPC (Soderlund et a?., http://www.genome.clemson.edulfpc/).  1997, 2000; Ness et a!. 2001;  A high-stringency automated assembly was first  performed in FPC to bin together clones with substantial overlap based on shared restriction fragments. In order to maximize the likelihood that each bin represented a region of contiguous DNA, or “contig”, a minimum of 85 to be binned together.  -  90% shared restriction fragments was required for clones  The automated fingerprint assembly resulted in the creation of 276  contigs in the JEC21 database and 261 contigs in the H99 database. Additional contig integrity was achieved by manual interrogation and editing of each contig via tools within the FPC software program, using fingerprint similarities to refine clone order and clone overlaps. Clones with fingerprints that appeared to be contaminated (comprised of DNA from more than one clone) or partially digested clones were removed from the database during the editing process. Following the refined positioning of clones within all contigs, clones at the ends of each contig were compared against all other clones within the FPC database at a reduced minimum required fingerprint overlap (approximately 50% shared restriction fragments) to identify potential joins between contigs. Potential joins between contig ends were manually examined and permitted only where the joins did not result in inconsistencies in the fingerprint data. Upon completion of these manual edits, the JEC21 map contained 2,322 clones, the H99 map contained 2,529 clones, and each map had been assembled into 20 sequence-ready contigs. The availability of fingerprinted BAC data from two closely related strains allowed a test of whether contigs could be assembled with orthologous clones from each genome. Specifically, FPC was used to analyze the combined set of fingerprints from both strains. No contigs could be generated that were composed of clones from both strains. Thus, the genomes of the serotype A  28  strain H99 and the serotype D strain JEC2 1 are sufficiently divergent to preclude analysis of synteny based on HindlIl restriction digestion patterns. Finally, BAC clones comprising a minimally overlapping tiling set were manually selected for each contig in both databases. Great care was taken to ensure that shared restriction fragments could be identified in the fingerprints of overlapping clone pairs. The selected tiling path clones represent a collection of overlapping clones covering the genomes of JEC2 1 (165 tiling path clones) and H99 (163 tiling path clones). These tiling sets will therefore be useful for assembling and finishing the genomic sequences of these strains. An automated algorithm was used to compare the restriction fragments of overlapping clone pairs in the tiling clone sets selected for each contig. The unique fragments for each tiling path clone were identified and their sizes summed to estimate the overall size of the contigs. Specifically, the algorithm performed the following for each contig: 1) to the total contig size, added the sizes of all the fragments in the left-most tiling path clone in the contig; 2) identified from the next left-most tiling clone in the contig all unique fragments (not shared with the previous clone) and added them to the total contig size; and 3) repeat (2) until all tiling clones in the contig have been processed. Shared fragments were as defined by the FPC parameters such that two fragments are considered the same if their calculated mobilities are within 7 mobility units of each other. For the JEC21 map, the contigs range in size from 184,760 bp (6 clones) to 1,748,127 bp (321 clones). For H99, the smallest contig (84,272 bp) contains two clones and the largest of 1,356,533 bp contains 246 clones. Correlation of shotgun sequence data with fingerprinted BAC clones allowed the contigs to provide a framework to assemble the existing shotgun sequence data for both strains. In order to facilitate the alignment of the physical maps with the emerging genomic sequence data, the ends of the fingerprinted BAC clones were sequenced and the traces were contributed to the shotgun sequence databases for both strains. The genomes for two serotype D strains, JEC2 1 and B3501A of C. neoformans have now been completed and published (Loftus et a?., 2005) and the serotype A strain, H99 is near completion at the Broad Institute of Harvard and MIT. The physical maps were a significant contribution to these efforts. BAC end sequence reactions were performed for both ends of all 3,072 clones from each fingerprinted BAC library, for a total of 6,144 BAC attempted BAC end reads per strain. A total  29  of 4,772 (78%) successful BAC end sequences were obtained for JEC21 BACs with an average read length of 540 bp. Of the successful reads, 4,186 were derived from clones that had fingerprints in the map. Of the fingerprinted JEC21 BAC clones with successful BAC end sequences, 1,939 had both ends represented in the dataset (3,878 total end reads, or 93%) and 308 clones had a single associated end read. For H99 clones, 4,908 (80%) successful BAC end sequences were obtained with an average read length of 560 bp. Of these successful reads, 4,390 were derived from clones that had fingerprints in the H99 map. For the fingerprinted H99 BACs with end sequences, 1,957 had sequence represented from both ends (3,914 total reads, or 89%) and 476 had a single associated end read. Since the maps were produced prior to a finished genome, JEC2 1 BAC end sequences derived from mapped BACs were correlated with the JEC2 I whole genome shotgun sequence assembly generated at TIGR, representing nominally 3.5-fold coverage of the C. neoformans genome and including the BAC end sequence data.  Each of the BAC end sequences was  compared against the complete set of genomic sequence assembly contigs using the BLAST algorithm (Altschul et al., 1990). Using this methodology a total of 7,643,886 bases of TIGR shotgun sequence were unambiguously correlated with the fingerprint contigs, or 48% coverage of the physical map. Finally, the hybridization experiments provided an important contribution to the use of the physical maps of JEC21 and H99 in the comparison of gene synteny between strains, the initial placement of markers on genomic assemblies of total sequence and the preliminary assignment of specific contigs to electrophoretically separated chromosomes. The results for these experiments and their contribution to the maps are detailed below.  30  MATERIALS AND METHODS A. Filter Preparation and Layout. High Density filters containing C. neoformans BAC clones were purchased from ResGen (Huntsville, AL). The organization of the clones on the filters can be found in Appendix TIc.  B. Probe Design. The Overgo method, as developed by Dr. J. D. McPherson (Ross et al., 1999; Volirath et al., 1999), and Overgo Maker (http://www.genome.wustl.edu/gsc/overgo/overgo.html) were used to design 123 probes for hybridization to the fingerprinted BAC clones. The sequences for the hybridization  probes  originated  from  known  C.  neoformans  genes  in  GenBank  (www.ncbi.nlm.nih.gov), putative genes identified in the JEC21 genomic database at the SGTC; http://baggage.stanford.edulgroup/C.neoformans/),  expressed  sequence  tags  (EST5;  http://www. genome.ou.edulcneo.html), karyotype markers (Spitzer and Spitzer, 1997), and BAC end sequences (http://www.bcgsc.bc.ca). redundancy  by  searching  The 40mer overgo probes were checked for  against  (http://baggage. stanford.edulgroup/C.neoformans/)  the and  JEC21 the  genomic H99  EST  database database  (http://www.genome.ou.edu/cneo.html) with the BLASTn algorithm. The pooled probes were purchased in a 96 well format (2 x 96 well plates of 24mers) from Life Sciences, GIBCO-BRL (now Invitrogen, Inc.). The 25 additional overgo probes (2 x 25-24mers) for 26S rDNA, BAC clone end sequences and karyotype specific markers were purchased from the Nucleic Acid and Protein Service Facility (NAPS) in The Michael Smith Laboratories (formerly the Biotechnology Laboratory), University of British Columbia. Sequences for the PKA2 and HIS3 genes were on a 4.5 kb fragment (PK42; pCD49) and a 604 bp cDNA in a TOPO TA vector (HIS3; pMJB54) supplied by Dr. Joseph Heitman from Duke University. Probe sequences can be found in Appendix ha.  C. Hybridization Protocol. The Overgo protocol was used for hybridization (Ross et al., 1999) except that free nucleotides were removed with a nucleotide removal kit (Qiagen) and filter washes were performed in 50 mL of 4 X SSC/0.1 % SDS, 1.5 X SSC/0.1% SDS and 0.75%SSC/0.1% SDS at 55 °C. Filters were exposed to film for three days at —80°C.  31  The plasmid-derived DNA fragments were labeled with an Oligonucleotide Labeling Kit (Amersham Pharmacia Biotech., Inc., Piscataway, NJ). The positive control was labeled as per the Research Genetics High Density Filter manual by a Random Priming Reaction. A flowchart of the hybridization strategy for pooled probes can be found in Appendix lib.  D. Data Collection and Analysis. All data was organized and processed in Microsoft Excel workbooks. Autoradiographs were read by eye with a 384 well grid. Data from the autoradiographs was interpreted according to the Research Genetics, Inc. manual. Each BAC clone was spotted twice on the filter. The positive clones were identified by a unique spotting pattern and field location. Filter layout and spotting patterns can be found in Appendix TIc. Results for probes were manually deconvoluted, and compared for each column and row. Clones found at the intersection of a column and row were assigned to the probe at that address. Ambiguous clones which had an anchor point; 1 columnl>1 row or >1 columnll row were resolved manually. Results for all positive BAC clones to individual probes can be found in Appendix lid.  E. Data Integration. Integration of markers into physical maps was performed by the mapping group at the Michael Smith Genome Sciences Center (MSGSC) (Formerly the British Columbia Genome Sciences Center).  F. Southern Analysis of HindilI Digested DNA and CHEF Gel Separated Chromosomes. Southern blot preparation and hybridization was performed as per Sambrook et al., (1989). Genomic DNA was digested for 4 hours at 37°C with HindIII, separated on a 1% agarose gel and transferred to a nitrocellulose membrane overnight in 20X SSC. The CHEF blot was provided by Dr. Joseph Heitman. The 26S rDNA probe was labeled with P 32 using the overgo protocol (Ross et al., 1999) as in section C. Hybridization Protocol-above. The 26S overgo probe (Appendix ha) was used for hybridization to the HindlII and CHEF blots. The HJS3, plasmid derived DNA fragment (pMJB54) was labeled with an oligonucleotide labeling kit (Amersham Pharmacia Biotech., Inc., Piscataway, NJ) for hybridization to the CHEF blot.  32  _________  ____________  _____  RESULTS A. Summary of Hybridization Experiments. Hybridization experiments were performed to place markers for known genes, ESTs and BAC ends onto the maps to identif’ corresponding contigs between the maps and to examine the conservation of synteny between the strains. Hybridization data from probes derived from BAC end sequences were used as additional evidence for the identification and evaluation of potential contig merges. A summary of the marker data from the hybridization experiments is presented in Table 1. Three sets of probes were used to identify hybridizing clones arrayed as a set of 9,216 BACs from the JEC21 library and 6,528 BACs from the H99 library on a high-density filter. An example of an autoradiograph hybridized to a row pooi of 12 probes can be found in Figure 1. First, a set of 96 overgo (Ross et a!., 1999; Materials and Methods) probes (40mers) was employed in a pooled format to rapidly match genes and ESTs with contigs. Second, overgo probes to BAC end sequences were employed to fill in missing data in the cross-reference analysis of the contigs. Finally, overgo and plasmid-derived probes for specific markers linked to the electrophoretic karyotype (Spitzer and Spitzer, 1997) were also employed in an attempt to match contigs with specific chromosome-sized bands.  Note that more BAC clones were  available on the high-density filter than were fingerprinted. Overall, 82 and 102 markers were placed on contigs for JEC21 and H99, respectively. Results for individual probes can be found in Table 2.  For all markers, a total of 1603 JEC21 and 1572 positive BAC clones were  identified. Probes gave a more successful hybridization result to the BAC clones from the strain whose sequence was used to design the probe. Table 1: Summary of the hybridization of selected markers to BAC clones of JEC2 1 and H99.  Probe Origin  Hybridization format  Genes/ESTs  Strain  Probes successful/attempted  Positive BAC  Average BAC clones/probe  Total Positive  Pooled  JEC21 H99  79/96 88/96  1175 1307  14.9 14.9  56 76  BAC Ends  Individual  JEC21 H99  14/15 15/15  351 428  23.4 28.5  13 15  Karyotype markers  Individual  JEC21 H99  14/14 11/14  505 280  36.1 20  13 11  Probes were hybridized to a total of 9216 JEC2 1 and 6528 H99 BAC clones. Pooled probes were employed in the Overgo format described by Ross et al. (1999). Unsuccessful probes failed to hybridize or gave ambiguous results.  33  CD CD C) CD  C.) C  + CD CD C) CD  Rows A-P Figure 1: Example of an autoradiograph showing the results of a row pool of 12 overgo probes hybridized to a high-density BAC clone filter. Multiple 384 well plates are spotted in each field 1-6. Each BAC clone is spotted twice per well. A unique spotting pattern (Appendix Tic) indicates the originating plate number for the positive clone. The filter orientation is determined by the positive control probe that hybridizes to the filter edges. Identifying marks include the reference marks on two corners where one is absent in the top left corner and an extra mark is present on the bottom right corner. Library identification marks are found on the right vertical edge of the filter. A 384-well grid is placed over each field and the originating 384 well microtitre plates for each positive clone are manually recorded in Excel workbooks. 34  Table 2: Summary of hybridization experiments for individual probes.  8 BAd Clones Hybridioed Name  Gene Fonetion  CPA2  CYCLOPI-IILIN A REGULATOR OP TRANSLATION (CAMP rag.)  CPAI  CYCLOPHILIN A REGULATOR OF TRANSLATION (cAMP rcg.)  PLB1  JEC2I  1-199 3  Strain Origin —  H99  26  3  SECRETED PHOSPHOLIPASE B  6  8  1199  CBPI  CALCINEURIN-BINDING PROTEIN  3  3  H99  L1AI partial  P450 LANOSTEROL 1-4 ALPHA DEMETHYLASE  CAPS9  SEROTYPE AID CAPSULE GENE  0  ERG1I  LANOSTEROL -4 ALPHA DEMETHYLASE  23  CNAI  CALCINEURIN A CATALYTIC SUBUNIT  o  UREI  UREASE  5  20  MAT ALPHA  PHEROMONE PRECURSOR  0  0  PMAI  PLASMA MEMBRANE H (+) ATPase  25  Ii  ATCC63S2  STE12 ALPHA  STERILE 12 KINASE  2  0  H99  GPBI  0-PROTEIN BETA SUBUNIT  0  19  H99  MP GENE GRAS  MATING RELATED OROTATEPHOSPHORIBOSYLTRANSFERASEIOROTIDINE MONOPHOSPHATE PYROPI-IOSPHORYLASE  29  26  STE2O  STERILE 20 ALPHA KINASE  0  1  1199  RASI  SMALL GTP BINDING PROTEIN  0  5  H99  CAP 10  CAPSULE ASSOCIATED PROTEIN  37  17  04500  RHOI mRNA  RHO FACTOR  0  0  B3501  LAd  DIPHENOL DXI DASE (LACCASE)  24  6  010  OPAl  0-PROTEIN ALPHA SUBUNIT  ADE2  PHOSHORIBOSYL AMINOIMIDAZOLE CARBOXYLASE  GPD  GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE  22  TORi  PHOSPHATIDYL INOSITOL-3-KINASE  PRRI  PKBPI2 MACROLIDE BINDING PROTEIN  TOPOI  TOPOISOMERASE  ‘PC’  INOSITOL PHOSPHORYLA CERAMIDE SYNTHASE  H99  FICSI  GL[JCAN SYNTHASE  1-199  MDRI  MULTI DRUG RESISTANCE PROTEIN  MI-106  ACTIN  ACTIN GENE  1199  THY SYN  THYMIDYLATE SYNTHASE  B3501  CAP6O  CAPSULE ASSOCIATED PROTEIN  B4500  CELl  CELl PROTEIN  B4500  TEFI  TRANSLATION ELONGATION FACTOR I-ALPHA  CHSI  CHITIN SYNTHASE  MAN OH  MANNITOL DEHYDROGENASE  9  PREI  PROTEASOME SUBUNIT  2  CAP64  CAPSULE ASSOCIATED PROTEIN  16  GAL7  UDP-GLIJCOSE-D-GALACTOSE-I-PHOSPHATE URIDYLTRANSFERASE  IJBIQ  UBIQUITIN CARBOXY EXTENSION PROTEIN  H99  ‘3 CBS 132 Ii  WSA-21 JEC2I  CBS 804 H99  JEC2I B350I 33  B350I  0  0  B3501  9  33  H99  19  20  1199  M 1-106 19  41 13  35  H99  ATCC 6352  9 BAC Cones Hybridiaed Name  Gene Fonclioo  CNIJB14  JEC2I  HOE  Stroio Origin  POLYUBIQUITIN  2  15  ATCC 6352  TRPI  PI4OSPHORIEOSYLANTHRANILATE ISOMERASE  0  0  HIS3  IMIDAZOLE GLYCEROL PHOSPHATE OEHYRATASE  9  CNRE-I  REPETITIVE DNA ELEMENT  E  I  NMT  N-MYRISTOYLTRANSFERASE EXONS I-I I  10  3  L2 10425  ARP  AOP-RIEOSYLATION FACTOR EXONS 1-7  4  5  L210425  TRP2  TRANSLATION ELONGATION FACTOR 2  10  22  IPI  ANTIPHAOOCYTIC PROTEIN  26  35  MPDI  MANNFrOL- I-PHOSPHATE DEHYOROOENASE  24  19  D11AI  DELAYED TYPE SENSITIVITY ANTIGEN  31  36  RHOI  RHO FACTOR  0  0  PIJKCI  PUTATIVE URCI KINASE  12  10  JEC2I (U moydis homolog(  17  JEC2I (U erojeh.s homalagi  PASAI  PUTATIVE ASPARTATE SEMI-ALDEHYDE DEHYDROOENASE  26  PHSPIO  PUTATIVE HEAT SHOCK PROTEIN Spornhe  21  0  JEC2 I (S pornbs’ komoIog(  PHSP78  PUTATIVE HEAT SHOCK PROTEIN Sposihe  22  II  JEC2 I (S pen;ho homalag)  PHSP9O  PUTATIVE HEAT SHOCK PROTEIN 0. foo;penico  10  I  PHSPI04  PUTATIVE HEAT SHOCK PROTEIN Secrevisiac Hsp78  7  12  JEC2I (0. /ao;po ice hoosolog) 3EC21 (S corey/nec Hop70 hossolog)  PATPI  PUTATIVE TRANSCRIPTION FACTORS pee;hc  7  I  JEC2I (S porohe hornolog)  PMTS1  PUTATIVE RIBOSOMAL PROTEIN LO  2  0  JEC2I (S penihc homalog)  PPVPI  PUTATIVE PROTEIN TYROSINE KINASE N. crane  4  2  IEC2I (N. eresse homolog)  PPYP2  PUTATIVE PROTEIN TYROSINE KINASE N. crone  4  alaOOen.fl olo0Scn.fl  RET giil197059  (201351)  ribosomal proleio  L3 -I 360 3.Ie-33  ESTgoIIPIDIe2O6572 (Y00841) core prolcia II -3 166 4 9c-I I  olalOco.fl  EST bbs1575 17  olallee.rI  EST goIIPIDIcII9O443  (557516) macil phosphorihosyl Icansfcrasc -2 311 4.Ia-27  albO4cnsl  iIII97O59 9 EST  nlIsO7cn.rl  eIIPIDIdIOI4552 0 EST  alc0lcn.1l  EST goIIPIDJoI23I300  alcO2cn.ft  i(2029925 9 EST  slcO3cn.fl  0IIPIDIe330964 9 EST  (A200I342( Polstioc S-phase-specific ptoleio +3 359 IKe-OS (201351) ribosossal proleio L3 +1 650 4.Ic-65  4  JEC2I (N. crease homolog)  3  I  H99  7  9  H99  II  6  H99  12  32  H99  6  4  H99  (D89194( similar to Ral ATP cilralo [(aso -3 224 6.4e-I0  2  13  H99  (AJ003I97(adooiso oacloo6dc traoslocasa -t  207 I.3e-24  10  12  H99  (AC002291) Sitoilarso Dns.I (heal shock protois) +2 206 I.Oe-15  32  17  H99  (Z90762) [sIts acid ovolkelase alpha sabosil -I 206 2.3o-l4  12  9  H99  4  11  H99  sle04en.rl  EST 9 i13420603  alcO6en.fI  EST gel(PIDI0I29I65I  (AL023290) kypolkelical pmlcm ((ABC Irsosporter) 4-2 327 3.Sc-20  0  IS  HOE  alc09en.rI  oIIPIDIcII327O7 (Z99I63( kypolhclisal prose (L-aIlo-tkrceeieealdolasc(. +1 260 I (10-21 0 EST  6  21  H99  alctlcnSf  EST geIIPIDI4IOI4I33 (0077351 ekosemal protelo LI4 -I 260’0.Oe-22  (AP075709( LsFA (Psoodomooss polida) (rokydrie) +1  343 I Se-30  4  25  H99  oilS 120243 (AP004672( ribosamal pmseie L41 -I 523 l.3e-49  I  4  HOE  ofdOKcn.fl  ESTgiI2I49ISO (U02536( faltyacidamide kydmlase+I 211 ISo-IS  I  0  H99  ald08rn.rI  t(2099797 (U9b695( deosyoridiocsripkasphatasc -3 349 3.6r-31 0 EST  0  II  H99  nldO9cn.fl  EST gol(PID(4I0I45I7 (009150) similarlo Saccharamycco cer... -3 113 l.4c-I6 2  2  II  H99  sIdlfcnSl  ESTgoIIPIDIo2634O9 (Z79602( K09E9 2 (Cacoadsabdilis elogaos( -I 227 7 4e-I8  22  23  H99  oleOlcn.rI  i1054566 (X07371) cmkamyl phosphale oyolhmc -2 330 I.9e-27 9 EST  9  3  H99  0  12  H99  12  22  H99  sldK0cn.fl  EST  aleO4rn.ff  ESTgoIIPIDIe334O27 (Z90529) Imoscriplico ieiñalios fhclor lIE (TPIIE( +2  ole06cn.ff  0IIPIDI4IO32200 (AB00077O(callalasc +3 122 3.3o-06 9 EST  171 9.4c-I2  36  8 BAC Clones Hybridleed Name  Gene Fnnctiun  nlfOlcn.fI  ESTgil I 841864 (1)871011) nucleic acid binding protein (gtycmc-ricls) -3 ItO 2.Se-05  1199  E5Tgij2668565 (1)81804) translation elongation factor-I 663 I .7o-64  1199  alfO2cn.fI ulIO7cnSl  EST  JEC21  gi11330376)U58758) ZKI 127.5 (Cuenodsabditis elecansl -3  59 I Sc-IC  algO2ca.fl  ESTgnIIPIDIcI249769 (Z99163) hypothetical protein (turnorsoppressor) +1  algO3cn.rI  iEST 9 I2088722 )AF003 139) strong siosiladey to 60S ribosomal protein -l  algO4cn.fI  EST bbs1t74324  pepninngeoA +3  373 9.00-34  56 4.4e-I0  ESTciItO4O756 (X79206) ptd e20572t IS pombol +3 531 l.6e-50  algO8cnSI  EST gi1832919 (X7t664) 0141-11 [Saccharomyccs core... -l 214 6.6e-17  ulgllcn.fl ulhOlcn.fl nlbOlcn.fI nllsl2cn.fl 185 rDNA (intergenic)  EST n 0 IIPIDIeI 173375  (Y13942) OTN Reductese A turoefaciens] -3  EST gn11P1D1e1285364  (AL022299) potatiyc cytoclsrome ct -3 330 3.7e-29  14  1199  15  27  N99  0  0  1-199  0  17  1-199  24  13  1199  0  1199  3  6  1199  )A0004530) prorcirr arginine N-ruothyl transferaso. -2 143 I.Ic-08  28  38  1199  OIIPIDIdIO25092 8 EST  (A00t0049)tmnsaldolase -2 395 4.5e-SO  >gi13481 l6Igh)L2264t.IIPIORRI0S  265 rDNA  >g)61 I0442IgbIAFl89845.11AP109045  PKA2  dsfoe@drike.edu pCt)49 rebate with dcl (I mglml)  H153  dsfosNjdukc.edo pMJB54 eDNA in TOPO TA mlcasc mills EcoRl  Spil6  1199  )D8927t) racicosoore assembly protein +3 213 ICe-tO  ESTgnIIPtDIdtOI4629 EST gnIJPtDjdtO222O2  143 I .3c-08  Strain Origin  1-199  290 6.7c-25  algO8cn.rl  at gO9cn.f1  1499  >prl 153 I609b)AA05 1839.UAAO5 1039 Cn0016-5 Cryptneocens ncoformaoa  SpiZS  >gil 153 t697)pbIAAOSlO47.IIAAOS 1847 Cn0025-5 Crvprococcas ocofomraos  5 3 Spi  >gi)ISS t707)gb(AAO5 1057 IIAAOS 1857 Cn0035-5 Cryptococcan neofomsans  Spi6  cpu 153160 lIgbIAAO5 1031. flAAO5 1831 Crr1111116-5 Cwptococcus scofonrrairs  Spi7  cpu 153 t602ipbIAAO5 1832. IIAAO3 1032 Cn110117-5 Cryplococcrrs uoofonssans  Z 3 Spi  coil 153 I7O4lgbIAAOS 1854. IIAAO5 1854 Cn0532-5 Cryptecoccus noofnrmeas  SpISS  >gil 15317 I9gibAA05 1869. IjAAOS 1869 Cn0055-5 Crypcococcus neefnmmans  5 Spil  coil 153 I688I NAAOS 1838. IIAAOS 1035 Cn0015-5 Cryptococcus oonfonrruns 9  Spi29  >gij 1531701 lgblAAOS 1851 .IIAAOS 1851 Cs0029-5 Cryptococcss srofonrraos  H083109.F  BAC END Sequence www.begsc.bc.co  11004E17.R  BAC END Sequence www.bcgsc.be.co  118153H04.F  EAC END Sequence svsvw.bcgsc.6c.eu  3  14  76  0  77  II  47  28  31  37  42  37  26  28  19  16  26  26  38  24  21  0  22  24  17  29  37  20  36  28  40  57  30  26  44  30  36  29  1199 CES582 CES882  Spitzcr aud Spitaor. 997 ESTs usasi as bars utypo maabem Spiseer and Spitecr. 1997 ESTs used as bervetype markem Spitaer and Spiteer, 1997 ESTs used as karyotype oserbems SplIcer ced Spiseer, 1997 ESTs trsed em katyotypc merbers Spitzrr asd Spitaer. 1997 ESTs used as karynrype mmmarbem Spitzermtd Spiteor. 1997 ESTs esed as barvoevpc usaskers Spilaermsd Spitoor, 1997 ESTs ased en kars’orvpe madrers Spitaeread Sprsmr, 1997 ESTs used as bereotvpe moebem Spiteer aud Spiseer, 1997 ESTs speed as barvotvpe merkcm 1199 H99 1199  11003H22.F  BAC END Sequence www.bcgsc.bc.ca  l1003M21.F  EAC END Sequence www.bcgsc.bc.ca  11003M21.R  BAd END Seqscsce snsnw,bcgsc.bc.ca  HOO2KII.F  BAC END Sequence wsvw.bcgsc.bc.ca  11003C03.F  EAC END Scquence wmnsv.bcgsc bc.ca  11807684.R  EAC END Sequence wss—sn.begsc.bc.ca  H801024.F  EAC END Sequence sswss.bcgse be.cu  1199  140152K09.F  EAC END Sequence www.bcgsc.bc.ca  1199  1100280SF  EAC END Seqoencc svww.bcgsc.bc.ca  H004008.F  EAC END Sequence www.bcgsc.bc.ce  Total Clanea Nybridiaed  37  7  13  28  32  26  33  44  26  24  35  tO  23  12  19  1603  1571  1199 1199 1199 H99 1199  1199 1199  B. Integration of the Markers into the BAC Fingerprint Maps. Summaries of the 20 assembled contigs for each strain can be found in Tables 3 and 4. These tables also list the number of markers that mapped to each contig by hybridization and the estimated total amount of DNA represented in each of the assembled contigs for both strains. With regard to genome size, the estimates of 15.79 Mb for JEC21 and 15.55 Mb for H99 are at the lower end of the range (15 to 27 Mb) estimated from electrophoretic karyotyping of different C. neoformans strains (Perfect et at., 1989; Wickes et at., 1994; Boekhout et al., 1997). The genome sizes estimated from the maps are likely to be underestimates because there may be areas of the genomes that are not represented in the BAC libraries. These may include areas that are difficult to clone or maintain in E. coil such as telomere or centromere sequences or areas with an unusual distribution of Hindlil sites. Of course, estimates of genome size from electrophoretic karyotyping experiments can also be confounded by problems with chromosome size determination and the co-migration of different chromosomes. These issues were largely resolved with the completion of the serotype D genomes (Loftus et at., 2005) The fingerprint maps are available in FPC format at the web site of the MSGSC (www.bcgsc.bc.ca). In addition, the maps can be viewed with Internet Contig Explorer (iCE) and the BAC end sequences are available at this web site. An example of an FPC output for contigil of in the JEC21 map containing the MATa locus, is shown in Figure 2.  38  Table 3: Summary of the contigs in the fingerprint map of JEC2 1.  Contig Number 1 7 11 5 8 10 16 18 4 9 19 6 12 20 13 15 3 17 2 14 Total  Contig Size (bp) 1748127 1484186 1434639 1164507 1070654 1038718 975364 970748 853084 732482 708498 697149 587336 503950 420308 407895 379639 222874 202053 184760 15786972  Number of Clones 321 196 250 185 167 163 154 129 128 108 71 98 68 91 65 52 49 12 9 6 2322*  Contig Depth (a) 20.16 14.87 19.23 17.04 17.73 16.13 16.59 14.57 15.83 16.01 11.09 15.09 11.95 19.13 17.14 14.22 14.05 5.40 4.67 3.40 14.21  Number of Tiling Path Clones 20 14 14 13 12 11 10 10 9 9 7 7 6 6 4 4 4 2 1 2 165  Number of Markers 7 8 11 4 6 6 4 4 5 3 11 11 2 3 4 1 2 0 0 2 82**  *Number of singletons = 3 11 markers hybridize to >1 contigs (a) The contig depth is the sum of the actual fragment sizes for all of the fingerprinted clones in the contig divided by the calculated size of the contig. **  39  Table 4: Summary of the contigs in the fingerprint map of H99.  Contig Number 5 3 12 19 17 1 10 8 20 7 13 4 11 9 2 16 14 18 15 6 Total  Contig Size (bp) 1356533 1221982 1204660 1199093 1192998 1128612 1080077 958050 957898 854305 843814 746210 689677 631074 601000 236418 197615 190795 175905 84272 15551099  Number of Clones 246 190 228 204 246 212 218 161 143 155 106 92 92 90 87 26 13 8 20 2 2539*  Contig Depth (a) 20.12 16.62 20.37 18.02 22.16 20.30 21.75 18.41 16.15 19.81 13.56 13.46 13.99 14.75 14.92 11.40 7.21 4.53 11.77 2.01 15.07  Number of Tiling Path Clones 15 14 12 11 12 11 12 10 9 8 9 9 7 7 7 3 2 2 2 1 163  Number of Markers 19 14 10 11 14 17 7 7 7 16 8 8 9 10 3 2 1 1 1 3 102**  **48 markers hybridize to >1 contigs (a) The contig depth is the sum of the actual fragment sizes for all of the fingerprinted clones in the contig divided by the calculated size of the contig.  40  Figure 2: FPC display from the map of strain JEC21. Contig 11, containing the MAT locus, is shown on the left. Markers hybridizing to BAC clones in this region are shown above the contig. Green boxes indicate tiling path clones. Gel images on the right show some of the fingerprints of overlapping clones in the contig. Horizontal lines represent positive shared bands between adjacent clones.  41  Hidden: IBuriedi  1Q07J04+  J003P03*  3006L08  J002H17+  3002M24*  ,j006H23*  JOOILIS*  30081<08*  3005004  J003C22*  3006P24*  3004C06+  3006L14+  MF—GEHE STEI 2—ALPHA STE20  3007F1 2*  1008H12*  3007308+  3001803*  3006M13  3008009*  3007E1 2*  3002319+  3006K01  30031104*  JOOSF2O*  3007K18*  DHA1  SpiO7  3008P09*  H003H22.F  J002824*  J008D03*  J0.5±J2.4 *  3008306+  3008109*  3007102*  J007N24+  TEF2 H004E17, R  3006806+  H003H04,F  3006110*  3004L07*  Spi55  SpiO6  !Con1igure Display! Clone:! !Edit Contig! ITrail.,.! Clear Fill! Merge! JAnalysisIIsnCtgCheck IsnCtgStepl Merge CtglB. Merge Ctg4. Merge Ctg43, Merge Ctgl7, Merge Ctg39. Merge Ctg258. Ctgil of JEC21_master Clones 51 of 250L Markers U. of 11, Sequenced i5i  Whole! Zoom: jj2,0  IClear Trail!  IBandi !RedraI IRernove All!  QHighGreen  ITraill  —  —  —  —  =-..  —  —  —  ——  —  —  ——  —  —  ——  ——————  ——  __  —  -  —  =—-——  _.__  —  ---  —  —  —_  —  —  —  —  1100 1200 1300 _ 1400 1500 ——• i600 1700 -—--—1800 1900 2000 2100 =‘=——— ----2200 _j =,——=— —————-—= 2300 2400: :z ZE E—— 2500 =.= =—=— 2600 = —— 2700:,:— 2800” 2900 == 3000 3100_ = 3200 3300 3400 3500 = = 3600 3700 3800 3900 4000  I _a_;izize=::=I=___  3004C06 3008K08 3008P05 3008F09 a e,J008H12 J007F12 J008F23 800  liJ  4000  IMove! Remove!  clip! 800  GreyRamp! Whole! Zoom4ij j6!  C. Comparison of the H99 and JEC21 Fingerprint Maps. Shared markers were found for 17 and 18 of the 20 contigs for JEC21 and H99, respectively (Figure 3). Note that no markers were found for contigs 2 and 17 of JEC21, but markers were found for all 20 contigs in strain H99. Hybridization with two different probes for C. neoformans rDNA sequences revealed that BAC clones carrying rDNA genes were found in contig 7 for JEC21, but were not present in the 6,528 clones from H99 BAC library. This result may indicate that the rDNA sequences from H99 have a different organization of HindlII restriction sites that precluded cloning of the region. DNA blot analysis of complete HindlII digests of H99 and JEC2 1 genomic DNA confirmed that the H99 rDNA contained fewer HindlII restriction sites (Figure 4), which may explain its inability to be cloned in pBe1oBAC1 1 using the protocols employed for library construction. Lists of the sequences that were used as probes for each marker are provided in Appendix ha. As shown in Figure 3, the hybridization data and the fingerprint information allowed us to match 18 of the 20 H99 contigs to 17 of the 20 JEC2 1 contigs. Markers were used for the comparison only in situations where they could confidently be mapped to a single contig. The comparisons of marker positions between the maps revealed considerable conservation of synteny between the strains with several clusters of markers showing identical order. In addition to the overall similarities between the two maps, the conservation of marker order was particularly striking for contigs 13 (JEC21) and 3 (H99), for contigs 19 (JEC21) and 9 (H99), and contigs 11 (JEC21) and 5 (H99). As indicated earlier, the hybridization probes for genes  known to be in the MATh locus identified contig 11 in JEC21 (Figure 3; 5A) and contig 5 in H99 as arising from the mating-type chromosome (Figure 3; SB). The MATa region has been the focus of targeted sequencing (Lengeler et a!., 2002; Karos et a!., 2000) in part because of the association of the a mating-type with virulence (Fan et a!., 2005; Nielson et a!., 2005a and b; Kwon-Chung et a!., 1992). The combined FPC and hybridization data also revealed several examples of rearrangements between the two genomes.  Specifically eight markers were  identified whose positions did not agree between the two maps relative to flanking markers (Spi25, Spi35, alb07cn.rl, ale04cn.fl, H003C03.F, URA5 and H003M21.R; Figure 3). The positions of some of these markers suggest the presence of an inverted region between the genomes (e.g. 5pi25 and Spi35) while the positions of others suggest translocations (e.g. 43  alb07cn.r 1 and al e04cn.fl). These results provided the first comparative view of genome organization between strains representing different varieties. The comparison of the locations of specific sets of markers between the two maps also suggested that certain contigs may be regions of the same chromosome.  For example, the  comparisons of shared markers between the maps suggest that contigs 1, 3 and 13 could be regions on the same chromosome for JEC21 and contigs 3 and 17 could be joined in H99. Similar connections are suggested for contigs 10 and 15, and contigs 7 and 9 for JBC2 1. For H99, contigs 4 and 18, 12 and 15, 1 and 19 and 7, 8 and 16 could be joined; the mapping data also suggest that contig 6 could be joined to contig 5 that contains the MATa locus. Overall, these joins would reduce the number of contigs to approximately 15 for the H99 map and 17 for the JEC21 map. For comparison, the reported range for chromosome number for the majority of strains of C. neoformans was between 11 and 14 (Boekhout et al., 1997) and is now confirmed to be 14 (Loftus et al., 2005).  44  Figure 3: Conservation of synteny between the genomes of JEC2 I and H99. The 20 contigs for each map are shown with the markers found on each contig. Note that only markers that mapped to contigs in both strains are shown. Contigs are represented as vertical lines with the contig numbers above. The relative positions of the markers within the contigs are approximated based on hybridization results. Where the relative positions of adjacent markers in a contig could not be reliably interpreted they are grouped by a square bracket. Corresponding contigs from JEC21 and H99, based on marker content and order, are placed adjacent to each other for comparison. Differences in marker locations between the two strains are also indicated. No markers were found for the JEC21 contigs 2 and 17; these contigs, along with 14 and the 1199 contigs 6 and 14, did not have shared markers between the genomes. The boxed and shaded markers also were used to relate the contigs to electrophoretically separated chromosomes (Fig. 5).  45  _______ JEC21  ___________________________ ___________  H99  13  H99  JEC21  3  8  H004001&F  H(x)400aF  alhl2cnfl alaO5cii.f1 albO4cri.rl  alhl2cn.fl alaO5cn.f1 albfl4cn.rl  10  16 I  I I  algi Icn.1l algO4cn.f1  aI1 Icn,fl algo4cn,11  i  GPD  GPD  aklllcn.fl  IPI  IP1  20  I  I  alcO4cn.f 1  1  H99  JEC21  H003M21.F  I  I-1003M21.R  I  TOPOI  H003M21.R  a1i1knf1  6 /i FKS1 MDRI  FKSI MDRI C}AL7 atgrncn ri  K CAP64  I CAP64_  CAP1O]  GAl 7 algO3cn.rI  18  11  aleO6cii.i1  a1c(cn.f1  alcO4cn.fl CELl {CA[N3O  alcO9cn,rl  alcO9cn.rl  I I  LJREI  11003C03.F  3  /9  CAPIO  ‘  19  CAP1 (1 I}/  18  H003C03 1-1002K 1SF  albOlen.rl PASA1  PASAI  Ii  PLBI a1lcnf1 aliO2cn.i1  I  9II  I I  H007G04.R 1131O9.FI 1RG11  15/  alcllcn.fI  A5  }/  1T002K09.F  11  } 110031-104.F H004E17.R  Jr H004E17R TEF2  110031122,F  H003H22.P  DHAI  IMF-C,RN’ I STEZOI  PHSP78  MPDI P1JKCI  CPAI  II  PHS8  M1’DI PUKC1  20 NMT  IMF.GENEI  1sTh20  I  1-10(J2K11.F  I  Hl)02K09.F  PATFI  H003H04.P  TEF2  I  PPYP2  alc03cn.f1  11001024.F  13  8 CPAI  al cO3cn.[1 H001024.F  12  HOO2K11.F  16  alh02cn.f1  11007G04.R  lRG II  12  alh02cn.fl  alffl2cn.fI Ho00Io9.r  I  H(X)2K15.F albO7cn.rI  PLB1 aicoicn.n  PMAI  I  2  46  I  Contigs without shared markers JEC21  H99  2*  6**  I  I  7*  14  I  ii 14  I *No markers mapped to contig  **Contig6may bcparlofcontig5 PMAI  =  500kb  1kb ladder  A  B •4— origin  1221 5090 4072 3054 2036  1018  506,517  Figure 4: Determination of Hindill fragment sizes for 26S rDNA. Southern Blot of Hindlil digested genomic DNA from C. neoformans. 26S rDNA probe. Size markers were 1 kb ladder from Invitrogen, Inc. Fragments carrying rDNA for JEC21 were at the upper size limit of the ladder; the exact size can not be determined but the fragment can be approximated in the range of 6,000-12,000 bp. Fragments with H99 rDNA were larger than the upper limit and noticeably larger than the JEC2 1 rDNA fragments, indicating fewer Hindill sites adjacent to rDNA in the H99 strain. A. .JEC21 B. H99  47  D. Relationship of Specific Contigs to Chromosome-sized Bands From the C. neoformans Electrophoretic Karyotype The positions of specific markers on the contigs were also compared to the published locations of the same markers on electrophoretically-separated chromosomes and the meiotic map. For this analysis, the electrophoretic karyotypes of two progenitors of JEC21, B3501 and NIH12, were used to represent the chromosomes because these strains were used for previous hybridization experiments (Figure 5A; Wickes et al., 1994; Spitzer and Spitzer, 1997). The patterns of chromosome-sized bands for these strains appear to be similar or identical to the pattern of JEC21, as determined by Lengeler et al., (2000).  The hybridization probes also  included the genes URA5, CAP64, CnLACJ and STE2Oct that have been placed on a meiotic map by Forche et al., (2000). The JEC21 contigs that hybridized with markers previously assigned to specific chromosomes are shown in Figure 5A.  Our results indicated that the three largest  contigs (1, 7 and 11) from JEC21 contained the same markers that map to the three largest chromosome-sized bands in the two other serotype D strains. The chromosome represented by the third largest band in these strains contains the MAT locus and this chromosomal location has also been established in JEC2I (Lengeler et al., 2000). The rDNA markers are present on the second largest chromosome-sized band in strains B3501 and NIH 12 (Wickes et al., 1994). Similarly, we found that the rDNA probes hybridized to the second largest band on a blot of separated chromosomes (Figure 6 B and D) and to contig 7 in JEC21 (Figure 5A). These results were in agreement with the hybridization data obtained by Wang et al., (2001) for the CPA] and CPA2 genes; these genes hybridized to the second largest band in JEC21 and were found with the rDNA on contig 7 (Figure 5A). However, it was found that the HIS3 probe hybridized to a band equivalent to chromosome 7 (Figure 6 F and H) in contrast to the reported location of this gene on band 11 (Wickes et a!., 1994). This result indicates that there are differences in the locations of some markers for JEC21 when compared with progenitor strains B3501A and N11112. Interestingly, it was later found that a chromosomal duplication event had taken place in JEC21 when compared to B3501A (Fraser et al., 2005). The duplication included 62,872 identical nucleotides and 22 predicted genes.  This duplication  occurred in the contig 18 (which corresponds to chromosome 4 at TIGR) of JEC21 where this study showed that HIS3 was located (Figure 3 and Figure 5A). This duplication in JEC21 may explain the differences in the marker placement on the karyotype when compared to the 48  progenitor strain B3501A. The sequence for HIS3 was retrieved from GenBank (U04329) and searched against the TIGR JEC21 database using the BLASTn algorithm, surprisingly, there was no significant hit. It is difficult to discern whether HIS3 resides in or adjacent to the duplicated region, however the lack of HIS3 in the TIGR database may explain its exclusion from the published list of duplicated genes in JEC21 if it does reside in that region. These observations suggest that caution should be exercised for some of the comparisons because of possible differences in the karyotypes between JEC21 and the progenitor strains. In this regard, several reports have described the variability of the karyotype in C. neoformans (Perfect et al., 1989; Wickes et al., 1994; Boekhout and van Belkum, 1997). Loftus et al., 2005 reported that the C. neoformans genome is rich in transposons, many near candidate centromeric regions and these features may contribute to karyotype instability.  However, in addition to  correlating contigs with the largest chromosomes, the hybridization data may provide insight into the contigs that represent the smallest chromosomes; this information may have utility for the analysis of chromosome structure in C. neoformans. For example, both contig 4 and the  th 10  chromosome-size band of JEC21 hybridize with the Spi29 marker and have similar sizes (0.853 Mb versus 1.02 Mb, respectively). Thus, contig 4 may represent most of chromosome 10 if the karyotypes are equivalent for B-3501 and JEC21 for this band (Figure 5A). The estimated sizes of karyotype bands on Figure 6 were based on the NIH12 karyotype by Wickes et al., (1994). They reported a chromosomal size range of 0.770 Mb-3.87 Mb for 12 electrophoretically separated chromosomes. A more recent meiotic map was published by Marra et al., in 2004 that reports a size range of 0.80 Mb-2.3 Mb on 14 chromosomes for a total 20.2 Mb genome. Finally, with the publication of the complete annotated genomes for strains B3501A and JEC21 (Loftus et al., 2005) those estimates have been further refined to reach an estimate of a 20 Mb genome. As mentioned, the complete sequence has now been published for the serotype D strains B3501A and JEC21 (Loftus et al., 2005). In light of this, markers used in our correlation of karyotype bands were retrospectively matched to the final chromosomes of JEC21 using the TIGR database (Figure 5A). Specifically, the sequences used to generate the probes were searched using the BLASTn algorithm against the TIGR database. The correlation was generally good although there were again examples where the placement of a marker differed in JEC21  49  when compared to the B3501A karyotype. These results were in agreement with those noted from the initial correlation of contigs to karyotypes. CAP64 was indeed present on chromosome 2 (contig 1 of the JEC21 physical map); the mating locus genes: MF gene (pheromone), STEJ2 and STE2O in addition to the SpiO6 and Spi55 markers were found on chromosome 4 at TIGR (matching contig 11 in this map). The marker Spi32 was mapped to contig 4 of the JEC21 map and found on chromosome 10 at TIGR.  This marker is an example in which the JEC21  placement did not correspond to B3501A. In the karyotype studies for l33501A, Spi32 was mapped to the third largest band (and the same band as mating type genes), while it was located on contig 4 not 11 (where the mating type genes are located) in the JEC21 physical map. This is a good example in which the physical maps correctly located a difference between the organization of the JEC21 and B3501A genomes.  Further support for matching contig 4 to  chromosome 10 is provided by the Spi29 marker that also mapped to contig 4 in the physical map and is found on chromosome 10 in the TIGR annotation. This marker was mapped to the th 10  band (Wickes et al., 1994) and  6 t h  band (Spitzer and Spitzer, 1997). This result indicates that  the Spi29 marker was possibly on the equivalent chromosome in JEC21 and B3501A. It was also found that the SpiOl probe hybridized to multiple contigs while Spitzer and Spitzer (1997) found that this marker is located on the smallest chromosome in B-3501. In the TIGR database, SpiOl is indeed located on one of the smallest chromosomes, number 13, suggesting that the results in the initial mapping studies were due to cross hybridization of the SpiOl probe. Two additional markers on the JEC21 map that did not correspond to the progenitor strains were URI45, found on contig 15 and chromosome 7, where the marker was found on the 4” largest band of the Spitzer study and the  4t  5 t h  or  6 t h  largest bands of the Wickes study, and PKA2 mapped to  contig 16 of this map and chromosome 9 at TIGR where it was located on band 5 of the Spitzer study and band 7 of the Wickes study.  It should be noted that the actual number of  chromosomes in these serotype D strains has been resolved at 14, so it is likely that these earlier karyotype studies do not have the correct resolution of the chromosomes. Therefore, some differences in marker placement may be an affect of unresolved karyotype bands and not necessarily different chromosomes when comparing the progenitor strains with the TIGR annotation. A limitation of karyotype analysis in C. neoformans is the possibility of doublets and triplets that are difficult to resolve. In addition, there is variation of karyotypes even within serotypes (Meyer et al., 1999; Kidd et al., 2004).  50  The markers for rDNA and the MAT locus are found on the largest chromosome-size band in the serotype A strain N1H371 (Wickes et al., 1994). The MAT locus is known to be on the second largest karyotype band in H99 (Lengeler et al., 2000) and our hybridization data link this chromosome with contig 5 in the BAC map (Figure 5B). Results with the CAP64 and Spi35 markers also suggest that contig 17 represents part of one of the largest chromosomes in 1199; this conclusion is supported by the comparison of the conservation of synteny (Figure 3) because contig 17 of H99 shares two other markers with contig 1 of JEC21. We noted earlier that the rDNA markers did not hybridize to any of the clones in the 1199 BAC library; the rDNA cluster is therefore not represented on the contig map. However, hybridization of the rDNA probes to electrophoretically separated chromosomes did locate the sequences on the largest band (Figure 6 A and C). Furthermore, the location of the rDNA region between contigs 8 and 16 in H99 is suggested by the comparisons of the shared markers; i.e., these contigs carry the markers Spi 16!H002K09.F and CPA] that flank the rDNA on contig 7 in JEC21 (Figure 3). For H99, the URA5 and HIS3 probes each hybridized to two contigs although clones from one of the two  contigs (19 for URA5 and 11 for HIS3) were more frequently detected.  Also hybridization  experiments to electrophoretically separated chromosomes (Figure 6 E and G) showed that HIS3 hybridized to a single chromosome in each strain. These results suggest that cross-hybridizing sequences may be present for these markers.  51  A Wickes et aL, 1994 Bands NIHI2  Spitzer and Spitzer, 1997 Bands B-3501  1(3.87 Mb) 2 (3.34 Mb)  3 (2.50 Mb)  2 3  4,5,6(1.61 Mb)  7(135Mb) 8 (1.29 Mb) 9(1.18 Mb) 10(1.02Mb) 11(889 Kb) 12 (770 Kb)  }  Spiker Band (B-3501) 1 2  Contig Size (bp) 1,484186 1,748,127  Mapped JEC21 BAC Clones 13 2  Unmapped JEC21 BAC Clones 29 0  TIGR CHR No Hit 2  7 (9)  1,484186  16  60  Not Represented  7(9) 1 (4) 1 (7) 11 (1) 11 (1) 11 (1) 11 (6) 11 (10) 4 (5) 11 (2) 15 (4) 16(12) 3 (2) 4 (5) 18 (4) MULTIPLE  1,484186 1,748127 1,748,127 1,434,639 1,434,639 1,434,639 1,434,639 1,434,639 853,084 1,434,639 407,895 975,364 379,639 853,084 970,748 N/A  18 5 9 2 1 1 8 11 5 3 4 14 2 6 6 3  59 21 10 3 0 2 18 27 16 19 25 33 15 31 25/9 23  Not Represented No Hit No Hit 4 4 4 4 No Hit 10 4 7 9 No Hit 10 No Hit 13  Unmapped H99BAC Clones 21 11 0 0 16 6 1 2 2 12 10 0 9 10 15 17 8 12 5  Marker Spil6 CAP64  HindUl Map Contig (Clones on Contig) 7 (12) 1 (2)  2  rDNA18S intergenic  2 2 2 3 3 3 3 3 3 3 4 5 6 6 7 8  rDNA26S Spi25 Spi35 Mato (MF gene) Make (STEI2) Matu (STE2O) SpiO6 SpiO7 Spi32 Spi55 URA5 PKA2 Spi15 Spi29 HIS3 SpiOl  B  Marker Spil6 CAP64  Hind Ill MapConfig (Clones on Conhig) 16(12) 17(7)  Conhig Size (bp) 236,418 1,192,998  MappedH9g BAC Clones 16 5  rDNA 18S intergenic rDNA26S Spi25 Spi35 Matu (MF gene) Mata(STE12) Mata(STE2O) SpiO6 SpiO7 Spi32 Spi55 URA5 PKA2 Spil5 Spi29 HIS3 SpiOl  NOTREPRESENTED NOTREPRESENTED 3(11) 17(8) 5(1) NONE 5(1) 5(11) 5(8) NONE 5(14) 19(12)20(3) 20(10) 17(10) 4(10) 11 (17)5(4) MULTIPLE  N/A N/A 1,221,982 1,192,998 1,356,533 1,356,533 1,356,533 1,356,533 1,356,533 N/A 1,356,533 1,199,093;957,898 957,898 1,192,998 746,210 689,677;1,356,533 N/A  0 0 12 10 1 0 1 14 14 0 15 16 13 12 12 25 6  Figure 5: Relationship between electrophoretically separated chromosomes and the contigs of the JEC21 and H99 maps. A. Diagrammatic representation of the electrophoretically separated chromosomes from the serotype D strains B-3501 and NIH12 (Spitzer and Spitzer, 1997; Wickes et al., 1994). The panel on the right shows the contigs of JEC21 that hybridized to the same markers used by Spitzer and Spitzer (1997) and Wickes et al. (1994) to identify specific chromosomes or groups of chromosomes. B. Locations of chromosome specific markers on mapped and unmapped clones and specific contigs of the serotype A strain H99. The number of BAC clones that hybridized with each marker is also presented for both the mapped BAC clones and the additional clones (unmapped) that were present on the high-density filter.  52  -  A  B  C  D  E  F  G  H  Figure 6: Genomic Location of 26S rDNA and HIS3. For JEC2 1 the corresponding bands to reported serotype D karyotypes are related to the JEC21 karyotype in addition to chromosome size estimates from Wickes et al., 1994. Probe from 26S rDNA hybridized to a CHEF gel of chromosomes CHEF gel A. H99 B. JEC21 and Blot probed with 26S rDNA C. H99 D. JEC21. 26S rDNA is present on the largest CHEF fragment in H99 and the second largest fragment in JEC2 1. CHEF gel E. th H99 F. JEC21 and Blot probed with HIS3 G. H99 H. JEC21. HIS3 is present on the 7 11 CHEF fragment in ( th) th ( 5 1h) 7 H99 and the fragment in JEC2 1.  53  SUMMARY The BAC fingerprint maps described here for strains JEC21 and H99, along with the sequences of the ends of the mapped clones, provided a partial framework for the completion of the genomic sequences of these strains. The map with the minimum tiling set of BAC clones for the genome and the end sequences of the BAC clones contributed to the genomic sequencing effort for JEC21.  The sequences for two serotype D strains JEC21 and B3501A are now  published in Science (Loftus et al., 2005). The genome for the serotype A strain, H99 has been completed at the Broad Institute of Harvard and MIT.  The maps also provided the first  comparison of the conservation of synteny between the genomes of C. neoformans strains from the A and D capsular serotypes that represent varieties grubii and neoformans, respectively. Furthermore, the maps provide the opportunity to use arrays of the minimum tiling sets of BAC clones to make comparisons between genomes from different isolates from the same or different varieties.  This approach has been used successfully to explore genome variability in the Mycobacterium complex (Gordon et al., 1999). Five more mapping projects have been initiated and successfully completed through collaborations of the Kronstad laboratory and the MSGSC using similar techniques. The robust analyses of these first maps for serotypes A and D provided a strong platform to guide further efforts in the mapping of C. neoformans and C. gattii genomes.  54  CHAPTER THREE: Serial Analysis of Gene Expression (SAGE) of C. neoformans under Iron Limited Conditions  INTRODUCTION The SAGE technique, originally developed by Velculescu et aL, 1995, provides a snapshot of the mRNA transcript abundance in a given cell state. The set of transcripts (known as a transcriptome) in a SAGE library is unique to the mRNA in the cells isolated for that library. Data output from a SAGE library is in the form of 10 base pair tags, each of which is 3’ adjacent to the 3’ most NlaIII (CATG) site in a cDNA. The abundance of unique SAGE tags may be compared from cells grown in different conditions. The corresponding transcript for each unique tag gives an indication of expression for that transcript in that cell state.  It is this step of  identifying a transcript and subsequent gene function from a SAGE tag that proves to be rate limiting. The SAGE analysis has become more efficient and accurate with the completion of sequencing, followed by assembly and annotation, for the JEC21 and B3501A genomes (Loftus et al., 2005). H99 genome sequence resources are available at Duke University and the Broad Institute of Harvard and MIT, in addition to EST sequencing projects at the University of Oklahoma, Duke University and TIGR. At the onset of this project the first sequencing project at Stanford University was in its infancy. SAGE data analysis at this time was slow, laborious and often yielded limited results. The value of the genomic resources developed within the time frame of this project have been clear in the efficiency of many analyses, both large scale (such as SAGE analysis) and on a gene by gene basis (Chapter Four). SAGE has been successfully used for a number of analyses in the Kronstad Laboratory and multiple libraries have been completed, sequenced, analyzed and published. For example, libraries were constructed to investigate thermotolerance in C. neoformans. JEC2 1 and H99 libraries were prepared from the mRNA of cells grown in vitro at both 25°C and 37°C (Steen et al., 2002). Also the MicroSAGE protocol was optimized for C. neoformans by Dr. Barbara Steen in order to construct SAGE libraries from the mRNA of C. neoformans cells that were isolated from the cerebral spinal fluid of rabbits. A modified protocol was necessary due to the low number of cells isolated from this in vivo source. These libraries provided a view of in vivo C. neoformans transcription that revealed clues to important virulence genes (Steen et a!., 2003). Libraries were also constructed from the mRNA of cells grown in vitro on low or replete iron media for the serotype D strain, B3501A (Lian et  55  al., 2005).  These libraries provided a view of differential gene transcription involved with capsule synthesis and identified a set of iron regulated genes. It was in these libraries that the SIT] gene (characterized in Chapter Four) was initially identified. Additional libraries have been completed for cAMP mutants in the serotype A strain H99, for wild-type H99 under iron replete conditions, for cells from in vivo sources such as lungs of mice and macrophages in culture. These data are currently being analyzed and will contribute, in conjunction with the SAGE analysis detailed in this work, to large scale transcriptome analyses in C. neoformans. Multiple studies have also been initiated to characterize genes identified in the SAGE analysis thus far and this genetic follow-up work contributes biological relevance to these studies. Chapter Four represents an example of this approach. At the time of map construction (described in Chapter Two), it was estimated that 810,000 genes existed in the 23 MB C. neoformans genome, by comparison to the fully sequenced S. cerevisiae genome which has 6,200 genes in a 12 MB genome (http://genome www.stanford.edulSaccharomyces/; Velculescu et a!., 1997). Cryptococcus has a greater number of introns per gene in comparison to Saccharomyces, and given an estimation for the average length of a fungal gene at 1000 base pairs, a smaller percentage of genes were predicted in C. neoformans, 35-43 % vs. 52% of the genome as coding sequence respectively. The completion of two serotype D genomes (Loftus et al., 2005) further refined these estimates to a genome size of approximately 20 MB and 6,500 intron-rich gene structures (revealing a smaller genome and fewer genes than initially predicted). The SAGE library constructed and analyzed in this work investigates the transcriptome of the serotype A strain H99 in low iron conditions. Low iron is an important nutritional signal for gene transcription in the fungus, resulting in the elaboration of the polysaccharide capsule, a major virulence factor. In addition, the acquisition of iron, particularly in low iron conditions, is paramount for the survival of virtually all organisms. Pairwise comparisons were also included in this analysis to compare the low iron transcriptome with those from cells grown in minimal media (YNB) at 25°C and 37°C (Steen et a!., 2002), in addition to a comparison with an in vivo library from rabbit CSF (Steen et a!., 2003). For simplicity in these comparisons, the libraries will be referred to as 37°C low iron (cells grown at 37°C in low iron medium), 25°C (cells grown at 25°C in YNB), 37°C (cells grown at 37°C in YNB) and in vivo (cells isolated from rabbit CSF).  56  MATERIALS AND METHODS A. Determination of Growth Conditions. a. Fungal strains. The serotype A strains H99, H99Apkal and H99Apkrl were provided by Dr. J. Heitman (Duke University).  b. Media and growth conditions. Low iron medium (LIM) was prepared as described with 20mM HEPES and 20 mM 3 (Vartivarian et al., 1993). The water used for LIM was treated with Chelex-100 resin NaHCO (BioRad) to chelate iron. LIM+Fe was prepared by addition of ferric EDTA to 100 tM (FeEDTA;Sigma EDFS). Five mL of yeast extract peptone dextrose medium (YPD) was inoculated with a single colony and grown overnight at 30°C in a gyratory shaker at 250 r.p.m. 100 pL of culture was transferred to 5 mL of yeast nitrogen base (YNB) medium and grown overnight at 30°C. Cells were washed four times with sterile water and transferred to flasks containing 50 mL of LIM or LIM+Fe. The cultures were shaken at 250 r.p.m. at 30°C. Cells for SAGE library construction were harvested by centrifugation after 6 hours of growth in LIM flash frozen in an ethanol bath and lyophilized overnight at -20 °C. c. Capsule microscopy. For measuring the elaboration of capsule in low iron (LIM) and iron replete (IR) media, cells were grown as detailed in the section on growth conditions except that cells were grown in LIM or JR and samples were obtained prior to flash freezing. Cells were stained on a microscope slide with an equal volume of India ink (10 pL: 10 pL) covered with a glass slip and incubated at room temperature for one hour. DIC microscopy was performed on a Zeiss Axioplan 2 microscope. Cells were viewed at 1 000X in oil immersion. B. RNA Extraction. Lyophilized cell pellets were pulverized with glass beads for 10 minutes and the cell powder was resuspended in 15 mL of Trizol extraction buffer (Invitrogen). RNA was isolated according to the manufacturer’s recommendations with the additional step of LiCl precipitation at 4°C, following the standard ethanol precipitation step. Total RNA was dissolved in 1.0 mL of  57  lysis-binding buffer from a Dynabeads mRNA DIRECT kit (Dynal). Beads were prepared as per the instructions of the manufacturer (Dynal), and mRNA was added to the beads. Poly(A)+ RNA was isolated as described in the microSAGE method (Datson, et al., 1999 and version 1 .Oe; www.sagenet.org).  C. Construction of a Serial Analysis of Gene Expression (SAGE) Library. The SAGE library was constructed as per the microSAGE protocol (Datson, el al., 1999). SAGE primers were purchased from the Nucleic Acid and Protein Service Facility (NAPS) in The Michael Smith Laboratories, University of British Columbia. A total of 300 PCR reactions (35 cycles) were performed to amp1if’ the ditags during the construction of the library. The first step of SAGE library construction was the isolation of total RNA. This was achieved using the Trizol extraction method (described above). The RNA was checked for quality by running total RNA on a 1% agarose gel and observing intact rRNA bands, the mRNA appears a smear (Appendix III;Figure lila). Following RNA isolation, mRNA is captured on oligo-dT beads and cDNA is synthesized. The cDNA is completely digested at the 3’ most NlaIII site (CATG) and only the 3’ most portion of the cDNA remains attached to the beads. Linkers are then ligated at the NlaIII site. The linkers contain the enzyme recognition site for the type II enzyme BsmJI and primer sequences for the PCR amplification step. The cDNA is then released from the beads with BsrnfI. This enzyme cuts downstream of its recognition site leaving a unique 10 bp fragment from the transcripts adjacent to the NlaIII site. The fragments were blunt ended with Kienow enzyme and ligated to form ditags.  These ditags represent unique tags from two unrelated  transcripts. Note that sequence data was later checked for duplicate ditags that contain tags of the same two transcripts. A high number of duplicate ditags indicates an artifact of PCR in library construction because the tags should independently sort at the ligation stage and should not be present in the same ditag multiple times. Note that duplicate ditags were not abundant and therefore not a concern in the data from the library constructed here. Following ditag construction, the tags were PCR amplified to produce a 102 bp band. The PCR conditions were optimized to identify the most robust number of PCR cycles and the optimal dilution of ditag. Reactions were run on a polyacrylamide gel for analysis (Appendix III;Figure Ilib). 35X cycles of PCR and a 1/50 dilution of ditag was found to be optimal. 300 reactions were performed and pooled for the construction of the H99 low iron library. The bands were separated from residual  58  PCR products on a polyacrylamide gel (Appendix III;Figure Ilic). Bands were cut from the gel, pooled and purified. Linkers were then cut from the tags with NlaIlI. A 26 bp band containing only the SAGE tags was then isolated by electrophoresis (Appendix III;Figure hId). Tags were concatemerized and run on a polyacrylamide gel (Appendix III;Figure ITIe). Various sized sections of the concatemers were cut from the gel and ligated at the SphI site of the vector pZERO for sequencing. One hundred colonies were screened by PCR to assess the insert size and frequency. The insert size range was 400-800 bp with an average of 600 bp and a 10% frequency of empty clones. Figures for all stages of SAGE library construction can be found in Appendix lilA i-vu.  D. Sequencing and Data Processing. The SAGE library was sequenced, tags extracted and initial data processed by the Sequencing and SAGE groups at The Michael Smith Genome Sciences Center (MSGSC). The library of clones was sequenced using BigDye primer sequencing and analysis on an ABI PRISM 3700 DNA analyzer. Sequencing chromatograms were processed using Phred software (Ewing and Green, 1998;Ewing et al., 1998) to achieve accurate base-calling. CROSS_MATCH software was used to detect and remove vector sequence (Gordon et al., 1998). Tags 14 bp (10 bp unique + 4-bp NlaIII site) were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative Phred score. Only tags with a predicted accuracy of 99% or greater were used in this study. Duplicate ditags and linker sequences were removed as described in the original SAGE protocol (Velculescu et al., 1995). P values for comparison of tag abundance between libraries were determined using the method developed by Audic and Claverie, 1997. These statistically significant differences between tag abundance in different libraries were based on the probability that a difference in tag numbers was due to random fluctuation.  E. Tag Identification. For preliminary assignments of tags to genes, EST databases and genomic databases were used. An EST database is available for the H99 strain at the University of Oklahoma’s Advanced Center for Genome Technology (http://genome.ou.edu/cneo.html) [funded under cooperative agreement UOl Al 485 94-01]. When an EST was not available for a tag sequence, genomic  59  sequence was searched at the Duke University Center for Genome Technology; Duke University Mycology Research Unit (DUMRU) (http://www.dumru.mc.duke.edu!), or the Broad Institute of Harvard and MIT (http://www.broad.mit.edu!annotationlfungi/cryptococcus_neoformans/). Tag sequence assignments were only reported if they could be unambiguously identified as matching either EST or genomic sequence. Gene assignments were recorded if they had significant similarity (<e ) to known sequences using the BLASTx (Basic Local Alignment Search Tool) 5 algorithm against the non-redundant database at the National Center for Biotechnology Information (NCBI-www.ncbi.nlm.nih. gov). Tentative gene assignments (including the species with the closest ortholog), expect values, percent similarity and positives were recorded in Excel spreadsheets  and were  assigned  Gene  Ontology  terms  (GO  term)  where  possible  (http://www.geneontology.orgl). Tags were only given a gene assignment in the analysis if they were found at the 3’ most NlaIII site in the putative open reading frame or in a 3’ untranslated region. Detailed information about the tag annotation process can be found in Appendix IIIvii. Data from additional libraries used in pairwise analyses were provided by Dr. Barbara R. Steen (Steen et al., 2002 and 2003).  60  RESULTS A. Determination of Growth Conditions for SAGE Library Construction Initially, growth conditions were carefully optimized for three strains of C. neoformans, H99 wt, H99 iXpkal and H99 Apkrl. Different media were used to investigate phenotypes controlled by the cAMP pathway as a prelude to SAGE analysis. These included medium that was low in glucose and contained the substrate for the production of melanin (Niger Seed and DOPA), or low iron medium (LIM) to induce capsule synthesis and the expression of iron regulated genes (LIM and IR). Ultimately, LIM was chosen for a number of reasons. First, libraries were underway for the serotype D strain B3501A in low iron and iron replete conditions. Constructing the H99 libraries under the same conditions would provide for an opportunity in the future to compare annotated transcriptomes across the two serotypes. Secondly, iron is important in capsule synthesis and growth in low iron a necessary adaptation for survival of the pathogen in vivo. In this context, the Apka] mutant lacks capsule and the Apkr] mutant has enhanced capsule formation. Finally, a sufficient number of cells could not be isolated from the melanin media even in large volumes of medium and the melanized cells that were obtained were too rigid for efficient RNA extraction. A hallmark of C. neoformans under low iron conditions is the elaboration of the capsule. Therefore, cells were grown in low iron and iron replete media and assessed for the capsule by DIC microscopy at 0, 3 and 6 hours (Figure 7). Cells were grown in the same manner as used for the libraries for the strain B3501A (Lian et al., 2005), and harvested at 6 hours when a marked difference was noted in capsule size. The far right panel in Figure 7, shows the cells grown in LIM at 6 hours for H99 wild-type (A), H99 Apkal(B) and H99 iXpkrl (C); these photographs show cells from the population used in final library construction. The capsule is apparent in the images as a halo surrounding the cells as a result of exclusion of the India ink stain. For wildtype cells of H99, the capsule is induced in LIM and repressed under iron replete conditions (Figure 7A).  As mentioned, the Apka] mutant lacks the catalytic subunit of the cAMP  dependent protein kinase (PKA) and produces minimal capsule in low iron conditions (Figure 7B); the Apkr] lacks the regulatory subunit of PKA, leading to a constitutively active signal from the catalytic subunits. This leads to a hyperencapsulated cell in both low iron and iron replete conditions (Figure 7C).  61  There is still some uncertainty with regard to the most appropriate time for the isolation of cells to investigate the response to the low iron environment. The physical elaboration of the capsule is likely the result of important transcriptional changes relating to the response to low iron and the expression of capsule synthesis genes. For the experiments described here, the decision was made to keep the conditions consistent with those used for the libraries from B3501A to ensure continuity across the SAGE analysis and to allow for cross serotype comparisons in the future. The wild-type library designated “3 7°C low iron” is presented in this work, the libraries for the tpka1 and the Apkrl mutants of H99 were later completed by Dr. Guanggan Ru and will be part of a larger future analysis that will include supporting functional studies of specific genes.  B. Summary of SAGE Library Construction (supporting data for library construction in Appendix III). The SAGE library was constructed according to the MicroSAGE protocol (Datson et al., 1999). The modified procedure was chosen because it was optimized for a small number of cells. LIM is a minimal medium and cells do not grow to high density. The MicroSAGE protocol increases the efficiency of mRNA yield by immobilizing the poly-adenylated RNA on magnetic beads containing oligo-dT.  The cDNA is synthesized directly on the beads and cDNA is  recaptured by placing the tubes on a magnet. In addition, MicroSAGE was already being successfully used in the laboratory prior to the construction of this library.  After library  completion, colony PCR was performed on 100 clones to assess insert size and percent of empty clones. An example of colony PCR results is found in Appendix III;Figure Ilif. The average insert size was 600 bp with a range of 400-800 bp and a 10% occurrence of empty clones. Approximately 3000 clones were sent to the MSGSC for sequencing (resulting in 19,278 high quality tags).  62  C) C)  C  B  A  Low fron  fron Replete Low fron  3 Hours 6 Hours fron Replete Low fron  Cells were grown in ElM or IR medium and isolated at 0, 3 and 6 hours. Cells were stained with India ink and viewed by DIC microscopy under oil emersion at 1000X. A. H99, B. H99iXpka] and C. H99i.\pkrl  Figure 7: Elaboration of the polysaccharide capsule in low iron or iron replete medium.  fron Replete  0 Hours  C. SAGE Tag Annotation. The SAGE data were analyzed in collaboration with MSGSC. This analysis involved the following: i) extraction of SAGE tags from raw sequence (MSGSC), ii) calculation of the abundance of each individual SAGE tag occurrence (MSGSC), iii) identification of corresponding transcript for 100 most abundant SAGE tags and 100 most differentially transcribed SAGE tags for each library (Kronstad Laboratory). A detailed explanation of tag processing can be found in Appendix IIIvii. The first step to identify the originating transcript from a SAGE tag involved determining whether an EST was available at The University of Oklahoma (http://www.genome.ou.edu/cneo.html) EST database. An EST is ideal for SAGE tag identification because it contains only the RNA coding sequence of the gene. However at the time of these analyses many tags did not have corresponding ESTs. This may be in part a result of different media conditions used to construct the EST libraries and differences in transcription that would be present in a minimal media like LIM. Also, the number of ESTs available for the H99 strain is quite limited. More robust EST resources are now available at TIGR and will greatly alleviate this difficulty in SAGE analysis for serotype D strains.  If no EST was found,  genomic databases for H99 were searched at Duke University (http://www.dumru.mc.duke.edu!) or the Broad Institute (http ://www.broad.mit.edulannotationlfungi/cryptococcus_neoformans/). The corresponding sequence plus about 2000 bp either side of the tag were retained for further analysis. The positive contig and location was recorded in Excel workbooks. These sequences were then searched against the NCBI non redundant database using BLASTx analysis (http://www.ncbi.nlm.nih.gov/BLAST/) to determine whether they contained putative genes. The gene sequences were then analyzed to identify the location of the SAGE tag and to ensure that the SAGE tag was located in the 3’ region of the putative genes. Gene start sites, open reading frames, promoter regions and stop sites are identified within the putative gene in the Vector NTI program. Analysis thus far has indicated that the SAGE tag often exists in the 3’ untranslated region (UTR) of the genes. UTRs in C. neoformans have a length between 50-400 bp in genes studied at present by the SAGE analysis in the Kronstad laboratory. In addition, genes appear to share a 3’ consensus sequence of 5’-TTTGTTTT-3’ which may represent a polyA addition sequence. A portion of the tag annotation found in the analysis presented here were completed by Dr. Barbara R. Steen and included in a master Excel workbook database. Initially the tags from the low iron library were searched against this resource to identify  64  common tags that were already annotated (to avoid repetition of annotations). All additional tags were annotated during this work and subsequently added to the database. This database has proven to be a valuable resource for subsequent annotations of tags from new libraries.  D. Analysis of the Low Iron Transcriptome. The top 100 most abundant tags in the low iron library were annotated and assigned Gene Ontology (GO) terms where possible. Among the 100 most abundant, 49 were successfully annotated with corresponding EST or genomic sequence, had a significant BLASTx hit in the non-redundant database at NCBI (<e-5) and were successfully assigned associated GO terms. Six tags (6%) had a corresponding genome sequence match, and had significant BLASTx hits, but were most similar to a hypothetical protein. Another 29 tags (29%) had a corresponding genome sequence match but did not have significant BLASTx hits to known genes. The latter two categories may still warrant further investigation as they may be novel genes important in iron regulation or acquisition. Nine (9%) of tags did not hit EST or genomic sequence. One (1%) tag hit multiple sequences and therefore the correct transcript could not be unambiguously identified. Finally, six (6%) of the tags contained multiple adenosines suggesting that they were derived from the polyA region of mRNA. Of the tags that were assigned GO terms, 20 genes were involved with protein biosynthesis. Transcripts for ribosomal proteins are generally found to be highly abundant in SAGE data and the presence of these tags is an internal positive control in SAGE library analysis (Steen et al., 2002 and 2003; Lian et al., 2005). Two abundant tags matched genes that are involved in protein catabolism including a carboxypeptidase and metalloprotease. Seven genes were identified in the most abundant tags that relate to the response to stress. This result is expected because low nutrient availability in LIM may provide a stressful environment for the fungus. For example, four of the gene assignments were for heat shock proteins (two HSPJ2, HSP7O and HSP9O) and two were for genes for thioredoxin and glutathione peroxidase (HYR]), that have been studied in detail and implicated in virulence in C. neoformans (Missall et a!., 2005a-d; 2004). A glucose-1-dehydrogenase was also found to be abundant in low iron. The high affinity iron permease FTR] was highly abundant in the LIM library. The FTR] gene was identified in transcriptional studies of the B3 501 A strain (serotype D) and found to be  65  five-fold higher in LIM vs. IR media. These results were confirmed by Northern analysis (Lian et al., 2005). Four signal transduction genes were identified including a 14-3-3 protein and three GTP binding proteins. Signal transduction is known to play an important role in the regulation of multiple virulence factors in C. neoformans (Casadevall and Perfect 1998; Kronstad et al., 1998; Wang and Heitman, 1999; Aispaugh et al., 2000; D’Souza and Heitman, 2001; Waugh et at., 2002; Kraus and Heitman, 2003; Pukkila-Worley and Aispaugh, 2004; Hicks et a!., 2004; Zhu and Williamson, 2004; Balm et at., 2004; Idnurm et a!., 2005; Gerik et at., 2005). Five genes were identified to be involved in carbohydrate metabolism and one in nucleic acid metabolism. A chitin deacetylase, involved in cell wall synthesis, was also abundant. This gene has been studied in detail in our laboratory but no aberrant phenotypes were noted in a deletion mutant. Two genes involved with cell growth and maintenance were identified that encoded cyclophilin A and alpha-cop protein. There are two known cyclophilin A proteins in C. neoformans CPA] and CPA2 and this protein is important as the target of the antifungal drug cyclosporine A (CsA). Studies showed that Cpalp and Cpa2p play a role in cell growth, mating, virulence and CsA toxicity (Wang et at., 2001). Finally, three unclassified genes were identified: CIPC, an oxidoreductase and a gene for a phosphatidylglycerol/ phosphatidylinositol transfer protein. CIPC was the fourth most abundant tag in the low iron library and the second most abundant tag in the library constructed from cells isolated from rabbit CSF (Steen et a!., 2004). CIPC has been named HOT] in C. neoformans and characterized in our laboratory; however, no significant phenotypes have been identified to date.  66  Table 5: Most abundant SAGE tags for H99 cells grown at 37°C in low iron.  BLAST HIT  Sequence  Low Iron  TATATGTGTA  280 203  TGACTGTTTA  123  heat shock protein 12 Saccharornyces cereeisiae polyA tail heat shock proteIn 12 Sacchsromyces paston3lus  102  CipC Emericella nidulans  ATATGAAAGA  ATTGAGATGG  72  TAOTTGTGT  66  B-value  % identitY  1.OOE-06  I  % similarity  GO Term  45  61  Response to stress  9 e-04  55  73  Response to stress  1.OOE-08  52-70(3)  72-76  Unclassified  57  65  Protein biosynthesis  no significant BLAST results in nr database at NCBI no significant BLAST results in nr database at NCBI SAGE tag does not hit C. neoformans cDNA or genomic sequence  TAAPATTGCT  66  GAAAAAAAAA  50  TrGTAAkAA  48  poiyA tail no significant BLAST results in nr database at NCB1  AGTACTCTTC  43  ribosomal protein P2 Podospora anserina  1.006-26  GCAGATCTAT  38  ribosomal protein RPL39  ANNOTATED  Protein biosynthesis  38  translation elongation factor I (TEF1)  ANNOTATED  Protein biosynthesis  CGACAGACCG  no significant BLAST results In nr database at NCBI heat shock protein 90 homolog Candida albicaris SAGE tag does not hit C. neoformans cDNA or genomic sequence  ATGCACMTA  37  TGTTATCGGT  37  MGCCCCTTG  37  AGGATGAGM  37  ACAATACCTA  36  hypothetical protein Agaricus bisporus SAGE tag hits more than one sequence contig  GGAGACCAGG  36  no significant BLAST results in nr database at NCBI  TMiCGCATM  36  GCATTCTTTA  35  CMCGATGAT  34  TATGATAGTG  34  TAGCGATCAC  34  AGMCTCAAA  34  ATAAAAAAAA  33  TCTTTGATGT  31  ACWCGATA  31  TATATATGCA  31  A&AGAAGTT  29  ATATGACATA  29  ATATGTATCG TASAAAkkAA  28 28  TGATGGAAGC  28  2.006-32  60  70  2.006-21  46  64  hypothetical protein Schizosaccharomyces pombe  4.006-04  27  50  405 ribosomal protein all Schizosaccharomyces pombe  8.OOE-54  72  80  Protein biosynthesis  no significant BLAST results in nr database at NCBI SOs ribosomal protein L7a (L8) Schizosaccharomyces pombe  3.OOE-82  66  79  Protein biosynthesis  no significant BLAST results in nr database at NCBI 40S nbosomal protein S6B Schizosaccharomyces pombe  4.006-75  65  74  Protein biosynthesis  no significant BLAST results in nr database at NCBI ADP,ATP carrier protein Neixospora crassa  1.006-118  77-79 (2)  84-88 (2)  Cellular respiration  hypothetical protein Microbulbifer degradans 2-40 heat shock protetn 70 Cryptococcus curvatus SAGE tag does not hit C. neoformans cDNA or genomic sequence no significant BLAST results in nr database at NCBI no significant BLAST results in nr database at NCBI polyA tail ubtquinol-cytochrome-c reductase Sctiizosaccharomyces pombe  Response to stress  9,006-05  30  49  4.OOE-56  61  68  Response to stress  0.002-0.22  36-66 (3)  54-88 (3)  Cellular respiration  67  Sequence  Low Iron  BLAST HIT  E-value  % Identity  % similarity  GO Term  TATACCTATG  28  4Cc ribosomal protein s3ae (Si) Schizoseccharomyces pombe  2 006-85  68  81  Protein biosynthesis  0006.00  68  79  Csrbohydrste metsbolism  4.006-23  79  92  Protein biosynthesis  TAGTGTCCCG  28  TAGCUAGGA TGAA.AAAAAA  27 26  6-phosphogluconate dehydrogenaee Aspergillus oryzse no significant BLAST results In nr database at NCBI polyA tail  CTTTGSATCA  26  60s ribosomal protein L3OIL3OA Schizossccheromyces pombe  MCTTGATTG  26  CAGAGATGTG  26  GAAAGCCMG  25  no significant BLAST results in nr database at NCBI no significant BLAST results in nr database at NCBI SAGE tag does not hit C. neoformans cDNA or genomic  CATATTGAGT  25  uracil phosphorlbosyltransferaee Nesrospora crsssa  2.006-37  65  84  Nucleotide metabolism  25  chitin deacetylase Schizophylluw commune  5.00E-08  41  60  Cell wall  24  thioredoxin Coprinus comatus  2.OOE-24  47  69  Response to stress  5.006-56  52-58 (2)  61-64 (2)  Protein biosynthesis  7.OOE-18  45  67  Unclassified  GACATTrTGA TCAGAAGTTt3 GGCCGACCTG  24  ATTGAATGTA  24  ARCGGTTATG  24  ribosomal protein Li Schizosecchsromyces pombe oxidoreductase Clostridium perfringene ribosomal protein L3 Spodoptere frugiperda  1.006-44  65  75  Protein biosynthesis  23  40S ribosomal protein S28 Neurospore cresse  1.006-59  80  85  Protein biosynthesis  22  cytoplaemic ribosomal protein 612 Podoepore eneerine  4006-43  60  67  Protein biosynthesis  CAGCAeJTTA  22  no significant BLAST results In nr database at NCBI  TCOTCTGAAG  21  3006-98  61-66 (2)  78  Protein biosynthesis  CATTCCTTCA  21  CAGATCTTCT  21  1006-140  72  85  Signal transduction  TCATAGGTAC  21  TATTCATA4C  21  guanine nucleotide-binding protein Neurospore crease no significant BLAST results In nr database at NCBI 60S ribosomal protein L37 Seccheromyces cerevisiae  9.006-34  79  87  Protein biosynthesis  21  hypothetical protein Schizoseccheromyces pombe  4.006-52  41  61  21  ADP,ATP carrier protein Neurospore crease  1.OOE-118  77-79)2)  84-88(2)  Celluler respiration  20  60s acidic ribosomal protein p1 Schizoseccheromycee powbe  6.006-22  48  61  Protein biosynthesis  AAATGGTTTG  20  no significant BLAST results in nr database at NCBI  CACATTGATA  20  2.006-80  72  81  Protein biosynthesis  TCTAAGTATA  20  TGGATGGGCA  20  GTCGTAGAGT  20  4.OOE-48  69  81  Carbohydrate metabolism  TATGATTTTA MGGACTCTC  TATMGAGGT TTCGGCAAGG ACTACGTTCT  TCTCTTCCGT  19  TATTACAGCT  19  TAGAATAGAG  19  GACATATGAA  18  6ltS ribosomal protein L6E Schizossccheromyces pombe neoformans cDNA or genomic sequence  ribosomal protein LISA Xanthophyllomyces dendrorhous no significant BLAST results in nr database at NCBI no significant BLAST results in nr database at NCBI enolase 3, (bete, muscle) Homo sapiens carboxypeptidase Penicilliumjenthinellum 60S ribosomal protein L6 Arebidopsis theliene  1.006-35  69  77  Protein cetebolism  5.006-08  41  60  Protein biosynthesis  no significant BLAST results in nr database at NCBI alphe-amylase Deberyomyces occidentelis  1.OOE-21  33  55  Cerbohydrete metebolism  1.OOE-123  96  98  Signet treneduction  3.006-66  50  66  Protein biosynthesis  3.006-36  34-43)2)  51-62)2)  Response to stress  MTTCGCTAT  18  TAACCCAeAT  18  14-3-3 protein Schizophyllum commune 60S ribosomal protein L7 Ceenorhebditie elegene  18  glucose 1-dehydrogenase Bacillus subtilis  CATTACTGCA  68  Low Iron  BLAST HIT  E-value  % Identity  % similarity  18  glutathione Peroxldase HYRI Saccharomyces cerevisiae  2.005-38  49  63  Response to  IS  cytochrome C oxldase subunit IV Sct,izosaccharomyces pombe  8.OOE-16  48  58  Cellular respiration  18  metalloprotease (MEP) Asperglllus fumigatus  5.OOE-27  39  52  Protein catabolism  18  SAGE tag does not hit C. neoformans cDNA or genomic sequence  AGTM 5 CTT  18  SAGE tag does not hit C. neolormans cONA or genomic sequence  CATATCATA CAkAAAAAAA  18 18  no significant BLAST results in nr database at NCBI polyA tall  MCCTTGCAT  17  2.OOE—47  56-68(3)  65-78(3)  Protein biosynthesis  GCATTGGCGT  17  25-29  28  48  CCMTGACGT  17  3.005-99  85  93  Signal transduction  CATCGTTACT  17  GTP-bindlng nuclear protein Spilp Sct,izosaccharomyces pombe .hosphatidylglycerolIphosphatidy linositol transfer protein Aspergillusoryzae  1.006-19  39  65  Unclassified  RhoZ GTP binding protein Ustilago maydis  7.OOE-17  48  65  Signal transduction  0  48-66  66-74  Carbohydrate metabolism  1.OOE-33  39  57  Vessicle transport  5.OOE-35  67  76  Carbohydrate metabolism  7.OOE-14  43  57  56  70  Sequence CACTTGTTA AGATGA5TGG TGTATGGTCT  TGA.AAATAAA  ribosomal protein L2DA Sctiizosaccharomyces pombe hypothetical protein (Homo sapIens)  MCCMTGTA  17  AAAAA°.A.AAG  17  CATTCGCATA  17  GAATAGTGGG  16  CCAATGATTA  16  no significant BLAST results in nr database at NCBI aconitase Aspergillus terreus  AATTTATGAT  16  no significant BLAST results in nr database at NCBI  AATTTTATCA  16  no significant BLAST results in nr database at NCBI  GCCACAGCCA  16  SAGE tag does not hit C. neoformans cDNA or genomic sequence  AAATCTTATG  16  alpha-cop protein Sos primigenius  GO Term  stress  polyA tail no significant BLAST results in nr database at NCBI  ATP citrate lyase Schizosaccharomyces pombe SAGE tag does not hit C. neoformans cDNA or genomic sequence  ATTTATCAA.A  16  GAGMAAAAA  16  CATTTTATGT  16  ATGATTTGAA  16  no significant BLAST results In nr database at NCBI no significant BLAST results In nr database at NCBI  CCCATCGTAT  16  plasma membrane iron permease Schizosaccharomyces pombe  CTTGTAGAA  16  hypothetical protein Neurospora crassa  5.OOE-06  TAATGGTGC  16  nbosomal Protein RPL22  ANNOTATED  CATTTCTTCA  16  no significant BLAST results In nr database at NCBI  HATCATCCT  16  no significant BLAST results in nr database at NCBI  TAGCCGCGA’  15  no signifIcant BLAST results in nr database at NCBI  69  Protein biosynthesis  E. Pairwise and Multiple Library Comparisons. a. Pairwise comparison of 37°C low iron vs. 25°C libraries. A pairwise comparison was completed for the low iron library (LIM), (designated 37°C low iron) constructed here against the library from cells grown at 25°C in YNB (designated 25°C) (Steen et al., 2002) (Table 6). Information for tag abundance classes can be found for each library included in the pairwise analysis show in Table 6. Total tags used in the analysis are 19,278 for the 37°C low iron library and 30,468 for the 25°C library. At the 99.9% confidence interval, 0.82% (123 tags) were more abundant at 25°C vs. 37°C low iron and 1.28% (193 tags) were less abundant for a total of 2.09% (316 tags) differentially abundant.  At the 99%  confidence interval 2.15% (324 tags) are more abundant, 2.22% (335 tags) were less abundant for a total of 4.37 % (659 tags). At the 95 % confidence interval 6.02% (908 tags) are more abundant, 3.38% (510 tags) are less abundant for a total of 9.4 % (1,418 tags). Tags below the 95% confidence interval are not considered to be significantly different. Therefore, 90.6 % (13,669 tags) are similarly expressed between these two libraries at alpha=0.05. A graphical representation of these data is found in Figure 8. The 100 most differentially abundant tags were annotated for 37°C low iron vs. 25°C and assigned Gene Ontology (GO) terms where possible (Tables 7 and 8). All data was normalized to the library with the fewest tags. There were 32 tags of the 100 more abundant (32%) and 31 tags of the 100 less abundant (31%) successfully annotated with corresponding EST or genomic sequence, had a significant BLASTx hit in the non-redundant database at NCBI (<e-5) and were successfully assigned associated GO terms. Note that differentially abundant tags in 37°C low iron vs. 25°C and 37°C low iron vs. 37°C were highly similar, that is differentially abundant genes in 37°C low iron vs. YNB media regardless of temperature had a similar profile. Therefore, genes of interest will be discussed only in the following Results: section Eb. (the pairwise comparison of 37°C low iron vs. 37°C libraries) and the Summary and Discussion.  70  Table 6: Comparison of SAGE data for strain H99 cells grown at (3 7°C low iron) vs. YNB (25°C) media.  Interval Count Total  Interval Count Total  More Highly Abundant 25°C Less Highly Abundant 2S°C Total  Similarly Abundant alpha=0.05 Total  Abundance 1. 4353 4353  Classes for 25°C (Number of Tags) 2-4 5-9 10-99 1976 644 492 5184 4078 12077  100-999 26 4776  1000+ 0 0  Total 7491 30468  Abundance Classes for 37°C low iron (Number of Tags) 1 2-4 5-9 10-99 100-999 6602 2161 441 209 4 5424 6602 2760 3784 708  1000+ 0 0  Total 9417 19278  Differentially Abundant (Number of Tags;% of Tags) 999 % 99.0 % 123 0.82 % 324 2.15 % 193 1.28 O/ 2.22 % 335 316 2.09% 659 4.37 °h Tags 13669  Percent 90.60 %  71  908 510 1418  95.0 % 6.02 °h 3.38 % 9.40 °h  Crjptococcus (H99) 5.0  I  I  I  I  99.9% .  99% 95% . Sij. U  1 e02 5.0  2  U  I  U  U U  0 leOl 5.0  Ii  4. +4. + 4.+ 4+ .+ ++ ++++ + + + ++ 4+ + +..+--+ +++-+-+++,-+ +  + +  +  +  +  +  +  +  +  +++++++4+  +  +  +  +  +  ++++4  4.  +  +  +  +  +4.4.4.  U  —— — — •U  +  +  ++++..u  _. — I  5.0  U.  I  leOl  5.0  1e02  5.0  25oC  Total:15087 Figure 8: Expression profiling comparing relative transcript levels of SAGE tags from strain H99 cells grown in YNB (25°C) and at (37°C low iron) media.  The trumpet shaped lines represent the separation of the data by confidence interval. Dark blue crosses inside the lines indicate tags that do not show a significant expression difference. The grey line represents the 95% confidence interval, the yellow line 99 % and the black line 99.9%. The green dots indicate tags that show a difference that is significant with >95% confidence. Tags in the top left quadrant of the graph represent tags more abundant at 37°C in low iron and the bottom right quadrant represents tags more abundant at 25°C. The total includes the number of tags used in the analysis. The image was produced in Discovery SPACE www.bcc.bc.ca. Singleton tags were excluded.  72  Table 7: Differentially abundant SAGE tags from H99 cells grown at (37°C low iron) vs. YNB (25°C) media. * data sorted by ascending p-value Snquenue  25 u C  25nC Normalized  Low bun  Fntd Difference  TA1’ATGTGTA  31  19.5  285  143  T4.&8ATTI3CT  0  00  56  unique to LIM  TTAIOTIUTGT  5  3.2  66  25.9  ATATGWGA  23  14,5  102  7.0  TTGTAMA&5  0  0.0  48  uoiqun to LIM  ARGCCCCTTG  0  00  37  unique to LIM  TGACTOTTTA  57  36.1  t23  3.4  ACMTACCTA  0  0.0  36  uoique to LIM  I3I3AGACCAI3G  0  5.0  38  unique to LIM  ACARCTCAOA  2  1.3  34  26.9  ATTGAGATGG  27  17.1  72  4.2  GMAGCCA6G  0  0.0  25  unique to LIM  TATATATGCA  3  1.9  31  163  AGTACTCTTC  it  7.0  43  6.2  ACMACGATA  4  2.5  31  122  ATIG6ATGTA  1  06  24  379  TCTPAGTATA  0  00  20  uniquo In LIM  TGTTATCGGT  9  5.7  37  6.5  CTTTGGATCA  3  1,0  26  13.7  AT6A5ASAnA  7  44  33  7.5  GACATATGM  0  0.0  18  uniqon In LIM  GMAOAAW  21  13.3  50  3.0  CCASTGATTA  5  0.0  18  unique to LIM  QCCACAGCCA  0  00  16  unique to LIM  ATGAT1TCA6  S  0.0  16  unique In LIM  TCGI3GGCTGC  0  0.0  15  unique to LIM  TAOCAATGTA  0  0.0  15  unique to LIM  GACATTCTGA  0  0.0  15  unique to LIM  MCTIi3ATTG  5  3.2  26  8.2  MASATCATC  0  0.0  14  uniqun to tIM unique to LIM  BLAST HIT titan Snook Protets 15 Saruhtnumyuns unleviniun SAGE tag dnnn not hit C. snntnnmans nDNA or snnsmin no signiflnant BLAST results in or database at NCBI C1pC Emnhnnlln nidnlnnn so signiflnant BLAST rnnuttn is sr databasn at NCBt SAGE tag dons not hit C. seufsrznans nDNA or gennmis Heat Shook Protein 12 Saunharumynnu pastodanun ,  SAGE tag hits morn than one nnqnnsoe nnntlg SAGE tag does nno hit C. nsmtnznsans nDNA or genemin 405 rihosnmal protnis 0GB Subizusauubarornyuns pnrnbn no slgsifioant BLAST results in nr datahann at NCBI SAGE tag dunn not hit C. nsolnrmans nGNA on genomin heat shook peooein 70 Cryptunnonus uurvatun dhosurrral protnis P2 Pnduspum annnnira hyputlretinal pmteis Miurobulbifnn dnqraduns 2-40 aloshol dehydrugenase Banillun nnblilis no significant BLAST nasulls in nr delebeun at 0091 hnat shnuh protein 90 hnmnlng Candida albinans Bits rihnssmnt protein L3OIL3OA Suhizosauoharumyuns pombe on signiguant BLAST results in or database at NCBI alpha-amylane Dnhnryomyuns nuuidnnlslis polyA tail aunnitasa protein Beninroidas fragile SAGE tag dons not hit C. sooturmans oDNA or ganom’m so siqnit’mant BLAST results is nr database at NCB1 SAGE tag dons not hit C. seotnrsnans nDNA or sennm’m Putaoine S.AIdP-aotiuatnd, garrsna subunit family Subizunauubnrnmyuns pnmbe SAGE lag doss not hO C. nentnrmans nDNA or gennm’m ns signiitsant BLAST resells in sr database at NCBI Tag hits In wrung nrinetatinn on BLAST hit SAGE tag does nnt hit C. neotnrmans oDNA or gesomin shitin dsanstylase Suhizuphyllum nnmmnnn no signiBnast BLAST rnsnlts in nr database at NCBI AlP nitrate lyasa Ouhiznsannhannmynns pnmbn en signifloant BLAST rnsutts in nr database nt NCBI hypothntinat protein Aqaninus bispoms SAGE tag doss not hit C. neotormans nDNA nr 5550mm hypothetical pmtsmn Plasmudium 50018 ynnh SAGE tag doss not hit C. seofurreans oDNA on gnnnmlo SAGE tag dons not hit C. seotnrrsans nDNA on gesomlo dOs rihosomal protein sli 5. ynmbs SAGE tag does sat hit C. nantarmans nDNA on gennsdo protnasnme nsmponnnt P052 hnmnioq Suhinusauuharnmyues pumbe Tag hits is mrnsg nrlentatins on BLAST hit Bnta.tnbntin nntantor D Homo salnnns nn signifloant BLAST resuBs is nr database at NCBI no slgniooant BLAST result is nr databasn at NCBI  TATATGCGTA  0  0.0  14  GACATTTTGA  8  3.8  25  6.8  CA1TTTATi3T  1  0.9  16  25.3  ATITATCW  1  0.9  16  25.3  MTTTATGAT  I  0.6  16  20.3  Ai3GATGAGM  15  95  37  39  CTTASAGTM  2  1.3  19  142  Gi3TCAGTCISA  0  0.0  13  unique In LIM  TATATGTGCA  0  0.0  13  unique In LIM  TGACTi3TCTA  S  05  13  unique In tiM  GCATICT1TA  IS  95  35  3.7  AI3TTTCTTGT  0  0.0  12  unique to LIM  ATIATMCGA  0  0.0  12  uniqun to LIM  ATATTCATAA  0  0.0  12  unlqun to LIM  TATACGTGTA  I  0.5  14  22.1  CATATI3TGTA  1  0.6  14  22.1  AOTTACTGQT  0  0.0  Ii  unique In LIM  CATTATATAT  0  0.0  Ii  uniqun In LIM  tag hOn 2 nnnttgs at DUKE  unique In LIM  Wrung orientation nt SAGEtag to BLAST 50  TAOITCGTGT  S  0.0  II  73  E-nalue  to identity to similarity  GO Term  1.505-06  45  61  Renponne tu s0uus  1.005-08  5270 131  72-76  Unulunnified  S n.04  55  73  Rnnpnnue In nonss  4 050-75  55  74  Proteir Bionynthosis  4.565-50  61  56  Snspunse lu utroun  1.OOS-26  57  65  Pruinir Biunynthnuis  7.WE-26  9.005-OS  30  40  4,005.22  44  53  Carbotiydneln mulabolism  2.WE-32  00  70  Rnnyunun to utrnnu  4555.33  79  52  Protein Biuuynlhosiu  1.OOE-21  33  55  Carbohydrate mntubulium  3.OOE-06  56  75  Carbohydrate metabolism  1.560-37  34  58  Orqnnl Iransdununr  5.000-00  41  80  Cell wall  0. OOE-3S  67  76  Carbnhydraln metebnlism  2.OOE-21  40  64  1.060-17  12-77121  50-09121  0.WE-04  72  85  Protein Blnsynsresis  2. 000-27  50-95121  74-95(2)  Protein Catebulium  5.000-44  32  47  Cell gnnoth andlor maintsnanuo  4.OOE-13  33  49  2SoC  Sequence  25cc  Normalland  GTATGA°AGA  0  0.0  II  unique to LISA  CATCMCCTG  0  0.0  11  unique to LISA  MAuAAAeAG  3  1.9  17  So  CAGTTATTM  2  1.3  15  11.9  Low Iron  Fold Difference  TrGTrAGATr3  2  1.3  15  11.9  MCTGTTGTA  1  0.9  13  200 7,1  CATATOATAA  4  2.5  18  CTTGTAGA°A  3  1.9  16  9.4  CTATGCACAA  0  0.0  10  unique to LIM  TTATGGGTAO  0  0.0  10  unique to LIM  TATGCA°A°A  0  0.0  10  unique to LIM  TGGPA°A°A0  0  0.0  10  unique to LIM  CT1’TGAGTCC  0  0.0  10  unique to LIM  TATATGTGTG  0  0.0  10  unique to L1M  A°ACTTTCM  0  0.0  IS  unique to L1M  CTTCCTGTM  S  0.0  10  unique to L1M  TTTCGTCMT  0  0.0  10  unique to LIM  TATMOTGTA  0  0.0  10  unique to LISA  TATTAOAGCT  9  32  19  9.8  TATGATTTTA  9  0.1  23  4.5  TATATAOTTG  I  0.9  12  19.0  TATOTMOCA  1  09  12  190  MCGMTGTA  4  2.5  17  6.7  TATTCATMC  7  4.4  21  4.7  TGP.OAOAkOA  Ii  7.0  26  3.7  CAOTTACAGT  3  1.9  19  7.9  ATOTATACCC  3  1.9  15  CA&k°ACATC  3  1.9  15  7.9  GTTTTATGGA  3  1.9  15  7.9  T,OAIOAOAOA  13  8.2  29  3.4  TOAGATTGCT  0  0.0  9  unique to LIM  TGCOA°A°A0  0  5.0  9  unique to L1M  GMTMTAGA  0  0.0  9  unique to LIM  TDTATGMTT  S  0.0  9  unique to LIM  MTGTGCCAT  0  0.0  9  unique io LIM  T°ATCATAGC  0  0.0  9  unique to LIM  CATTTGCATA  0  0.0  9  unique to LIM  TTTTMGCTA  0  0.0  9  unique to LIM  TATTCTACAC  0  0.0  9  oniqoo to LIM  TTGTATGATA  S  0.0  9  unique to LIM  ATATCCTOCA  S  0.0  9  unique to LIM  -  7.9  r  [  BLAST HIT  B-caine  tag bite 2 oontigs at DUKE Bod2Op Oaooharonnyoeo oeneolnlan OS ribosomai protein S23 Nooroupora onaooa SAGE log dens not hit C. nnofnrmans oSNA or gnnomio aiginate iyasn Hatotin dinous htnnai Genemio Hit too oiona to S End of Contig SAGE tag does not hil C. naofnrnnans oSNA or gnnomio SAGE tag dens not hit C. nnafamqans oSNA or gonomlo SAGE tag hits mam than one saqoenne oentig SAGE log dens not hit C. naoformans oSNA or genemio 905 nibonomai protein LB Anabidopsin thatana dOS elbonomai protein S25 Naunonpota or0000 no nigoifroant BLAST results in or database at NCBI ooosneond hypothntinai protein. yeast Candida oibioaon Bho2 GTP binding pmlnln Untilaqo may40 fibS nibonomai proteIn L37a Saooharornyoan oereoisiae SAGE lag dens not hit C. neofarmans oSNA or gnnomio hypothetical protein Neur0050na onaooa Translation Eiongatias Faotor3 nytonhroma ci pr000rnor Neuronyona oraooa Heal shoob prolsis 60 Oaooharowyosn oareoloiaa peivA tall Long-ohain-taBy-aoid-CoA rgaso-rho protein Arobidofuin thaliane SAGE tag does not hit C. nnolnrmass oDNA or gasomio so oignifinant BLAST reeoits In or database al NCBI no nigni9naot BLAST results in no database at NCB1 Wrong odsntatien of SAGEtag to BLAST 55 hypothotloal iorntuis  GCTGTAGTAT  S  0.0  9  unique to LIM  CAGAACGACG  S  0.0  9  unique to LIM  TAGGTTI3TCT  0  0.0  9  oniqoe to LIM  TTCGAGAGCT  S  0.0  9  unique to LiM  dihydrotiaoonoi 4-eaduotasa S0000anomyoen onrnoioiae  TACATATATT  2  1.3  13  10.3  ATATT7CTAT  I  0.6  ii  17.4  I  5.8  ii  174  A°ATCTTATG  4  2.9  16  83  CACATIGATA  7  4.4  20  4.0  TGA°.°ATASA  6  3.8  18  4.7  GO Term  eoioA tail no significant BLAST results in or database at NCBi No eignitioant BLASTu hits near tag no significant BLAST rasoito in or datahasa at NCBI no signifinani BLAST resaits in er database at NCBI no significant BLAST resaito in nr databana at NCBI  Neuronpona ora000 no significant BLAST reunil in or databasa at NCBI Wroog onlonlatlon of SAGEtag to BLAST hfi pradiotad protein Neuronpora onanna Wrong annotation at SAGEtag lo BLAST h9 SAGE tag does not hit C. naotormann oDNA ergonomic no signifroant BLAST result lo or database at NCBi SAGE fag does nol hit C. naoformann oDNA or ganomlo SAGE tag does net hit C. oaafoomaoo nSNA or genomlo  CATATAGA°A  % density % similarity  SAGE lag dccc not hit C. seoformaos oDNA or ganomio SAGE tag dccc not hit C. oeofermans oDNA or gnnomio  no nignifloasl BLAST nosuito in or database at NCBI nobaryolin translation Initiation faotor 4A2 Mon monouiuo so significant BLAST mnoits In or database al NCBI aipha.oop proloin Bos yrimiqanios ribonomal prolnin L13A Xanthophytiomyoas dnndroAooo SAGE lag does sot hit C. aeetormaos oDNA or ganemlo  74  7.OOE-tt  44  57  Call growih and/on maiotonanoe  3.009-45  59  61  Protein Biosyothesiu  2.0GB-IS  30-40  45-93  Carbohydrate mntabolism  5.000-OS  41  60  Protein Biosynthesis  lOSE-OS  80  85  Froteio Biooyothaoio  S.OOE-09  27-29  41-46  7.OOE-17  48  60  Siqoal inaosdooSon  9,00E-34  79  87  Pnoteio Siooynihaols  1.000-to  26  43  3.000-55  04  67  cellular neopiraiioo  4.000-56  99  54  Oe000noetouSoos  9.000-29  34  49  Lioid and Sterol Meiaboi’om  0 000-W  31-56  43-75  0.OOE-12  45  07  9. 000-05  7  7  I.OOE-08  31  53  ResE0000 lo o0ooc  7.000-23  77  90  Protoin Biooonihnsin  1.000-33  39  57  V000iola Oansyod  2.000-80  72  51  Pnoiain Biosyoihaoio  ANNOTATED  Pnoieio Siooynthaols  Table 8: Differentially abundant SAGE tags from H99 cells grown in ‘[NB (25°C) vs. (37°C low iron). * data sorted by ascending p-value Sequence  250 C  250 C NormalIzed  Low Iron  Fold Difference  1]CAGCAGGC  490  310.0  II  28,2  CTCAGCGATG  384  243.0  9  27.0  CATTCGCATA  342  210.4  17  12.7  GCCANCGCCG  213  134.8  I  134.0  GCTCTCCAGG  189  110.8  I  119.6  CATCTGTTCC  187  118.3  3  39.4  ATAAGC1TTC  162  102.9  6  17.1  GTTICCGCTG  ISO  94.6  5  19.0  TCTDGTCGAG  131  82.8  2  41.4  CGCGDMAGG  167  109.7  9  11.7  GTGGACACGA  132  83.0  3  27.0  AGCGAGCACT  124  78.9  2  39.2  ATATGACATA  232  146.8  29  5.1  GTATTGACCC  119  79.3  2  37.6  GCTGCCTACA  96  62.0  0  ooiqce to 25  ATGATCGGGC  119  72.0  3  24.3  ACGDTGGCM  95  60.1  0  unique to 29  GGTTACGCCQ  93  58.8  0  unique to 28  TAGCCGCGAN  162  102.5  15  6.8  CGACAGACCG  239  148.7  36  3.9  GCGTTCTCGG  87  85.0  0  uoiqueto2s  CCGCGACCGT  102  64.9  4  16.1  MTGMTCTT  132  83.9  11  7.6  AaAzACGCGT  109  69.0  6  11.5  OTCGGTGGTA  149  86.6  14  6.3  ACTCAGGTTG  91  07.6  3  19.2 28.3  ATGCATTTCD  83  92.5  2  ACCGTCGITG  75  47.5  I  47,5  GCTCGCOACG  77  48.7  2  24.4  GGTATCCTCG  62  39.2  0  unique 1020  ACGOCCGTTA  61  38.6  0  ueiquo to 20  AGCOCTOCTO  61  30.6  0  uoiqoo to 20  CATCACTCTT  107  07.7  11  6.2  CACGTTCACG  64  40.5  1  40.5  TTCGGCPAGG  137  86.7  21  4.1  MCOTCTGCC  80  50.8  5  10.1  CCTCANCGGC  54  34.2  0  unique 1025  CGTGTCANGC  54  34.2  0  unique to 25  TCTTTCCOAG  65  41.1  2  20.6  AT0000TCCC  53  33.0  0  unique to 25  GCTGGTTTGA  93  33.5  0  unique to 25  TGGTGGGMA  53  33.5  0  unique to 28  ANGCCCGfl’O  59  37.3  1  37.3  OCTTTTGCCC  59  37.3  1  37.3  CATCACGCTT  62  39.2  2  19.6  GTTGGCMCG  50  31.6  0  unique to 25  ‘  BLAST HIT  potatine motel traeaporter Schizosaooharomyoes pombe oytokiee ieduolog-glyooprsteie Filobaoidiella reotormano no significant BLAST meulte leer database at NCBI Cyolophille A SAGE lag doee not hit C. eeotnrmans oDNA or genowlo no slgnllloaet BLAST reeslle leer database at NCBI maeeltot-1-phusphate dehydrogesase so slgsifloaet BLAST resells leer database at NCBI Histoee 54 protele Moo wuoculos so sigeifioaet BLAST resells leer database at NCBI euolaeslde dlphosphate-eugar hydrolase Snhizosaooharomyoes so elgnifloaet BLAST beetle ieee database at NCBI so slgeifloant BLAST results in er dalabaee at NCBI phosphebetolase Thermosyooohoyooyos elortatos 5TP eyethase ohgomyole seeeltloity ooeterral protein N. onassa SAGE tag does not hit C. eeeformaes eDNA or genomle en sigeifioaet BLAST results In er database at NCBI mitnohondrial malate dehydrosesase 0. no sigelfloent BLAST results leer database at NCBI Translation eleogatlee factor I (TEPI) tranealdolase Soh’oosaooharomyces rombe hypothesoat protein Neurospona crassa no sigeifioaet BLAST results in or dalabese at NCBI myo-ieoe9ol i-phosphate synthase Neorospona onassa Ft ATFase beta subuns lyoyyeromnoes taotis Nuotose biphesphale aldolase Porecoooidioides brasitiensis no significant BLAST results in or dutahase at NCBI tñosephosphate isomerase Asporgillos oryzee Ribesomal Protein Lt Tenopus laenis 03 smell nuoleolar riboeeolaoprutein prolate IMP3 S oeran’s’ao SAGE tag hits more thae oea sequeeoeooetig Hlstsse HI; Hhotp Saooharomynes conenistee pyrenata deoarbooylase Candida glabrata thioredoole perooldase S. pombe ADPATP oaerlar protein Nosrospora onassa probable membrane protein YLRIS2o RIRBNcDNA Moe masoolos no slgoifioaet BLAST results In cc database at NCBI glyoeraldehyde3-phesphate dehydrogeoase Fitobasldiolta ATP synthase gamma ohale Snhiz0500ohanomyoeo pombe glutamate eoaloaoetate traesamieaee Dante rndo eterol-C5-daeaturase Mon mosoolso no slgoifloaet BLAST resells leer database at NCBI hypothetloal protele Sohtaosaoohanomycos pombe seed maturation protele PM27 Gtycioe mao related to Ypt-ieteraotleg protele TIP2 Nesrospora orassa related to aide-hole radestase TPRI NeurosBsra orasea Unhoewe Streot000500s a9alaoriao ATP syethase G chaIn, mitoohoedrial Sacyharonnycos SAGB tag does not hit C. oeotormaes oDNA or genomlo  E-nalue  % identity  % similarito  6.000-52  36  50  DO Term  Transpod  ANNOTATBD  Unolassifled  ANNOTATED  Signal Transduction  ANNOTATED  Carbohydrate metabolism  9,008-30  75  80  Chromosome organ’oohon and biosynthesis  7.008-26  35  St  Nucleobase Metabolism  2.000-32  46  61  Carbohydnato motaboliom  6.00849  43  63  3 OOE-37  65  79  Canbohydrato metabolism  t.009-lt4  68  78  Carbohydrate metabol’sm  4.008-09  48  74  2.008-67  54  70  3,00B-03  80  91  Cellolar Respiration  1.008-40  70  82  Carbohydrate metabolism  ANNOTATED  Fnotein biosynthesis  Carbohydrate metabolism  9.008-50  52  72  Carbohydrate metabolism  5.OOB-102  72  82  Protein biosynthesis  9 008-50  59  74  Cell qromlh and/or mainleoaooo  9.008-05  45  61  Chromosome organization and biosynthesis  5.008-26  52  66  Carbohydrate metabolism  2.008-63  BI  76  Respoesa to stress  l.OOB-llo  77-79(21  84-88(21  Cellolar Respiration  3 OOE-04  35  51  Transport  5.008-42  62  75  Unyl055ibed  4.008-63  96  100  Carbohydrate metabolism  t.OOE-66  47  70  Carbohydrate metabolism  8.008-98  56  72  Amino acid metabolism  7.000-17  62  73  Lipid and storol metabolism  -  -  CAGACGTANC  49  31.0  0  unique to 25  CCGAGACMC  60  389  2  190  ACACGTCTOO  92  50.2  10  5.8  CACCTCANOC  58  36.7  2  18.3  75  8.OOE-06  36  60  2.000-07  29  46  Unclassified  9.OOE-30  44  58  Cell growth and/or maintenanco Carhohydnata metabolism  9.OOE-13  38  59  2.OOE-07  42  57  7.OOE-05  31  50  Cellulan Respiration  Sequence  25cc  25 a C Normalized  Law Iron  Fcld Difference  TAGCGATCAC  159  100.6  34  3.0  CCTGATCGCG  44  27.6  0  unique Ic 20  OCOOACMCT  50  31.6  1  31.6  TCCTGGCANG  43  27.2  0  unique to 26  CACCAGGCAT  42  26.6  0  oniqun to 25  TCGOTCGTGT  42  26.6  0  unique to 25  AOGCTTOGAQ  42  26.6  0  oclque to 25  TGC4ANCGCG  73  46.2  7  6.6  GGCCTCGGTT  57  36.1  3  12.0  ACCTTGA000  47  29.7  1  29.7  TCTGCATCAT  47  29.7  1  29.7  TCTGTCGAGG  52  32.9  2  16.5  TCAGANGTTG  123  77.6  24  3.2  ANGCCTGACG  67  42.4  6  7.1  OCC4ACTCTC  55  34.8  3  11,6  TA’rfcCGGTC  59  37.3  4  9.3  TCCACCAYI’A  39  24.7  5  unique to 25  ANGCCCOACT  39  24.7  0  uniqun to 29  ATCGGTACCC  58  36.7  4  9.2  CGAGOTATCA  38  24.0  0  unique to 25  OAGMGCGTG  37  23.4  0  unique to 25  AGCANOGAGG  64  40.0  6  6.7  UCTCCTCTTA  73  46.2  9  5.1  CAGMCCCCG  58  36.7  S  7.3  BLAST NIT nc siqsifioant BLAST results in nr database at NcBI unnamed ponteis product Podospora rnnndnn sccteostde diphcsphate binasn Emndcnlla nidulacs profilin Malon 0 domeslioa PS Goscypium hiroutom predicted protein Naurosporu oranno nn significant BLAST rnsottq in nr database at NCBt pnrooisnmat membrane protein Pmp2Op no significant BLAST results in sr database at NCBt SAGE tag dnns not hit C. ncntormans cGNA or gnncmio hypotbntioal protein Borkholdnria tongorum dbcsomal prcteis S12 Branohiostoma bobbed Thiorndoois Copdnun ucmatus nc signifinest BLAST results In nr database at NcBI RNA-bindtng protein AoRNBP Ambystoma meoioanum arqlnincosocinatn nynthasn Stuohatomyonc unrevisirn mangannsn sspnrooide dismotase Phanoruchanto chrynonyorium Lg-Hspl2p Sacoharomycen pastorianon phosphate transport protein S. cereolsian NADPH-dopendnnt atdnhydn redoutase Sporidiobotos oolmonioolor Ribucomal Protein RPL2IA S. onrnuisiao Acyl-CnA-binding protein Chactophraotos villosus ATP synthasn alpha chain N. orassa hypothntlcat pratnin Neorospora orossa ribosumat protein L24 Etuo’unromuons lactic SAGE tag hits more than one sngonnon contig no significant BLAST resolts in nr database at NCBI Wrong onientntlun SAGEtag tn BLAST hit hypothetical prctnls Nnurospora upacca MIS rlbcsomal protein 926 Soh’oophylluw commune nc significant BLAST rnnolts io nr database at NCBI 6-phcsphcqlocooatn dehydrcgecasoAspnrqilloo oryzan hypothnhcal protnin Sch’oosacoharomyoos pombe brain prntnin (3F505) Caerortrabdiss etogacs nc significant BLAST rnsclts is nr database at NcBI SOhA Acpnrqillos nidolans nc signifloast BLAST results is nr databose at NCBI nc signlgoost BLAST results in fir database at NcBI  0-cube  % identity % nimitarity  GO Term  5.000-32  30  2.000-46  57  77  Nuolnobace Untnbutsm  0.OOE-22  42  60  coil growth and/or maintenance Unotassiled  54  1.000-20  44  69  9.008-00  29  38  1.000-21  40  52  Response to stress  3.000-06  31  46  2.050-43  56  81  Protein biocunthesis  2.OOE-24  47  69  Response to chess  3.000-14  60  78  Cell growth andtor waietccacco  3.SOE-04  g  83  Amino auid metabolism  4.SOB-73  08  76  Response to ctress  0.001  55  73  Response to strocs  4.OOE-97  00  71  Traespod  8.000-60  40  01  Carbuhydratn metabolism  4.000-52  54  76  2,508-10  47  65  Lipid and sterol metabolism  1.000-66  77  84  Cnlbolan Respiration  2.000-58  76  86  2.OOB-34  54  07  Protcin biosonthecis  -  ASGGGTOOTG  69  437  9  4.9  GAGGAGGAGG  47  29,7  3  9.9  OOCCcAGACA  31  19.5  0  unique tn 25  CCCcGTcAcT  31  19.6  0  unique tn 29  CCTCGTATCG  30  19.0  0  unique to 25  ANGCGAflTt’  71  44.9  11  4.1  TCGTTATCTI’  28  18.3  0  uniqun to 29  ACGGCCAuAC  29  18.3  0  uniqun to 29  GACGACTCTA  47  29.7  4  7,4  CCACCARTGC  35  22.1  1  22.1  O4ATAGT000  83  52.5  18  3.3  ATGCACCCAT  28  17.7  0  unique to 25  TCCGACCACT  28  17.7  0  unique to 25  CCOc1TITGC  28  17.7  0  unique to 25  CAATTCG  28  17.7  5  unique to 29  GMGTAGAuA  42  266  3  99  MGMGACCG  27  17.1  0  unique tn 25  GTCCOANGcA  32  20.2  1  20.2  GCCATcTTCA  44  27.8  4  7.0  GACTCOACGA  26  16.5  0  unique to 25  ANGGAGATTC  26  16.5  0  uniqun to 25  TGC1’TCTGTG  26  165  0  unique ro 25  CGANGACTCA  26  16.5  5  unique to 25  GARTGGMTG  31  19.6  1  19.6  OATGGCAGUG  31  19.6  I  19.6  OTCAAGANGc  25  15.8  0  0010cc to 25  Potyohiqoiin mitnchcnddat ATPase alpha-ssbcnit N. cracsa SAGE tag dons not hit C. nnntcrmanc cGNA cr genomlo cpmbtcsis-rntetnd protein Laccoria bioolor pro-mRNA splicing tautor S. pombn hppcthntical protein Nnorospora crassa utathris tight chain Sch’oosacoharomucec pombn SAGS tag hits morn than cnn seqonncnocntig DNA Unwinding Factor DUPO7 Xnnopos landis gtyccgen branching enzyme Acpnrqilbon orqzae SAGS tag dcns not hit C. nnotcrmans cSNA cr gnnnmtc potatioe protein *--, ----.  76  Protein hiosyethosls  1.OOE-13  47  03  5.000-20  56-79131  66-97131  Protein bioopnthesis  0  g  79  Carhohcdmte motabulicm  g.OOE-21  47  81  0008-14  6.000-23  38  47  45  63  Uculassirad  77  84  Ccttutrr Respirstino  ANNOTATED 1.000-56  Unclassified  protein uatabollnm  3.005-56  35  40  Ueolassiled  7.008-52  45  60  Cr11 gnowth and/or maintnnnnue  9.000-30  44  50  0.001  32  49  3.000-04  20  45  DNA reptuation  0.OOE-42  44  56  Carbohydrate metabolism  4.000-28  34  49  Tmacspoo  b. Pairwise comparison of 37°C low iron vs. 37°C libraries. A pairwise comparison was completed for the 37°C low iron library against the library from cells grown in YNB (3 7°C) (Table 9). Information for tag abundance classes can be found for each library included in the pairwise analysis (Table 9). Total tags used in the analysis are 19,278 for 37°C low iron and 38,988 for 37°C. At the 99.9% confidence interval 2.3 1% (370 tags) were more abundant in the 37°C vs. 37°C low iron and 1.81% (290 tags) were less abundant for a total of 4.13% (660 tags) differentially abundant. At the 99% confidence interval 4.78% (765 tags) are more abundant, 2.72% (435 tags) were less abundant for a total of 7.50 % (1200 tags). At the 95 % confidence interval 7.93% (1269 tags) are more abundant, 4.33% (693 tags) are less abundant for a total of 12.27 % (1962 tags). Interestingly, this library from cells grown at 37°C is less similar to the low iron library also grown at 37°C than the cells from 25°C. Tags below the 95% confidence interval are not considered to be significantly different. Therefore, 87.73 % (14,031 tags) are similarly expressed between these two libraries at alpha=0.05.  A graphical representation of these data is found in Figure 9.  The 100 most  differentially abundant (more and less) tags were annotated for 37°C low iron vs. 37°C and assigned Gene Ontology (GO) terms where possible (Tables 10 and 11). All data were normalized to the library with the fewest tags. There were 40 tags of the 100 more abundant (40%) and 55 tags of the 100 less abundant (55%) successfully annotated with corresponding EST or genomic sequence, had a significant BLASTx hit in the non-redundant database at NCBI (<e-5) and were successfully assigned GO terms. For the purpose of this study only these tags will be discussed. As mentioned in Results section Ba, (the pairwise comparison of 37°C low iron vs. 25°C), differentially abundant tags in 37°C low iron vs. 25°C and 37°C low iron vs. 37°C were highly similar. Therefore the genes will be discussed in this section only and the 25°C and 37°C libraries will be referred to collectively as “YNB.” Tags of interest that were more abundant in 37°C low iron vs. YNB were (Fold change): iron permease (16), HSP12, 60, 70 and 90 (5-62), glutathione peroxidase (12), CipC (10), chitin deacetylase (25), 5’AMP activated y subunit (15), Rho2 GTP binding proteins (17) Interesting tags that were less abundant in 37°C low iron vs. YNB were (Fold change): polyubiquitin (28), Ubiquinol-cytochrome-c reductases (3 8-42), high affinity copper transporter (30), CuJZn superoxide dismutase (17), carbohydrate metabolic genes (6-93) and a phosphate transport protein (11).  77  Table 9: Comparison of SAGE data for strain H99 cells grown at (3 7°C low iron) vs. YNB (3 7°C) media.  Interval Count  Total  Interval Count Total  Abundance Classes for 0 37 (Number of Tags) C 1 2-4 5-9 10-99 4183 4183  841 5467  1899 4996  642 14799  100-999 48 8383  Abundance Classes for 0 37 low iron (Number of Tags) C 1 5-9 2-4 10-99 100-999 6602 2161 441 209 4 6602 5424 2760 3784 708 Differentially Abundant (Number of Tags;% of Tags) 999 0/0 99.0 % 370 290 660  2.31 % 1.81 % 4.13 %  Similarly Abundant alpha=0.05  Tags  Percent  Total  14031  87.73 %  More Highly Abundant 37°C Less Highly Abundant 37°C Total  1000+ 1  765 435  1200  78  4.78 % 2.72 % 7.50 %  1160  7614 38988  1000+  Total  0  9417  0  19278  95.0 % 1269 693 1962  Total  7.93 % 4.33 %  12.27 %  99.9% 99% 95% Siq.  5.0  1 02 :  5.0  2  leOl 5.0  5.0  leOl  5.0  1e02  5.0  1e03  370 C  ®  Total:15993  Figure 9: Expression profiling comparing relative transcript levels of SAGE tags from strain H99 cells grown in YNB (37°C) and at (37°C low iron) media.  The trumpet shaped lines represent the separation of the data by confidence interval. Dark blue crosses inside the lines indicate tags that do not show a significant expression difference. The grey line represents the 95% confidence interval, the yellow line 99 % and the black line 99.9%. The green dots indicate tags that show a difference that is significant with >95% confidence. Tags in the top left quadrant of the graph represent tags upregulated at 37°C in low iron and the bottom right quadrant represents tags upregulated at 37°C. The total includes the number of tags used in the analysis. The image was produced in Discovery SPACE www.bcgsc.bc.ca. Singleton tags were excluded.  79  . I I  •  Table 10: Differentially abundant SAGE tags from H99 cells grown in at (37°C low iron) vs. YN’B (37°C) media. * data sorted by ascending p-value Suqunsoo  37cC  2? cC nunrnagaed  Lea Ian  Fold Dtttorcuce  BLAST HIT  B-nellIe  % Idanrtlny  to sbtdlaAny  GO Tmm  TATATGTGTA  0  0.0  290  unAnmo lu Lea Irum  Heat Shack Preteht 12 Se000aromooes oereuieiee  I ODE-OS  45  Al  Rescosue to streus  TOACTOITTA  4  20  123  02.2  S e.04  50  73  Roueaouo to stress  TAeASTTGCT  0  65  65  aniraluLewIrun  ATATOPuIuSGA  21  t04  102  u.n  1.020-SB  52-70(31  72-TA  Uratoqodted  ITGTA&4°A5  o 3  0.0 1.5  46 55  ainunlnLosnlrunn 30.7  O  00  37  uninue lu Lou Ims  CIpC Emeooete o.drdaes nu slgsltloanl BLAST results In er databaso at NC BI cubA tell SAGE lag dues net hIt C. neotettnaes eDNA cc genumle eeguunee  uniuun lu Lou Irun  SAGE leg hAs mere thee net segaenoE oentlg  unineo to Lea Irus 2.4  ou00000aoo  4500000110  Heat Shack Prulele 12 Sac000rurnoons eastoniorna SAGE tag does not hA C. neotetmoes eDNA He geeeede sequanee  ACMTACCTA  O  00  36  QQASACCAI3G  o ISA  o.o 03t  30 203  ATOCACASTA  I  0.B  37  74.A  CAS005TSAT  o  0.0  34  uninun to Lou Iruo  NeoN sore eeIoA tail Be slgsllleanl BLAST results In Cr dulabase at NCBI no elgnltloeet BLAST results in er database at NOEl  TATSATASTS  0  0.0  34  uniquu Ia Lou Ian  605 dbusumal prutein L7a Lu) Ootiausaooherumuoes combe  ACA&SCGATA TPAI,aAkaAS  o  31 28  unioLto Ia Lom Ian uoiquo to Lee Ian  Norrothetloal prelele Morabslbtendenradaus 2-40 eolmA tall nlgnltloanl BLAST results In ne nr databaseatNOEl 4n5 dbosamal peetein StE Sohensacoherumoces oombe  o  o.o o.o  TA0CTTOGGA  o  0.0  27  uninun le Lou Iron  A0ASCTCPAS  3  t.5  34  22.9  ATAT0ACATA  I  0.5  29  507  414-5045010  1  0.5  29  55.7  SCOTT CTIA TGAt.IA5ASA  4  o  20  oo  35 20  17.7 unbnseloLonnlrorn  TATATATGCA ATAeAnAa,aA  2 3  to 15  at 33  31.3 223  GpAa000450  o  o.o  25  uninun to Lea Ian  ATTGAeJGTA  o  o.o  24  Le0500loLewlrun  O0TACTCT’TC  11  5.4  43  7.5  ATATGTA000  2  to  2A  003  nu slgrdtloanl BLAST results In ttrdatabasealNCBl  n-s4  72  50  Patois biosonlhesm  42*-OS  01  60  Rnmro.mo to stress  eclduredsutaso Cleslnidkan cenlrieooes nlbusattnal pruleltt P2 Pudoseana ansan’na  7.660-15  45  57  Urclassioed  1030-26  57  65  Protein bkrsqnitrnoie  203k-no  GA  81  Protein bbenetdtnenb  4,000-04  27  00  10  28  253  35  30  104  hyputhelloal prencltr Sch’eosacch.mrnooos eomhe  AGOATSASM  O  40  37  9.4  TOTTA0000T  o  to  37  S.4  ASCTTSATTS  2  t.O  25  26.3  CASAGATGTS  2  1.0  20  263  1.0  26  263  25  203  TCTASSTATA  o  o.o  20  uniuue lo Lou lien  TASASTASA0  o  0.0  19  unique In Lea Iran  TATMOASOT  1  0.0  21  42.5  T0ASASTAIA  O  00  18  unique Ic Luo Iron  ASTOCSCTAT  o  0.0  IS  unique In Lom Ian  CTTMAGTM  0  0.5  15  rainue to Lea lea  ASATOASTOG  o  0.5  iS  raetLetuLealan  000ATATGAA  O  0.0  10  roetuctuLeatron  CAITACTGCA  0  0.0  10  uesuuotoLealruo  CPGCM’rrTA  2  1.0  22  22.2  TOA000MOC  6  3.0  28  5.4  ASCSASTSTA  0  05  17  undooetuLealtorn  TCA0000TAS  2  IS  21  212  -  Protein biouomhes’e  Patein biosenthenis  T  1.0  40  74  2  2  79  30  55  TATAC COATS  2  66  S.OOE-00  4.OOE-75  TAS0000TAO  GACAT]TTSA  3.00E-A2  nn nlgnltloanl BLAST tasulls In en database aINCBI SAGE lag dues nut hIt C. rtentotmans eDNA oe enumIo setsuaeoo des Hbcsaeral peeleln BlI S 00,4-re cubA laB boat shook ptulele TO Cttmloceucus crrualuu ealmb taB SAGE tag dues not hA C. nrnutetnuane eDNA me geriatric  dOs dbusaesat prolate s2oa (SI) SulAoosacoln.rumyces curIAe  CIFTOSATCA  UnoleseiSed  hypeshetloal protele Marcus biecorus heat shuck ptetcln BE Nomolug Csndid. elbicone elgnltlosot BLAST nnnolts In no nrdatabeseal 5081  2.000-21  40  04  2.000-32  65  70  Respunsr to stress  4,000-23  79  92  Protein biosordhosis  0,000-OS  41  65  Cell aell  4.000-s2  41  61  t OOE-123  96  9k  Ire slgsltlosnt BLAST results In nrdatabase at NCBI BOs Ahosunrol Erotoln L3BIL3AA Seldeuuucoharomcoee cumbe uhItIn deacetylase Sohionehollum commune tie nlgslBceet BLAST results In nt dalaheneat NCBI cc nlgnltloant BLAST nesslts In nrdatabaso at NCBI sypulhetloal proteIn Suhiaosaooharumocos cumbe BAGE tag dues nut hIt C. eeetonmann aDNA or gonumlu Id-OS pnoleln Schieaphyllam commune SAGE tag dues cut hIt C. neetorenaes eDNA at Baomndo sagueeee csouhteme c eeldasc rebunt IV Ssdiauuauukammcoos cumbe alpba-areytasuAmyA Eme.tota nidriaco SAGE lag does nut hA C. rnnmtoeesaars eDNA en Benetnlo suguence no slgnlscanl BLAST tnnuttsln erdatabaso at NCBI nedoutaue Schizuoaoulnunmnyonu emnbe 8hu2 GTP bArdleg pretcln tlsraaoumaydis  TATOCATASC  2  1.0  21  212  TAIOACASCT  1  55  lB  304  no slgnlsuanl BLAST mucus In nn database at NC RI NAS dbenumal pruleln LOT 000charumoces oooeoisbae sEC Abusumal peotele L5 ftuabidoouislbslisn.  ATTTATCAAS  5  0.5  18  unison to Lea Ian  ATP eltease lyase Sohioeeacohuromvcns remAn  CCCATCOTAT  0  0.0  16  ensue to Lou roe  nn slgsltluarnl BLAST results In nn database a500BI  80  S’mnal treosduGino  0000-t6  44  50  C meokodmi  2000-45  31-59  44-00  Cnnbolrodnele motabuBsrn  0002.022  36-66(31  54-50131  Cdltdar ascbnoce  7000-17  do  69  SarAlcorurixflon  9030-34  70  87  Protnin biosordheett  5.000-on  41  66  Protein blosorthesis  5.000-35  5?  76  Carb000drabe mntebolrsm  fiaquancn  32eO  07 nO nonnalleod  Lom Ims  Pold Gittomnon  BLAST SIT  fl-salon  NO Idoomp  NO similarIty  hypothntinal protain Nouroscora donna  5.006-CO  56  70  GG Tarm  SAGE tag does not hit C. neotonnans cONS on gnoomlc GCCACAGCCA  0.0  16  odoco to Los Iron  ATGATTTGM  0.0  16  onioco In Low Imc  GAGM067A0  0  0.0  IS  onhon to Low Iron  CTTGTAGW  0  0.0  to  oohon to Loo Iron  CATITTATOT  0  00  no slgnlfinant BLAST nosolto to nn datahaso at BCBI SAGE tag does not hit C. naotorntaos cOttA or gonomlo  AMTCTIATG  0  0.0  16  oni000loLoolmn  no significant BLAST tosolts In on datahase at NC El alpha-cop protoin Boo n6mine6us  1.066-33  31  57  Vnsniole lrannnort  CCASTGATTA  0  0.0  16  oninon to Low Imn  Aopergiiius lorrous  0  48.66  66-74  Carbohcdraln molohoism  TWTGGTGC  0  0.0  to  oninon In Loo Imn  fithosotnal Pnotnin BPLO2  ANNOTATED  on’moo In Loo Iron  Protein biosonthnsis  no significant BLAST macfin in or datahaseat host on signIficant BLAST rooctts in ordatahaso at ttoBt comA tall so significant BLAST resells in nrdatahaso at host SAGE tog dons not hit C. nontormans oGNA or gonomlo  MTTTATGAT  0  0.0  16  on’nlon In Low Iwn  TSGAT000CA cMAaAoMA  2 1  1.0 0.5  20 18  202 36.4  TAGCGATCAC  13  6.4  34  5.3  TCGGGGCTGC  0  0.0  15  enrIco lo Lao Iron  TTGTTAGATG  0  0.0  15  onlnooloLoolwn  TANGAATGTA  0  0.0  15  onmnooloLaolron  OAOTTATTAN  0  0.0  15  onlnon to Low Iwn  OWITCATT  0  0.0  15  06100 to Low Iwn  dlonnlaotnna hydmiaso family Snhioosaoclraromyonn pomho no nigniticant BLAST msatts in or database at NOEl  go significant BLASTo NIle  GANTGTACPA  0  0.0  15  oninoeloLoolron  OA&8NOATC  0  0.0  15  onl000 to Low Iwn  Potatlvo 5-AMP-actinatod gamma nahunit family Snhiaosaccharnmcoos nomho on signltinant BLAST resells is or datahaso at NCBI  1.000-37  34  56  Sional lrancdcwion  1.060-15  30  54  Awwalio oowcocnd dorrradatioo  3.006-55  54  67  Oolhdarrnsciranon  5.000-44  32  47  Coil nrowlh andlorwainlonanon  nytonhromn ci pmoutnor  CATTTATGM  0  0.0  14  enrIco bLow Iron  SAGE tag hits mom than non snqannoe000tlg no signinnant BLAST msults Is or datahasnat NOEl Beta-tsbslln selector 0  TMCATMTG  0  0.0  14  udoco to Low Iwn  TATACGTGTA  o  os  14  codon to Low lroo  AAAOATCATC  0  0.0  14  06000 Ic Low Iron  TATAT005TA  o  co  14  odaoa to Low Iron  TAOCATACTT  0  0.0  14  oniqoo to Low Iwo  CATATGTOTA  0  0.0  14  on’moatnLcwlron  CIIITIN SYNTHASE I ONhOTATEG SAGE tag dons oct hit C. nooformans oGNA or gonoinlo sogoanco no significant BLAST monolts in no databaso at NOEl no slgnlfioant BLAST moolts in nr datahaso at NOEl  M000TTATO  6  3.0  24  8.1  rihnsomal pmtaio L3 Scodonlnra Nuoinnrda  MmTATCA  I  0.0  16  324  TTATCATCOT  1  0.0  16  324  CAT100CATA  2  1.0  17  17.2  MCTGTTGTA  o  o.o  13  udoon to Low Iwn  TATATSTOOA  o  o.o  13  undoo to Low Iron  TATOTTITAC  o  o.o  13  06000 to Low Iron  0.0  13  oninoo to Leo Iwn  TGACTSTCTA  Coil oaII  1.000-44  65  75  Proloir bmnsordhnsis  2.000-51  42-70  60-74  Enmoy and motahotaw  2.OOE-36  41  63  Rnscomn Ioslrnss  2.OOE-80  72  61  Proloin biosynlhasis  65  84  Omnomo to Orcns  1,000-55  80  85  Proloin hiosoolhos’n  6.006-22  60-55  74-55  Prolnmn Ioldira and dooradotioo  6.OOE.S0  55-70  06-52  Nmlnobaso wolaholisw  5.006-07  51-60  71-71  Nmloobasn wolaboliam  no significant BLAST msolts In or database at NOEl no signlfioant BLAST macits in nrdatabasn at lost no significant BLAST macits in nodatahaseat host no significant BLAST macIts In nrdatahaso at 11051 SAGE tag doan oct hit C. naotntmats oGNA or gonomlo naltoonna oropnrphyrlnogon donarh000lana Dade mnrio SAGB tag dons not nib. noolormats sOfiA or gonomin san00000 SAGE tag dons oct hit C. nnolntmats nGNA or fi050mic  TA00000ATA  0  0.0  13  anmnun In Los iroo  CARCTTGTTA  3  1.5  to  12.1  CACAITOATA  4  2.0  20  10.1  GAOATTCTGA  I  0.5  t5  30.3  OTTTTATGGA  I  0.5  15  30.3  Saocharomycnscnmcislan  4.OOE.56  05  15  30.3  ANNOTATED  ATOTATA000  Glatathlonn Pnnooldaso hTfiI Samharomconscnmacisiae dhosomal proteIn LISA Xanlhonhsllomccns dondrorhooc SAGE tag does not hit C. saotormana cGNA cr genomin  TATGATTTTA  7  3.5  23  6.6  ATTATPACGA  0  0.0  t2  odnun In Low Iron  ATATTCATM  o  o.o  12  udoun to Low Iron  Translation Elcngatlnn Factor 3 dbg rlhnsomal paotnln gg boumnsooracrasns protoasoma componnot POPS homolog Sctdnosaochammooon nombe pnrnibln S.c3000dhancoloasn, 1 dsp Aansrgillcs Siminalm  AGPAT000AO  O  00  12  uoiqun to Low Iron  no sIgnificant BLAST msolts In nodatahaso at NOEl  0.0  12  uninun to Low Iron  TATGTSTAAT TATATAC’TTG  0.0  12  06000 to Low Iron  AGTTTCTTGT  0  0.0  12  uni000 In Low Iron  MTOTOOTAT  0  0.0  12  udquo Ic Low Iron  dbonaclooprntnln, F4lkn Sohiacoacohammycos nomhn no stgnifinant BLAST mnolts in or datahasnat NOEl SAGE tag dons not hoC. nonlormans oENA or gnnnmlc sngnanca no ntgnlfioast BLAST msolts is  81  Prolnin bionynlhnsin  I  Table 11: Differentially abundant SAGE tags from H99 cells grown in YNB (37°C) vs. (37°C low iron). * data sorted by ascending p-value Sequence  37cc  37 oC normalized  Low rca  Fold Difference  COACADACCD  1160  573.0  38  15.1  GOCCTCDGTT  430  212.6  S  70.9  GCCAACDCCD  359  177.5  I  177.5 147.3  CACGTTCACD  299  147.3  1  OCTCGCDACG  290  143.4  2  71.7  AACGTCTDCC  303  149.0  S  30.0  COCDD4AADD  294  146.4  9  16.2  BLAST HIT Translation eiongaooo tactor I 1TEPII no significant BLAST results in nr databaoe at NCB1 Cycicphflin A  ANNOTATED  Signal iraosducoon  2.000-03  01  76  N0500nse Ic oSess  0-902  72  62  Protein BiosyoArds  2.000-43  66  89  Pnotoin Biosynthesis  4.OOE-34  64  76  Protein Orosynthocis  3.000-03  80  91  Cellular respiration  9.009-56  52-50(21  6944121  9.000-39  75  gO  Protein EiosyoAnnis Chromosome organioa9on and biosynAesic  0.000-39  43  63  Collular recyinasoo  2,00E-32  46  09  Carbohydrale metabotsm  30  93  77-79  00  500 RIBOSOMAL PROTEIN L6 Xenopus laeyis SAGE tag does net Nit C. neolormans cDNA or gesomic  240  110.7  5  237  210  103.0  2  51.9  Ribosonsal protein S12 Bnanclrioctoma boloheri  98.4  1  98.4  180  92.0  0  unique to 37  OTCOOTOOTA  267  127.1  14  9.1  AN000TOOTO  229  1132  9  12.0  OCTCTCCAOG  171  84.6  I  84.0  ACOOCCOTTA  148  73.2  0  unique to 37 22.4  tag too close to end at canda  TAOGCCGTCT  101  89.9  4  OOCCGACCTO  286  141.4  24  9.9  TCTOOTCOAO  164  01.1  2  40.5  OCTOCCTACA  149  692  0  unique to 37  CTCADCOATO  199  97.9  9  10.9  OTATTOACCC  149  73.7  2  36.6  600 RIB050MAL PROTEIN Lot-A Saocharomycesoereuisiae Fl ATPasa beta subunit Ktuyvoromyoos iaoys en stgolficant BLAST results Is sr database at NCBt SAGE tag does eat hit C. oeotormans cDNA or genemic no significant BLAST resaits in sr database at NC6t SAGE tag does not All C. noolormans cBNA or genomic ribnsomat proteIn LI Sohioosacchanumyces oombe Histose h4 proteIn Mus musculus ATP syotNaso ollgomycin sasoitinity conlerral pretels N. urama cytobine isdscing-giyceproteln Filobaddirta oeotormans phosphoketoiaso Thanmocynoohocoocus alongalus  AT000CTCCC  127  62.8  S  uniquc to 37  Nypethetlcai protein Mionobolbilen degradons  3.000-17  TCTTTCCDAO  144  71.2  2  35.6  aiteroative onldase Cry010coccus neolonmans oar. grubi  Aoootatod  CAOANCCCCG  196  77.1  S  15.4  ADPIATP carriar protoin Neurosponaorassa  -ItO  CACOOCOCAT  185  91.5  11  8.3  Ribasomal protein Ldt  ANNOTATED  TCOOTCOTOT  012  55.4  0  unique to 37  GCTTTOCTOC  129  63.8  2  31.0  ATOATC000C  934  66.3  3  22.9  prediotod prstsio Neun0500ra crassa putatine hydrolase; dienelactene bydralase tamlly; possibly inoeioed In oblcrocatocol degradatIon S. combo SAGE tag does not Nit C. oaatarmans cDNA or gonomlc hypotNeticai proteIn Neurosoora orasna CelOn saporoolde dlsmutase Crypl000ccus nrolormans oar. gmbb mltocboodrlai malate deNydrogonase 0. hypotboticai protein Schiaosaocharomyoes pombe  TCCATCCOAT  140  69.2  4  17.3  CACOTCCACO  140  69.2  4  17.3  GOTTACOCCO  108  53.4  0  unique to 37  OCTTTTOCCC  917  57.9  1  57.9  CCTCTTCCTO  106  92.4  0  OACOACTCTA  136  67.2  4  16.8  OCOTTCTCOO  104  59.4  0  unique 1037  COTOTCAAOC  104  51.4  0  unique to 37  OOTATCCTCG  903  50.9  0  unique to 37  OTTOOCANCO  902  50.4  0  uniquetu37  OCCOTCCGAA  141  69.7  6  11.6  OTCAAOMOC  89  49.0  0  unique to 37  ACTCAOOTTO  122  60.3  3  20.1  CTCTTCCCCT  145  71.7  7  10.2  COADOTATCA  96  47.5  0  unique to 37  TCCTOOCMO  95  47.0  0  unique to 37  OCOGACOACT  lOt  49.9  I  49.9  TCTOCCTCCO  90  44.5  9  uniquetu37  CCOCOACCOT  117  57.8  4  14.9  dbosomai proteIn 513 4ibo; protnio Arabidoosistrataoa hypolNetloat proteio Neunospona uraosa  OCCOCTTCTO  85  42.0  0  unique to 37  ablqulnel-oytochromo-u roductase Neunosooracnassa  unique  0037  GO Term  Thioredoolo perooidase Schizosacuhunonnyces pombr  DTTTCCDCTD  199  % similarity  Protein Bi0500Aosis  TCTDTCDAOG AADCCCOTTG  % identity  ANNOTATED  no significant BLAST results in or database at NCSI no signtficant BLAST results in or database at NC6i  DADANOCOTO  E-vaiue  predIcted protals Neurosoora omssa conserved hypotbatical protein Sohiaosacoharomycns combo transaidniase Sohizosacoharomycos oombo so signiitcant 6LAST results in nr database at NCBI 03 small nucieoiar rlbosuoloopratein prelate 1MP3 S. oerovidae mtatad ta Ypt-Istoracting protein YIP2 Nourosouro crassa dbossrrnal pralels S6; 405 ribosomal protein 05 Homo sapiens putatlue protein Anobidoosis thahaoa Sucrose biphesphate aldatase Panauoocidioideo bradtends dbosomai protoie L37 Sacuharomyoesoereuisiae atdohydn reduolase INASPHI Sponidobolus salmonicolun prottils Malus a domossoa seciaoslde dlpNosphale klease Emerluelia oidulaos  82  ANNOTATED  Onclacsifird  Cellular respiradon Protein Siosyntheds  9.000-08  29  38  9.000-09  20  d7  Unolasolnad  6.000-95  76  89  6.OOE-g9  69-96  96-900  Ronponse to nErns  3.000-37  65  79  Cellular reooir0900  0.SOE-06  36  60  6.SOE-05  60  64  9.000-21  47  69  1.OOE-114  68  76  Carbohydrate moiabotsm  5.0DB-SO  50  74  Cr0 gruoth aodlom mdateoaoco  9.000-30  44  58  Call grooth andloro mdnieoaoce  2.OOE-75  71  84  Protein Biosyntheds  4D-28  34  49  lOSE-dO  70  g2  Carbohydrate motabo9sm  4.OOE-32  59  01  Protein Bionynorosis  0.005-60  40  61  Carbohydrate metabolism  0.OOE-22  42  60  Cell gruuu9r anryoro mdntenanoa  2.OSE-46  57  77  Nucleohaso metabolism  2.005-38  73-65  66-94  Protein Biosynthesis  4.005-09  48  74  2.000-75  68  80  Cellular resoiraSon  Seooasoa  37 nO  37o0 normalized  Low iron  Fold Oirrennnoo  S CT C CTCTTA  140  69.2  9  77  CACCTCMOC  99  49.0  2  245  SC CO 000TCG  70  38.6  0  unique to 37  CÁO CACACC S  78  38.6  0  uniqon to 37  TO CÁO CATTA  75  37.1  S  uoiqoo to 37  BLAST HIT ATP s09thann alplra Strain Honassa SAGE tag does oot hit C. nnnlnrmaos oDNA or gnnotnis hypnthe6oal protoio Magentospiriiioooognototeobourn obtqutsot-oytoohromn 0 rndootase oomptno ooboott Sohiooseoohorooyoes poobo moog anose soperootde dismotase Phaooooohooto oinysosoooom oytoptaomio ribosornat protoio S12 Podosporo ansedno no sigoificoot BLAST resoits in or database at NCBt Htstoroe HI; Hbnlp Saodcsomyons onreoteon SAGE tag dons ont hOC. nnotoomaos oSNA or gononds dOS dbosornat protein SO Sobizopbylom oonerooen trtnseploosptrate inonrorase Aopnogdoo ooyaan  E-oaloe  % Identttn  % sindlaeito  109646  71  64  3.006-tI  37  54  2.006-22  41  64  Cnlioiaroospirotor  4.006-73  66  76  Onsooese ta slress  4.006-43  65  67  Protein Biosynthesis  9.006-05  45  61  Cnraomosooe orgorloaton nod biosynthesis  1.006-70  67  76  Protelir Biosynthesis  9.506-50  52  72  Cwhobydoole enoloboison  GO  TerOr  -  AAGGA000TC  161  86.5  22  41  A000TTOOAG  73  36.1  S  onique to 37  AGO OCT OCTO  72  35.6  S  wiqsn to 37  TCCM000TA  69  34.1  S  oi4qoo to 37  ACCMGCTTO  8.4  41.5  2  208  ACC ST C 5170  76  37.6  1  37.6  MCTTOITNGA  65  38.6  2  158  ATOTACC CCC  65  32.1  5  uorqoe to 37  TACAOC000T  73  36.1  I  36.1  C CT C GTATC S  64  31.6  0  oniqon to 37  OCTANCOCCO  64  31.6  0  ooiqoe to 37  GAOOAGOAOO  84  41.5  3  138  COAATTATOO  62  35.7  0  oniqor to 37  AGCACCA&AO  61  30.2  0  redqsn to 37  ATCOO3TACCG  87  43.6  4  108  OAOITOTTGA  123  60.6  12  5.1  CAOATGGAGA  75  37.1  2  10.5  0CCAhCTCTC  60  39.6  3  132  CWTTTFCG  56  27.7  S  oniqon to 37  AAOATOCGAG  56  27.7  6  ooiqon to 37  CAGTACCAGG  67  33.1  2  16.6  GATOOCAI300  60  29,7  1  29.7  GCTO OTTTOA  52  25.7  S  ACC SC C OAT 0  58  26.7  CGMCC0000  75  37.1  4  93  OTOACOT1TC  50  24.7  0  ra000n to 37  C CT GATC SC 0  50  242  S  seiwieto3l  GGTATGAACO  do  24.2  S  uerrpoe to 37  CGOTGT7GAT  56  277  1  277  AGO GAOCACT PAOMOACCO  62 dO  30.7 23.7  2 S  153 uniqon to 37  TCTOAOCOCO  do  23.7  S  onigunta37  hypotnrottoat procein Neorosporo 000550 toypotho6sat protein Miooobotbiloo degrodaos no sigsifi000t BLAST resoits in or database at 6661 hypothetloat protein Noorosporo wosso poptidyt-protyt ots-traos isoworaso Neorospore 000550 SAGE tag hIts more mao one segoenoe condo pstaboe synaptobreoin Asoergtioshimiqosis 6166 artadty sepper transpoetor Sotrioooaooirewnryons poobe ptroopbate teansport protein S. ononoteon hppotttetioat protein Noorosoooa ooasso no signinoast BLAST rnsoits in or databaso at NCBt BNA-hteoeng protein OoBNBP Aodoysronoa nmoroarum Poiysbtgsiun spnaptobreotn hnmotogl Soh’oosaoohooooyoes p0060 beta-tebottn 2 Hy0000aaoiroos SAGE tag dons not hit C 000lormans sONA or genomio  0.005  32  43  9.05E-18  45  65  Co*rdorrnspirahon  1.006-13  47  63  3.006-77  45  OS  Cnll growth and/nm mainteneooe  I 006-11  57  61  Cell groooth ondloro maintenance  4.006-25  40  92  Trwrsowl  4,006-97  60  71  Tmnrneoel  5.00640  43  97  3,006-Id  60  76  Coo m000th ordloro oowetnraeoo  4.006-26  HI  72  Cell growth aedloro mortnnonoo  4.006-38  71  81  Cell growth aedloro noinlenenon  6.006-96  56  72  Amino cold mntabohso  S 006-42  82  91  Protein Biosynthesis  4.096-37  47  65  Transono  5.096-22  48  62  ANNOTATED  Protein Cotebolism  etotamato oaaloaoetate ooiqun  1037  287  ASOCTTTCTC  47  23.2  0  unrgueta3l  CO3ACCCCTCG  47  232  0  unigue Is 37  CCACCAATGC MTGCCGGM  54 59  26.7 29.2  I 2  26.7 146  AAGCCTGACO  79  39.1  6  65  OGAAGATCGC  45  223  S  oriqun to 37  TTCACCACCT  45  223  o  uoiqonto37  AOCT000CM  44  21.9  0  raiqse to 37  GACTCGACGA  44  21,8  S  tedqoets37  CCGCTTTTISC  44  21.8  o  uniqon to 37  GTGTTMGGC  43  213  0  seiqun to 37  ATCGACTTGG  43  21.3  S  seiqun to 37  TTCAGCAGGC  96  47.5  11  4.3  CC OTAAC GOT  60  29.7  3  9,9  74  36,6  H  6.1  Oanio redo spiioing taotor; rnsI7a ribosomal protein Sohizonaooharooryons pombe PIroHOp 0000irmonryons onrnoisioo hypothodoat protein hourosoora orassa onnamnd protoin peodoot Podospora ansnAoa Protein wIth 3 RNA binding domains Sobioosoostrwooyoos dhosomal protein S21 Ceoddo albroors  5.096-32  30  94  2.506-19  52  63  Nrwieobasn rentthobso  1.006-26  62  02  Protein Bicsysthnsis  no signifioont BLSST rosoita in ne databose et NCBi no significant BLAST rnsoits in nr database at NCBi SAGE tag bits mote then non segoense sontig protoasome sobonit iota Mis mosoulos brain protein (3PtOt) Ceeooohobdtrs nleqans  1.006-36  31-72  91-94  Protein Carabolism  9.506-14  30  47  Seolossrted  no signifioant BLAST resoita is nr database at NCB1 SAGE tag hits more than con sngsoenoe snntlg atiergon Hatesonoio symoodails  2.006-11  26  43  Undoes/fled  2.006-Sr  34  53  Carhohs&ate metabsism  9096-36  44  59  2.006-09  40-SI  57-70  UnniassAnd  6.006-52  35-36  90  Transpod  1006-16  47  69  dehydrogeoasa Ssnetomyoes ooolioolor hypotholtoat protew Noro0500ra wassa no significant BLAST rnsotts In sr database at hunt teanslormer sednndargintse-doh rihonooieoprnteln, pota000 Arabidopsis theliere SAGE tag hits more Ihon one segoenoe oontig potatioe metal ttanspottnr Sohioosaooharooyons pombo on signinoent BLAST renoits io or database at NCBi Aoyi-CoA-binding protein Chaetoohraotus orllosus  83  c. Pairwise comparison of 37°C low iron vs. in vivo libraries A pairwise comparison was completed for the low iron library against the library from cells isolated from rabbit CSF, designated as the “in vivo” library (Table 12). Information for tag abundance classes can be found for each library included in the pairwise analysis (Table 12). Total tags used in the analysis were 19,278 for 37°C low iron and 49,048 for in vivo. At the 99.9% confidence interval, 0.20% (44 tags) were more abundant in cells from in vivo vs. 37°C low iron and 0.1% (23 tags) were less abundant for a total of 0.31% (67 tags) differentially abundant. At the 99% confidence interval 0.80% (176 tags) are more abundant, 0.32% (71 tags) were less abundant for a total of 1.13 % (247 tags). At the 95 % confidence interval 5.42% (1188 tags) are more abundant, 0.68% (149 tags) are less abundant for a total of 6.1 % (1337 tags). Tags below the 95% confidence interval are not considered to be significantly different. Therefore, 93.9 % (20592 tags) are similarly expressed between these two libraries at alpha=0.05. A graphical representation of this data is found in Figure 10. Notably, there were few tags that were significantly differentially expressed indicating that the libraries were highly similar. Pairwise comparisons were annotated for genes that were more abundant in 37°C low iron vs. in vivo and assigned GO terms where possible (Table 13). All data was normalized to the library with the fewest tags. Only 16 tags with higher abundance in 37°C low iron vs. in vivo (16%) were successfully annotated with corresponding EST or genomic sequence, had a significant BLASTx hit in the non-redundant database at NCBI (<e-5) and were successfully assigned associated GO terms. The low number of annotations may be partially due to a low level of stress-related ESTs in the H99 database, and lack of a completely assembled H99 genome at the time of SAGE data analysis. For the purpose of this study only the fully annotated tags will be discussed. Interesting genes encode a zinc finger protein, a ubiquitin degradation protein, a putative 5’-AMP activated gamma subunit signaling molecule, a putative ABC transporter and a white collar protein homolog.  White collar protein, encoded by BWC] is  involved in light sensing and has been characterized in C. neoformans (Idnurm and Heitman 2005). Mating and haploid fruiting is normally repressed when light is present but null mutants of BWC] have been shown to mate regardless of the presence of light. It is possible that Bwcl has a repressive affect on mating and therefore may be more highly expressed in the presence of light. In this regard, it is plausible that this tag may be more abundant in 37°C low iron vs. in vivo due to a higher exposure to light during growth in vitro vs. in vivo. All other genes identified  84  are involved in protein synthesis, cellular metabolism and maintenance. Genes that were more abundant in vivo vs. 37°C low iron were annotated by Dr. Barbara Steen and will become part of a larger scale SAGE analysis in the future. Genes found in vivo but not in vitro will likely provide interesting targets for further study.  Table 12: Comparison of SAGE data for strain H99 cells grown at (37°C low iron) vs. cells isolated from rabbit cerebral spinal fluid (in vivo). Interval Count Total  Interval Count Total  Classes for in vivo (Number of Tags) 2-4 5-9 10-99 100-999 4183 1007 685 27 10426 6436 16651 5230  1000+ 0 0  Total 16207 49048  Abundance Classes for 37°C low iron (Number of Tags) 1 2-4 5-9 10-99 100-999 6602 2161 441 209 4 6602 5424 2760 3784 708  1000+ 0 0  Total 9417 19278  Abundance 1 10305 10305  Differentially Abundant (Number of Tags;% of Tags) 9g %  More Highly Abundant in vivo Iess Highly Abundant in vivo Total  44 23 67  0.20 % 0.10 % 0.31 %  Similarly Abundant alpha=O.05 Total  Tags 20592  Percent 93.90 %  qçfl  99.0 °/o  176 71 247  85  0.80 % 0.32 % 1.13%  1188 149 1337  % 5.42 % 0.68 % 6.10 %  Crptococcus (H99 99_9% . 99% 95% B Si!j •  1e02 5.0  leOl I.-. 5.0  5.0  leOl  5.0  1e02  5.0  1e03  in vivo  ) Total:21929  Figure 10: Expression profiling comparing relative transcript levels of SAGE tags from strain H99 cells isolated from rabbit cerebral spinal fluid (in vivo) or grown at (37°C low iron) media. The trumpet shaped lines represent the separation of the data by confidence interval. Dark blue crosses inside the lines indicate tags that do not show a significant expression difference. The grey line represents the 95% confidence interval, the yellow line 99 % and the black line 99.9%. The green dots indicate tags that show a difference that is significant with >95% confidence. Tags in the top left quadrant of the graph represent tags upregulated at 37°C in low iron and the bottom right quadrant represents tags upregulated in vivo.The total includes the number of tags used in the analysis. The image was produced in Discovery SPACE www.bcgsc.bc.ca. Singleton tags were excluded.  86  Table 13: Differentially abundant SAGE tags from H99 cells grown at (37°C low iron) vs. cells isolated from rabbit cerebral spinal fluid (in vivo). * data sorted by ascending p-value Soqeenee  11 otoo  St .1gw soreseozed  Late row  Fold Sfftereooe  BLAST lIT  OTTTGADTOO  0.0  10  wcquntotow’won  CTTOCTGTM  0.0  10  teiouototowwoo  TATAT000TA  1.2  14  11.6  CATCMCCTG  54  II  29.0  Wrong orioslanon SAGE tag to BLAST hB SAGE tag dons not htt C, newtortrtano oSIdA or 0000mb nonoenwe SAGE ta dons 60666 C. nnwlorrntars cOttA or g000rtdo noquesce SAGE rag doss not hOC. oeotoemaes cGldA on gewsedc seqoeoos SAGE rag hits noon. than one swqoeooe tontig SAGE tag does nut hit C. neotorrenets oEINA or 0060ndo nmwnson SAGE tag hAs mom than one sequence oonttg  CATTATATAT  OA  11  290  ATATCCTGOA  02  9  aqouotolow’won  CTATGCACAS  0.4  10  25.4  TATDCMw6PV6  54  10  254  ATCGAGACGG  0.0  9  unjoco to low woo  TASGAVIGCT  0.4  5  229  CAmGGTAT  0.0  7  uoiqeotolowiroo  TAATACGCGA  0.0  7  uoiqoo to low iroo  CCACTGCOGC  0.0  7  uoi000 to low iroo  ASITCUSAT  0.0  7  uoiqootolowiroo  G0-°ATATGSA  0  CATATDSAGT A9.OAWA6G  00  7  uoiooo to low iroo  00  7  unison to lowiroo  Wrong odentution SAGE tag 1w BLAST his SAGE tag hAs mom than onoseqasnon sonttg Expressed pmtetn-stmttar to RIKEN oGNA Nnhidoosis lhsliaoa WHtTE COLLAR I PROTEIN (WCI) Souwsituta utsssa  17  4.3  poltA wit  04  9  20.4  TGAGAGGW  0.4  9  20.4  TPAGPATGTA  at  to  4.9  TATACDTGTA  2.9  14  5.1  Wrong ndantaAoo SAGE tag to BLAST hA SAGE tag does got hit C, nsclorntaes oGNA or ganondo sequence suhuris lamtty Schwosaucharonycen eowbs Bwea4tahegn cwtaxtor S Hoowsaeieou 565 rittohontal pecteis LIT Suhtoooauultatuntycan owobe SAGE tag dose cot hit C. nooloenonts cOttA ow sonetrdw seqneewe  TAaAOCAOTC  2  CTI’WCACA  0  TWGAO.OAIt  06  9  hA  24  13  5.5  0.0  6  wdnwetobw’woo  0.0  6  uoinoo to low iwo  1’. stntttarty  aSSE-37  36  St  UteleooiOod  S.SSE-S4  4t  09  60000000 to tbht  GO Term  doom. otinowtion SAGE tao to ELAST hA SAGE tag dons not hit C. seolermane oGNA or nesendosnnaesss SAGE tag dons not hit C. neolormans oGNA ornenondcsenoesse  59  TGACTGTCTA  % tdrrdtty  Wrons edentatton SAGE son to BLAST 66 SAGE lag dons not htt C. iteolermens cOttA or neonede snnoenoe  TAOAGCCPAG  to  E-oabe  moons odmttation SAGE log to ELAST he topoisortwrona 5 ANNOTATED SAGE tag dons not hAG. sowlomonts oGNA or ennoedo emoeooe SAGE tag doss not hit C. nooleenraes oGNA or gotsondo sequ000e SAGE tag dons cot hit C. nooroeoaes oGNA or osnoardo smaetwe consomad hypuehotioct protais yeast Candito atoloam  mGGP.0ACC  0  0.0  6  urwtobwiow  1TCTAGTGTA  0  0.0  6  tr*twtolowfrow  000’TTI’GTCA  0  0.0  6  wnwtolownow  TATGTPAGCA  3  1.2  12  10.2  TTGTTAGATG  S  55  15  4.2  CAGCDTADTA  31  14  4.5  TTAODGC1TT  04  7  17.6  ho stenihoact BLASTo hAs nero tan hypothetIcal AlT-family protein hieDsacchatum9ces oomhn SAGE tag dons not hit C. seolormacs oGNA or genendo sequence  1006-37  34  56  Sigoat Trsosdtwtioo  5526-44  32  47  Cat month eodloe merarrence  A.-3d  55  66  Protoit sto4hesis  4.Wh-56  26-50  36-57  5.026-SO  27-29  41-46  2666-07  37-05  48-49  htwleobena metebothto  4.SSE-S6  ST-7t  76O2  Protoio synthesis  1.596-to  26-30  42-49  -  DTGTACGTAG  0,4  7  17.6  No slgntfeaet BLASTs hits near fag  CGATCDMDG  OA  7  17.0  Ne sigetficant BLASTo hAn near tag  ACCTCGDATC  0.4  7  17.0  6SS dhosomrt nmtois L34-B  ACCDTTGAOA  0.4  7  17.0  OTTGTAAacOA  0.4  7  t7.6  No etgotft006t BLASTt hAs sear tag SAGE tag does net hitC. nonlormans oGNA or owonmic sequenoe  ATGTATGGTT  04  7  17.6  00  6  102  TATATDAGTA  2  ATATGTATGA  00  8  10.2  CATADTGTAG  20  11  56  CATIGACACT  t2  0  76  Wrong odentatton SAGE tan to BLAST hit SAGE tag does not hit C. neoformann sGNA or qenonde sequence SAGE tag hits more than one seqoewse ocnttg SAGE rag hits mum than woe sequeooe 000iig  ATA.OATtAIA  0.0  5  aique to tow ‘eon  SAGE rag hire emra than one seqotece 000tig SAGE rag dons not hit C. seulurorans sENA or gerwmio sasoonoe SAGE rag hits mum that one seqsonoe eontig SAGE tag deco nwt he C, eenlonnons nENA or geewedc sntrnrrson  SACGTGATTG  0,5  5  wiqunwtow’eoo  doors eeiwosaAon SAGE leg to BLAST hit  4.660-ti  38  54  CATAGCAOCC  0.5  5  Lecque to tow ‘eon  Wrors edestation SAGE ton to BLAST he SAGE sag dwos sot he C. ntotorrnans oGNA or gwewodo setssmtoe SAGE sag dons not hA C. noolormans eSNA or genomtosegurnee pltmphattdytinoseoi hhiana-mmalnd hEster DtserAa towbtw SAGE tag dons nut hA C. neolornrans nSesA or geeondo seqomwe SAGE tag duos ont hit C. nsnloennans eShlA or oaeondo neqoenon SAGE lag duas tot hit C. neoeernonss oGNA or gorsoedo saguorwa giso Nnqor protein Onh’aosaoohawmyces cembe SAGE tag dccc not hA C. nAelermaos oSNA or genomic sequence SAGE tag dues not hA C, neofermans oSNA or aenomic smusnee  1000-167  47-65  02-75  2266-66  44  63  L6-d cod Sotot metabolism  i.66E-55  27-34  48-52  Pwwin cetsbnhon  TGCAOAAOOOA Ar1A,rrw’rn  3  TATGATASTA  1.2  9  76  12  0  76  0.5  5  toqw to bow ‘woo  as  5  utiour to tow iron  0  0.0  0  tatiocw to ton. ‘eon  0  0.0  0  tat our to bow ‘woo  0  GGTATATAGT  0  MTTGDATAC ATMCC1TTC AS1TCAGPAG  0.0  0  wiqtwtwbo’eoo  TTTWTACT  0.0  5  teiqurwbweoo  1TIGTCATTAS  5.0  5  0010.0 totow woo  OACTGCTA1TI  0,0  0  ooiour to tow too  TGGT500TTC  0.0  0  ooiouo to tow too  87  Sequctrnw  hi utoo  Is slow noenretteed  Low trwr  Fold Dilterenco  GTATCSACGG  0  0.0  5  unIson to low iron  GCGGTGTICG  9  0.0  5  Lrmtw In low ‘non  TCGGCACGGT  0  00  5  Lei000tolnw’non  WTGGTCTG  0  00  5  wniqwn to Ice iron  BLAST HIT  CATTATAGCT  0  00  5  unique to low Iron  GGAAACDTGG  0  0.0  5  unique to low iron  No elgelloerd BLASTu she new tee SAGE tug duos set hOC. eeelorerane oGNA or gnreoedc sneoweco SAGE lag does net 911G. nuetererano cGBA or grncndc seeueeoe SAGE tag does net hit C. enolormues rOSA or gencetic soquenon  CATTITCTIA  0  0.0  5  unique In mw iron  Wrowe oduetuttee SAGE tag to BUST hit  GACTGTTGTA  0  0.0  5  unioun Intro iron  No stontncunt Bt.AST0 hon eeoc 109 SAGE tog dnnn not 99G. neetonmees oGNA wr eunomtn uequeeoe GSA tepnteomoraee III Grosoplritumuleonouster SAGE tug does nut httC. nootuewern oGNA or qenuetmo sequ000e SAGE tag does nut hltC. neotnewons eGNA or qeeuwto soquunon ebtquflte testes dugrodatlun prwtetn Sohwoescchar00005e nowhe SAGE tag dews not 9IIC. eeodomrecrs cElIA or gmrue,4o suqAence SAGE tug dnns sot hi C. eootooeresrs oGl4A on geswndo sequence h5pwthnocal semeheane proteIn Neronepuro o’uoou  CTTGCTTTAG  0  0.0  5  unique Intro iron  CGTTACCGM  0  0.0  5  unique Intro iron  CTC000CTAO  0  0.0  5  uruoue to low irnn  ACCAACCT7G  5  2.4  11  4.7  TCC0.6CGACT  I  0.4  B  15.3  GCATICDTCC  1  0.4  B  15.3  TCTCTGTACC  I  5.4  6  15.3  4AITTCTGTT  1  0.4  6  15.3  AGTATGTAGC  I  5.4  6  15.3  GAT1GATCCC  I  TA  6  15.3  GCG1TICCTA  I  0.4  6  15.3  MTAGCTCAG  1  0.4  6  10.3  GMAO7ACTG  1  0.4  6  15.3  ACOACTCAOA  1  0.4  6  10.2  TTGTMG0.SA  I  0.4  6  10.3  ATCATC 0 A40r  15  3.9  14  3.6  TMTCATAGC  4  1.6  9  5.7  CAGMCGACG  4  1.6  9  5.7  CAO.AGGTACC  2  0.6  7  6.9  GACITTCMT  3  12  6  6.0  AGT0AMVI  3  12  6  6.0  GACATATGM  17  6.7  16  2.7  TGACAOSATG  0  0.0  4  urqotw In low eon  11TTGTCAGC  0  0.0  4  rerique to lore ‘eon  ATAGDTACGG  0  0.0  4  Lewrw to low horn  Wrong eeleetanoe SAGE tag to BUST hi SAGE tag dote sot hIt C. eootwrnraes oGleS or emeusde euesotwe tanoy peotete Golnl000anctruewer005 conrhn SAGE tog dens not lid C. neuloninane eDNA or genoetto sequence  Wrong odentetico SAGE 1001w BUST 69 hypothetIcal proteIn seuroAtore crouce SAGE tue dots rot hItC. seotoenuns cDNA or enromlo sequence putetioe ATP.htrdtsg cassette trenspofler orococ4dioidu hrusitisnn’n acelyltranetcreeu 500chorornooesoereuieiae SAGE tag dote sot 611G. eeutoesaes sItNA or qenuetic soeuecos elplru.acerylaswAuryA Emeticefle redriens SAGE lee dote nolh9C. neoloenrans oDNA en genundc sueueeoe SAGE tag dots nut hOC. nnolwnenuns oGNA ce osnorntc seeueece SAGE tog does sue 911G. neotoenane rENA wn ernondo sequence  CTMCTTTCA  9  00  4  rerhiuetolow’eon  9  09  4  reiuuetulow’eon  No sknrlllcald BLASTo hfle ecer tag SAGE leg dote cr199 C. seulusoetru oGNA orueeordo ewouwroe  DTGMTCGCS  0  05  4  uoioue to low ron  Wrneq cdrnlattcn SAGS lee to BUST 99  5ACTTDGGCA  0  0.0  4  unique to low iron  0GT1ACTGCA  0  0.0  4  unique In low iwn  ATACCTTCPA  0  0.0  4  unique to low iron  ITCATCATCA  0  0.0  4  oniqun to low iron  GA000TGATT  0  0.0  4  unlqun to low lore unique to low Iron  No stenlloent BLASTu hits neer leg SAGE leg dues oct 6IIC. esotosnees cGNA or gencmlc sequence SAGE leg hits now then one suquense roche SAGE leg hfls more then unu sequence ccsltg SAGE leg duns sot htl C. nenlnsnene cENA cross umtc sequence SAGE leg dote sot hit C. oeotosnees rOSA oneenomloeequencw SAGE leg dote set 611G. neolcenene oGNA or ennomle 510051cc SAGE leg dose eel 611G. seotormuns cGNA or genomlo sequence  ATATTGDATG  0  0.0  4  5  0.0  4  onique to low Iron  WCGGTCAG  9  50  4  unique to low iron  Irs Idnntiy  % niedleiky  l.OOE-19  25  37  I.OOE.62  40.100  64.150  DNA rudioosnn  400.22  313-IA  49.62  Prurojo ottuholiero  G Term  3000.16  41  04  2005.47  35.49  55.68  1.000.110  26.54  42.77  6,000.00  31.56  43.75  2,006.32  40  04  Ttensnmt  1 050.13  56  71  Cstt wattlcaAohudroto b’wuonthesis  2.35545  31.59  44.60  Cwholrodrelr nreueEotern  1.OSC.50  27  41  Cdl orowftr uodlnr esuirtoterwe  So togettleent Bt.ASTO hits Beer toe SAGE tog dote not 911G. newtwrwunn oDNA ne eeromto seguence SAGE toe dots not 9IIC. neotorenene cENA we ennomlo sequence  1TIGTTAku.A  GGAAGACCCG  [  E.oehw  SAGE lug dote not SOC. nuolnnnreee rOSA nr qeewedo eeeueeon SAGE tug dote rot 9SC. ewolonerees oG1IA or eenusedc sequ000n  88  d. Iron-regulated tags: Three-way overall comparison of the libraries 37°C low iron, 25°C and 37°C in YNB. SAGE tag occurrence was compared between the libraries from 37°C low iron, 25°C and 3 7°C. Data is represented in a Venn diagram (Figure 11). The low iron library has a far higher number of unique tags at 7,329 vs. 3304 for 25°C in YNB and vs. 4,210 at 37°C in ‘(NB. These results further indicate that the low iron nutritional signal has a greater effect on transcription than a temperature change in ‘(NB. A total of 771 tags were shared by all libraries, some of which likely represent essential metabolic genes. There were a higher number of tags shared by the 25°C and 37°C libraries (2,366) than 37°C low iron and 25°C (1,050) or 37°C (267) libraries. The same interesting observation that the 37°C low iron library is more similar to 25°C than 37°C was observed in this multiple library comparison.  89  low iron  A  c  Z5oC  37oC  Figure 11: Comparison of SAGEtag occurrences for cells grown in low iron (37°C), YNB (25°C) or YNB (37°C) medium. Occurrence of a tag in a SAGE library for A. 2 5°C, B. 37°C low iron and C. 3 7°C. Intersections of the Venn diagram represent shared tag sequences between libraries. Numbers represent occurrence of a tag and do not reflect relative abundance.  90  e. Comparison of multiple libraries including: 25°C, 37°C low iron, 37°C and in vivo. A multiple comparison of SAGE tag occurrence was performed between the libraries designated : 25°C, 37°C low iron, 37°C and in vivo The data are represented in a Venn diagram .  (Figure 12). In this comparison, the in vivo library from rabbit CSF has by far the most unique tags at 10,290, followed by37°C low iron at 5,388, 37°C at 3,737 and 25°C at 2,331. These results suggest that the in vivo environment induce multiple genes necessary for survival in this low nutrient environment; some of these may help the fungus thrive in the hostile host environment. It should be noted that these comparisons do not take into account library size and the in vivo library had far more tags (49,048 vs. 19,278) than the low iron library. This size difference may account for some of the difference in the number of unique tags. In total, 683 tags are shared by all libraries, again likely reflecting the many tags for potentially essential metabolic genes. Genes that are essential would presumably have some level of transcript present under any condition. This is similar to the number of shared tags in the comparison of low iron with 25°C in YNB and 37°C in YNB (771). This is not surprising because the shared tags likely represent tags that are essential for cell growth in any condition. The greatest number of tags shared by only two libraries is that of the in vivo and low iron libraries at 1,941 tags compared to 973 for in vivo and 25°C, 473 for in vivo and 37°C, 138 for low iron vs. 25°C, 108 for low iron vs. 37°C and 1,590 for 25°C and 37°C in YNB.  This global analysis of tag occurrence is  concordant with findings from the pairwise comparisons where the in vivo and low iron libraries are more similar than the libraries from cells grown in YNB.  91  A  Figure 12: Comparison of SAGE tag occurrences for cells grown in YNB medium (25°C), isolated from rabbit cerebral spinal fluid (in vivo), low iron (37°C) medium or YNB (37°C) medium. Occurrence of a tag in a SAGE library for A. 25°C, B. in vivo C. 37°C low iron and D. 37°C. Intersections of the Venn diagram represent shared tag sequences between libraries. Numbers represent occurrence of a tag and do not reflect relative abundance.  92  SUMMARY AND DISCUSSION A striking result in this SAGE analysis is the similarity of the low iron library to that of cells isolated from rabbit cerebral spinal fluid. Tag abundance compared between these two libraries show only 0.31 % difference in abundance at the 99.9% confidence level. This indicates that the libraries are nearly identical (<0.10 % at 9 9.9% would be considered identical). This result suggests that the low iron nutritional influence on gene transcription in C. neoformans may be relevant to conditions during infection. Further, the data suggest that that low iron medium (LIM) may provide a useful in vitro model for studying virulence-related gene transcription. Another interesting result was that the 37°C library for cells grown in low iron was more similar to the library for cells grown in ‘(NB at 25°C than grown at the same temperature, (37°C) in YNB. Cells are stressed under low iron conditions as partially indicated by the number of stress related genes that are highly or more abundant under these conditions. It is possible that strain H99, originally a human clinical isolate, is less stressed at 37°C than 25°C. Therefore, more stress-related genes are induced at the lower temperature and this response may more closely mirror the stress of low nutrient availability that is found in low iron conditions.  Not  surprisingly the same phenomenon was noted in pairwise comparisons of the in vivo library from rabbit CSF to the ‘(NB libraries (Steen, B.R. personal communication). Genes that were notable in relation to the response to stress in the low iron transcriptome encoded the heat shock proteins (HSPs). HSPs are involved in the modulation of apoptotic death (Garrido et al., 2001) acting as cellular chaperones (Parcellier et al., 2003).  HSP]2 is of  particular interest because it was highly abundant in 37°C low iron. There are two copies of HSP]2 in the strain H99. The first and third most abundant tags showed similarity to HSP]2 of S. cerevisiae (designated HSF]2-1) and Saccharomyces pastorianus (designated HSP]2-2) and had 280 and 123 tags, respectively. HSP]2-l and HSPJ2-2 were highly upregulated in the 37°C low iron library in comparison to the 25°C library (HSP]2-1 at 14.3 fold ; HSPJ2-2 at 3.4 fold). Both genes were upregulated compared to the 37°C library (HSP]2-1 was unique to 37°C low iron; HSPJ2-2 at 62.2 fold). HSP]2 was also found to be highly abundant in the in vivo library, with 637 tags corresponding to HSP]2-1 and 468 tags for HSPJ2-2 (Steen et al., 2003). The tags corresponding to HSPJ2 in the in vivo library were more abundant in comparison to the 25°C library (HSPJ2-1 at 31 copies ; HSP]2-2 at 57 copies) and the 37°C library (HSP]2-1 at 0  93  copies; HSPJ2-2 at 4 copies). These results suggest that HSPJ2 may play an important role in the response to stressful situations such as the low iron or the in vivo environment, but interestingly not to temperature for C. neoformans. In fact, SAGE tag numbers for HSPJ2 are lower at 37°C (HSP12-1 at 0 copies; HSPJ2-2 at 4 copies) than 25°C (HSP]2-1 at 31 copies; HSP]2-2 at 57 copies) in YNB, which is counterintuitive when considering the gene designation as a “heat shock protein”. The same trend was noted for one of the HSP]2 genes from SAGE temperature libraries for the strain B3 501 A (serotype D) (Steen et al., 2002). A second copy of HSPJ2 was identified in genomic DNA of B3501A, however no corresponding SAGE tag was identified in this SAGE temperature data (Steen et a!., 2002). HSPJ2 has been implicated in the response to many stressors in C. albicans and S. cerevisiae. The well studied process of vinification represents a number of stresses to yeast cells including heat, ethanol, oxidative, osmotic and glucose starvation stress; in all of these cases HSP]2 has been shown to be upregulated (Carrasco et al., 2001; Sales et a!., 2000). Transcriptional and biological studies in S. cerevisiae have identified HSP12 as required for the following processes: 1) biofilm formation (Zara et a!., 2002); 2) upregulated in response to cadmium stress (Momose and Iwahashi, 2001); 3) involved in barotolerance and cell wall integrity (Motshwene et a!., 2004; Chauhan et a!., 2003); 4) survival during near freezing temperatures (Kandror et al., 2004); 5) transcriptional response to the herbicide 2,4 dichlorophenoxyacetic acid (Simoes et a!., 2005); and 6) resistance to ethanol and acetaldehyde (Aranda et a!., 2002). C. albicans mutants that were resistant to the drug fluphenazine showed an increase in transcription for HSPJ2 in conjunction with the genes for the multidrug efflux pumps CDRJ and CDR2 (Karababa et a!., 2004). Finally, Hspl2p has been suggested as a potential medicinal component in yeast extract for the treatment of burns and wounds of the skin (Schlemm et a!., 1999). Alcoholic extracts of yeast have been used as a naturopathic treatment of these ailments for a number of years, biochemical analysis by this group identified a number of candidate peptides related to the stress response including Hsp l2p, copper and zinc superoxide dismutase and ubiquitin. Further analysis on the separate compounds would be necessary to identify which proteins carry definative medicinal attributes. HSPJ2 is regulated by a number of well-studied signal transduction systems.  In S.  cerevisiae, HSPJ2 is regulated by cAMP through the zinc finger proteins Msn2p/Msn4p (Ferguson et a!., 2005; Praekelt and Meacock, 1990) and by the two component signal  94  transduction system that includes the putative response regulator Ssklp and mitogen-activated protein kinase (MAPK) Hogip (Chauhan et al., 2003; Alepuz et a?., 2001). In the absence of a functional Msn2p/Msn4p transcription factor and PKA catalytic subunits HSPJ2 has been shown to be controlled through the heat shock factor Hsflp (Ferguson et al., 2005). There are many uncharacterized putative zinc finger proteins in the genome of C. neoformans that could be homologs of MSN2/MSN4. The HOG pathway has been characterized in C. neoformans where serotype A null mutants are attenuated for virulence (Bahn et a?., 2005) but the pathway has not been studied with respect to the stress response. The global regulator in the C. neoformans HOG pathway is thought to be Pbs2p rather than Sskl p as is the case in S. cerevisiae and C. albicans. A strong candidate for an HSFJ homolog is present in C. neoformans, on chromosome 2 of JEC2 1  (1 .4e-27  http://www.tigr.org/tdb/e2k1/cna1/)  and  H99  (6e-2 1  http://cneo.genetics.duke.edu/menu.html). Although a great deal is known about what induces and regulates HSPJ2, little is known about its specific function except that it is located in the cell wall and cytoplasm (Motshwene et a?., 2004; Sales et a?., 2000) and is proposed to be a cellular chaperone (Parcellier et a?., 2003). HSP]2 clearly plays a role in a wide range of stress responses in fungi as is apparent from the multitude of publications that identify the gene as upregulated in response to stressors and in the sensitivities of hsp]2 null mutants of S. cerevisiae to stressors. It is also clear from this SAGE analysis that HSP]2 likely plays a role in the response to low iron in C. neoformans. In C. neoformans, homologs are present or systems are characterized for the HSP]2-related signaling pathways found in S. cerevisiae and C. albicans, however, many of these pathways have not been well studied in C. neoformans with respect to low iron stress. It would therefore be very interesting to elucidate the function, localization and regulation of HSP]2 in C. neoformans with respect to low iron and other stressors such as glucose starvation, oxidants, high osmolarity, cell wall damaging compounds, ethanol, high pressure or antifungal agents. A number of genes that were more highly abundant in the low iron transcriptome have been further investigated in the laboratory. These include the iron permease FTR], a gene of unknown function HOT] (homolog of CIFC from Emericella nidulans) and a chitin deacetylase gene. Due to the similarity of the low iron library to the in vivo library, many of these genes had already been identified as potentially related to virulence by studies of the transcriptome from cells isolated from the CSF of rabbits (Steen et a?., 2003) or from iron regulation in the serotype  95  D strain, B3 501 A (Lian et al., 2005). The following chapter presents the results of a detailed functional analysis of the SIT] gene identified in iron regulation studies of the serotype D strain B3501A (Lian et al., 2005).  96  CHAPTER FOUR: Analysis of the Siderophore Transporter Gene SITJ INTRODUCTION Pathogens possess specific mechanisms that allow survival in the mammalian host environment including the ability to sequester iron that is tightly bound by host proteins. The low iron environment found in human serum, brain and cerebral spinal fluid also provides a nutritional cue for induction of key virulence factors (e.g. capsule formation) in C. neoformans. The work described in this chapter is specifically focused on the mechanisms of iron sensing and transport in C. neoformans. Fungi have both reductive and non-reductive mechanisms to acquire iron. In C. neoformans a high affinity, reductive iron system has been identified and characterized that includes the high affinity iron permease, FTR] and the multicopper oxidase, FET3 (Lian et al., 2005). In conditions where iron is scarce or tightly bound, more specialized mechanisms are often employed by organisms to acquire this essential nutrient.  Recently, a  siderophore transporter homolog was identified in a serotype D strain by SAGE; this gene was designated SIT] and it was found to have a higher transcript level under low iron vs. iron replete conditions in SAGE analysis (Lian et al., 2005). This relationship was confirmed in this chapter using Northern analysis.  Siderophores are small molecules that bind ferric iron with high  affinity. Due to this high binding affinity, iron bound to siderophores is largely unavailable to more generalized reductive uptake systems (such as that encoded by FTR]/FET3) and is therefore reserved for organisms with the ability to uptake such molecules. The siderophore bound iron can then be non-reductively transported into the cells by specialized ABC transporters. In C. neoformans, SIT] is a homolog of the S. cerevisiae ARN3/SIT] gene that encodes a ferrioxamine B permease from the major facilitator superfamily (Lesuisse et al., 1998). The high transcript level of SIT] in low iron vs. iron replete conditions and its possible role in iron acquisition in vivo led to the initiation of gene characterization studies of SIT] in C. neoformans as detailed in this chapter.  97  MATERIALS AND METHODS A. In silico Analysis of Sitip and SIT]. a. Determination of the coding sequence and exon-intron boundaries of CnSitlp. The coding sequence and exon and intron boundaries were manually determined in the nucleotide sequence by comparison of six frame translations of the C. neoformans SIT] gene to the S. cerevisiae homolog Sitlp/Arn3p (gi6320770refNP_010849.) protein sequence. Exons and intron locations were further resolved by identification of C. neoformans exonlintron consensus sequences: donor 5’-GT(XX)GY-3’ and acceptor 5’-YAG-3’ (See sequences in Appendix IV-Aa.). Intron and exon location and coding regions were later supported for the serotype D strain in the annotated genome at TIGR where the SIT] gene was given the identifier 181.m08534 and designated CNA07920. Note that the TIGR database is a JEC21 database and that JEC21 Sitip has 605 predicted amino acids, however closer inspection revealed that the stop codon had been included as an amino acid in the prediction and the published sequence (when downloaded) had 604 amino acids. No EST was available for SIT] in the University of Oklahoma databases for B3501A or H99.  b. Comparison of SIT] and Sitlp in serotypes A, B and D. SIT] sequences were identified in genomic sequence databases for the serotype D strain B3 501 A (http://www-sequence.stanford.edulgroup/C.neoformans/index.html), the serotype A strain  H99  (http://www.dumru.mc.duke.edu/)  and  the  serotype  B  strain  WM276  (www.bcgsc.bc.ca). Amino acid sequence alignments were performed with Clustal W. http://www.ddbj .nig.ac.j p/search!clustalw-e.html. The similarity tree was produced from the Clustal  alignment  W  file  using  the  Clustal  W  program:  http://www.ddbj nig. ac.j p/search/clustalw-e.html. Nucleotide similarity comparison from exon .  and intron sequence of SIT] was achieved by comparing sequences using the BLAST 2 sequences algorithm at NCBI (http ://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2 .cgi).  c. Identification of siderophore-related genes in C. neoformans. Siderophore-related genes were identified by searching known fungal genes involved in siderophore synthesis and usage (Haas, 2003) against genomic databases for C. neoformans using the tBLASTx algorithm.  Homologs were searched against the following databases:  98  Serotype D-at SGTC (http:Hwww-sequence.stanford.edulgroup/C.neoformans/index.htrnl) or TIGR(http://www.tigr.org/tdb/e2kl/cnall),  Serotype  at  A  DUMRU  (http://www.dumru.mc.duke.edu!) and serotype B at MSGSC (www.bcgsc.bc.ca). Only fungal homologs that had been fully characterized were included in the analysis. The genomic location of hits and expect values were recorded in an Excel spreadsheet.  d. Comparison of Sitip to fungal homologs. Sequences for fungal homologs were supplied by NCBI (www.ncbi.nlm.nih.gov) and GenBank Accession numbers can be found in Appendix IV-Ab. C. neoformans SIT] sequence was  from  the  D  Serotype  seciuence.stanford.edu!group/C.neoformans/index.html)  strain  B3501A  (Appendix  IV-Aa).  (http://www Amino  acid  alignments were performed with Clustal W http://www.ddbj .nig.ac.jp/searchlclustalw-e.html The similarity tree was produced from the alignment file using the Clustal W program http://www.ddbj .nig.ac.jp/searchlclustalw-e.html. Abbreviations of fungi are: An-Aspergillus nidulans, Ca-Candida albicans, Cn-Cryptococcus neoformans, Sc-Saccharomyces cerevisiae and Urn- Ustilago maydis  B. Strains and Growth Conditions. Cryptococcus neoformans strains used in this study are listed in Table 14. The serotype D strain B3501A was provided by Dr. J. Kwon-Chung (National Institutes of Health). The serotype D strain JEC21, JEC43 (ura5), the serotype A strain H99 and serotype D and A cAMP mutants were provided by Dr. J. Heitman (Duke University). The ura5 mutant of B3501A was isolated by plating the wt strain on 5-fluororotic acid (5-FOA) plates; the spontaneous mutants obtained were restreaked twice for single colonies on 5-FOA. LIM was prepared as described in Chapter Three. LIM+Fe was prepared by addition of ferric EDTA to 100 M (FeEDTA;Sigma EDFS). LIM+BPDA+Deferoxarnine was prepared by addition of bathophenoanthroline disulfonic acid to 550 tM (BPDA; Sigma B1375) and deferoxamine to 100 tM (Deferoxamine; Sigma D9533). For measuring growth in liquid media, 5 mL of yeast extract peptone dextrose medium (YPD) was inoculated with a single colony and grown overnight at 30°C in a gyratory shaker at 250 r.p.m. 100 iL of culture was transferred to 5 mL of yeast nitrogen base (YNB) medium and grown overnight at 30°C. Cells were washed four times with sterile water and  99  transferred to flasks containing 50 mL of LIM, LIM+Fe or LIM+BPDA+Defereoxamine at a concentration of 1 x 106 cells per milliliter. The cultures were shaken at 250 r.p.m. at 3 0°C. The growth of wild-type and mutant strains were monitored by measuring absorbance at 600 nm. Results were an average of three separate trials.  Table 14: Strains used in this study.  Name JEC21 JEC43  Genotype MATawt MATa JEC21 ura5 (FOAR)  je-si  MATa,Isitl::URA5 ura5  je-siRl B3501A b-ui b-s10  MATotAsitl::URA5 ura5 + SIT1::NAT MATcz wt MATa B3501A ura5 (FOAR) MATaAsitl::URA5 ura5 MAToAsit1::URA5 ura5 MATaAs1t1::UR,45 ura5 + SIT1::NAT MATczAsitl::LIRA5 ura5 ÷ SIT1 MATcz wt  b-s42  b-s42R1 b-s42R2 H99 h-si  h-s2 h-siRl cdc40 cdc 68 cdc 99 cdc 103 cdc 2 JF-13 cdc2+ PKA1 GPA1 cdc 7  MATczAsitl::NEO MATaASItI::NEO MATaAs1t1::NEO + SIT1::NAT  MATaJEC21pka1::ADE2ade2 MATa JEC21 pkrl::URA5 ura5 MATa JEC21 pka2::URA5 ura5 MATa 3EC21 pkal::ADE2 pka2::URA5 ade2 ura5 MATa H99 pkal::ADE2 ade2 MATcL H99 pka2::UP.A5 ura5 MATa H99 pkal::ADE2 ade2 + PKA1 MATccH99gpal::ADE2ade2 MATcz H99 pkrl::URA5 ura5  Serotype D D D D D D D D D D A A A A D D D D A A A A A  Reference J.Heitman J. 1-leitman This study This study J. Kwon-Chung This study This study This study This study This study J. Heitman This study This study This study J. Heitman J. Heitman J. Heitman J. Heitman J. Heitman 3. Heitman J. Heitman J. Heitman J. Heitman  C. RNA Isolation and Northern Analysis. Cells were grown as described for the measurement of growth in liquid media. Cells were harvested by centrifugation after 6 hours growth in LIM or IR, flash frozen in an ethanol bath and lyophilized overnight at -20 °C. Cell pellets were pulverized with glass beads for 10 minutes and the cell powder was resuspended in 15 mL of Trizol extraction buffer (Invitrogen). RNA was isolated according to the manufacturer’s recommendations with the additional step of LiC1 precipitation at 4°C following the standard ethanol precipitation step. Northern blot preparation ,  and hybridization was performed as described (Sambrook et al. 1989) and all hybridization experiments were performed with two independent preparations of RNA from cells grown in LIM or LIM+Fe. The hybridization probe was prepared with polymerase chain reaction (PCR) amplified 409 bp DNA fragment from exon 6 of the SIT] gene using primers: SITEXON6F  100  (GTTATGATCCAGTCTGCCGT) and SITEXON6R (TGCCGAGAAGCTCGAGAAGG). The probe was labeled with 32 P using an Oligolabeling kit (Amersham Pharmacia Biotech).  D. Construction of SIT]:: URA5 (serotype D) and SIT]::NEO (serotype A) Disruption Alleles. For serotype D strains JEC21 and B3501A, a PCR overlap strategy was used to construct disruption alleles (Davidson et al. 2002). First, DNA fragments containing the 5’ (742 bp) and 3’ (853 bp) regions flanking the SIT] gene were amplified from genomic DNA from strain JEC21. The primers adjacent to the gene contained sequence for SIT] and the URA5 marker 5’: SIT1A (ACTCACTTCCTCCGATTCAG) and SIT 1 C (AAGGTCGAGCAACTTCGCTCAGGACTAAGACGTTGGCAAG); 3’: SIT 1 D (CCCACCTCCTGGAGGCAAGTCGGTGCGCTGTTATATGAG) and SIT1 F (CTATGTCATCAGGTGAGTGG). The URA5 marker was amplified using hybrid primers for SIT] and URA5: SIT1 B (CTTGCCAACGTCTTAGTCCTGAGCGAAGTTGCTCGACCTT) and SIT1 E (CTCATATAACAGCGCACCGACTTGCCTCCAGGAGGTGGG) amplified from the plasmid pJHM973 containing the URA5 marker encoding orotidine monophosphate pyrophosphorylase. The three fragments were joined by overlapping PCR as per Davidson et al., 2002 using the primers SIT1A and SIT1F. For the serotype A strain H99, a modified overlap strategy was employed (Yu et al,. 2004) that increased the yield of the final construct. A DNA fragment containing the 5’ (1151 bp) and 3’ (1162 bp) portions flanking the SIT] gene were amplified from genomic DNA from strain H99. The primers adjacent to the gene were hybrid for the neomycin (NEO) marker: 5’: SITA1 (AATCCGCACTCTCTCCATCA) and SITA3 (AGCTCACATCCTCGCAGCCCAAGATGTTGGCAAGTGGA); 3’: SITA4 (TAGTTTCTACATCTCTTCATGTACCATAGCTGCGGCTG) and SITA6 (CTGTGTGCTGATAACTGTCG). The NEO cassette was amplified using hybrid primers for SIT] and NEO: SITA2 (TCCACTTGCCAACATCTTGGGCTGCGAGGATGTGAGCT) and SITA5 (CAGCCGCAGCTATGGTACATGAAGAGATGTAGAAACTA) from the plasmid pJAF1 containing the resistance gene for neomycin. Fragments were used in  nd 2  PCR reactions as per Yu et al. 2004. Nested primers were used in 3’ round PCR:  101  and 3 round  SITANF (TGCGCATGCTAAGAACTTCC) and SITANR (AAGGCCGAAGCGGAGAGGAT). Constructs were introduced into the appropriate parent strain by biolistic transformation (Toffaletti et a!. 1993). Transformants were screened by colony PCR using primers: SIT1UP (TCACTTCCTCCGATTCAGAC) and 6328 (GGTCGAGCAACTTCGCTC) for serotype D; and SITAUP (GGACAGAGAATTGCCTTCGT) and NEOPS (AGCTCACATCCTCGCAGC) for serotype A. Transformants in which the wild-type allele was replaced were confirmed by Southern analysis. One disruption strain for JEC21, two for B3501A and two for H99 were retained for analysis. To complement the disruption mutations, the wild type SIT] gene was reintroduced by biolistic transformation into one mutant of each strain background on the vector pCH233 that confers resistance to nourseothricin (NAT). Serotype D reconstituted mutants were confirmed by PCR using primers SIT1A and SIT1F (Appendix IVD) and Southern analysis with the enzyme Scal (Figure 21 D) to ensure the return of the wt band. Reconstituted strains were resistant to NAT and regained the ability to use a siderophore as a sole iron source (Figure 24A and B). The serotype D strains were also 5FOAR and unable to grow on uracil deplete medium (return of ura5- phenotype). For serotype A, reconstituted strains were resistant to NAT and regained the ability to use siderophores as a sole iron source (Figure 24C).  E. Plate Assays. a. Siderophore utilization assays were performed as described by Heymann et a!. 2002. Iron free medium plates (IFM) were prepared in the same manner as LIM with the addition of 20g!L of Bacto-agar and 550 tM BPDA chelator. 200 iL of 1 0 CFU/mL cells were spread on IFM. A disk of Whatman paper was saturated with 10 iL of 100 M deferoxamine then placed in the center of the plate. Plates were incubated for two days at 30 °C. Zones of growth were recorded by digital photography.  b. Melanin production assays: DOPA medium was prepared with 20g/L bacto-agar in 900 mL dH O (autoclaved for 40 minutes). Separately, 1 g of L-asparagine (Sigma A0884), 1 g of 2 glucose,  3  g  of , 4 P 2 KH O  0.25  g  -7H 4 MgSO 0 , of 2  102  200  mg  of L-DOPA  (3,4  dihydroxyphenylalanine Sigma D9628) were dissolved in 100 mL 2 dH O . The pH was adjusted to 5.6. One mg of Thiamine-HC1 (Sigma T4625) and 5 tg of Biotin (Sigma B4501) was then added. The mixture was filter sterilized and then added to the autoclaved agar after cooling to 50°C. The following modifications were made to DOPA medium prior to filter sterilization to test various parameters: 1. 4X [DOPA]: 4mM DOPA (Sigma D9628) was added instead of 1mM 2. 4X [DOPA]  +  glucose: 4mM DOPA (Sigma D9628) instead of 1mM  +  1.0% glucose  instead of 0.1 % was added 3. DOPA pH9: media was raised to pH9 with NaOH 4. DOPA  +  Fell Ascorbic acid: 100 iM of Fell Ascorbic acid (Sigma A0207) was added  5. DOPA  +  BPDA: 100 tM of BPDA (Sigma B1375) was added  6. DOPA  +  BPDA  +  : 100 i1 2 ) 4 Cu(S0 M BPDA (Sigma B1375) was added stirred for 1  hour; then 200 pM of Cu(S0 , was added 2 ) 4 7. DOPA  +  FeEDTA: 100 iM of FeEDTA (Sigma EDFS) was added  8. DOPA  +  Deferoxamine: 100 pM of deferoxamine (Sigma D9533) was added  9. 2,5 Dihydroxybenzene diacetic acid: 100 tM (Aldrich D10,920-7) was added in lieu of DOPA 10. 3,4 dihydroxybenzoic acid: 100 !IM (Sigma P5630) was added in lieu of DOPA 11. hydroxyquinone: 100 iM (Sigma H9003) was added in lieu of DOPA To test for melanin production, 10 pL of 1  CFU/mL cell suspension was spotted on plates.  Plates were incubated for two days at 30°C, then assessed for pigment. c. Temperature sensitivity assays: Low glucose asparagine medium (LGA) was prepared in the same maniier as DOPA without the addition of DOPA. DOPA medium was prepared as described in the DOPA assays. 5 tL of 10-fold serial dilutions of cells were spotted on either LGA or DOPA plates, initial inoculum was 1 0 CFU/mL. Plates were incubated for two days at 30°C or 37°C.  d. Cell wall integrity assays: Assays were performed as above except 0.015 % SDS, 300 tg/mL Congo Red or 30 pg/mL of calcofluor white was added to the media.  103  F. Microscopy. a. Differential interference contrast (DIC) microscopy was performed on a Zeiss Axioplan 2 microscope. Melanized cells were prepared by placing 10 L of sdH O on a 2 microscope slide. A loop of cells was scraped from a DOPA plate (4 days growth), resuspended in sdH O and covered with a glass slip. Cells were viewed at 1 000X in oil immersion 2  b. Transmission electron microscopy (TEM) was performed on a Hitachi H7600 TEM. The cells were grown as described for the DOPA plate assays on DOPA or low glucose asparagines (LGA) grown for 4 days at 30°C. The full 10 pL spot of cell growth from DOPA or LGA medium was resuspended in lmL 2 sdH O , vortexed for 10 minutes then centrifuged for 5 minutes at 13,200 rpm. Cells were fixed in 2.5% glutaraldehyde in 0.05M cacodylate buffer at 28°C under vacuum. Microwave (Ted Pella Microwave) processing was employed at 100W with 2 minutes on and 2 minutes off (repeated twice). Samples were washed with cacodylate buffer at 28°C (twice) for 40 seconds using power level 2. Cells were fixed in osmium tetroxide at 28°C under vacuum by microwave processing at 100W for 2 minutes on and 2 minutes off (repeated twice). Samples were dehydrated in increasing concentrations of ethanol as follows: 50%, 70%, 90% and 100% (the final step was repeated three times). Samples were infiltrated with Spurrs resin in increasing ratios of Spurrs;Acetone under vacuum power level 3 for 3 minutes: 1:3, 1:1 and 3:1. Resin was polymerized by baking the sample 0/N at 60 °C. Samples were cut into 70 nm sections on a Leica Ultracut T Ultramicrotome and stained with 2% uranyl acetate for 14 minutes and lead citrate for seven minutes.  G. Antibiotic Susceptibility Assays. Antifungal compounds were purchased from Sigma-Aldrich Corporation as follows: streptonigrin (Sigma S1014), amphotericin B (Sigma A4888), ciclopirox olamine (Sigma C04 15), glyphosate (Sigma PS 1051) and phleomycin (Sigma P9564). Antibiotic susceptibility assays for determining the minimum inhibitory concentration of antifungal agents (MIC assays) were performed as per the National Committee for Clinical Laboratory Standards (NCCLS) procedure for micro-broth dilution of yeasts (approved standard M27-A). For these assays, 96 well plates (Falcon 353072) were employed with a starting concentration of 16 ig/mL for all  104  compounds except phleomycin (which was 4 ig/mL). Assays were performed in Sabouraud Dextrose Broth (SDB) with a starting inoculum of 5 X  CFU/mL. Microtitre plates were  incubated for two days at 30 °C and growth was evaluated visually. The MIC assays were performed in triplicate.  F. Virulence Assays in the Murine Model. Cells were prepared by inoculating 5 mL of YPD with a single isolated colony and growing strains overnight on a gyratory shaker (250 r.p.m) at 30 °C. Cells were washed three times with sterile phosphate buffered saline (PBS), then resuspended in 5mL of PBS. Cells were counted with a hemocytometer and cell counts were confirmed by plating serial dilutions of strains on YPD agar with subsequent growth for two days at 30 °C and colony forming units (CFU) were counted. Virulence assays were performed by Anita Sham. For serotype D, the B3 501 A wt strain and the sit] mutants were tested initially using DBA1 mice (Jackson Laboratories, 16-20 g). Five mice were used for wild-type B3 501 and four mice were inoculated with the sit] mutant. The mice were inoculated by intranasal inhalation with 5 x 106 cells per mouse in 50 p1 of PBS. For serotype A, A!Jcr mice (NIH Program, 16-20 g). were infected intranasally with an inoculum of serotype A cells per mouse in 50 p1 of PBS. Ten mice each were used for wt, sit] and  5x sit]  +  SIT]. An additional experiment was performed using a ten fold smaller iiinoculum 5 x  cells in 50 p1 of PBS and 5 mice per strain. Animals that appeared moribund or in pain were euthanized. Protocols followed approved standards of the UBC Animal Care Facility.  105  RESULTS A. Characterization of SIT]. a. Comparison of SIT] and Sitlp in serotypes A, B and D. The Clustal W program was used to compare the nucleotide and amino acid sequence similarity and produce similarity trees of SIT] and Sitip from serotype D (JEC21), serotype A (H99) and serotype B (WM276) strains of C. neoformans (Figure 13 and 14). As found in previous cross serotype comparisons, the genes from serotype D and from A are more closely related to each other than either is to serotype B (Tanaka et al., 2005). There is also a notable insertion in the serotype B Sitip protein (605 amino acids) in comparison to serotypes A and D (604 amino acids). This insertion appears to take place in the carboxy-terminus at amino acid 600 where serotype B Sitip appears to have an additional glutamic acid residue (E) (Figure 13-in bold).  The SIT] gene was further compared across serotypes for nucleotide divergence in  separate exons and introns (Figure 15). As expected, the majority of nucleotide sequence divergence occurs in intronic sequence. Nucleotide similarity in exons is> 80 % in all pairwise comparison of strains. Intron sequence similarity varied widely, with little to no similarity in some introns for serotype B vs. A or D strains (e.g. introns 1, 4 and 5).  106  SerotypeD SerotypeA SerotypeB  MPDQLYPEYELQRAISNTKNADDSTAFGEKSPGVRRIEI IAASFTTWHRWVLFISVFFMS MPDQLYPEHELERAISSTKNADDSTAFGEKSPGVRRIEIIAACFTTWHRWVLFISVFLMA MPDQIJYPEAEIJERAI SNTKNADDSTAFGEKSPGVRRIELIAASFTTWHRWVLFISVFLNA **  .  Serotypeo SerotypeA SerotypeB  CNYGTJDGSVRYTYQAEALSELGTSAQVSTVTVVRS IVAAAAQPAFAICVSDYFGRVSILI I CNYGLDGSVRFTYQSEALSELGTSAQVSTVTVVRS IVAAAAQPGFAKI SDYFGRVS ILVI CNYGLDGSVRYTYQAEALSELGTSAQVSTVTVVRSIVAAAAQPCFAKVSDYFGRI SILVI  SerotypeD SerotypeA SerotypeB  SVILYVVGTIVTATSTNLAAFCGGSVLYQFGYTGVQLLVEVLIADVTSLRSRLLFSYI PA SVILYVVGTIVTATSTNLAAFCGGSVLYQFGYTGVQLLVEVLIADVTSLRSRLI FSYI PA SVILYVVGTVVTATSTNLAAFCCCSVLYQFGYTGSQLLVEVLIADVTSLRSRLLFSYI PA *********:************************  ******************:******  SerotypeD SerotypeA SerotypeB  TPFLINAWI SGNVASAVLTHSTWGWGIGMWAI IFPVTVIPLLFSLIQAEWRAHRKGLLRB TPFLINAWI SGNVASAVLTHSTWGWGIGMWAIIFPVTVIPLLFSLIQAEWRAHRKGLLRE TPFLINAWI SCNVASAVLTHSTWGWGIGMWAIIFPVTVIPLVVSLVQAEWRAHRRGLLRE  SerotypeD SerotypeA SerotypeB  I PSPLRTFGDRBMWADIFWQI DLVGLLLLAAVLALILLPFTLAGGVAS IWRTARVIAPLV I PSPLRTLGNRHMWADIFWQI DLLGLLLLAAVLALILLPFTLAGGVGS IWRTARVIAPLV I PSPLRTLGDRHMWADIFWQVDLMGLLLLAAVLSLILLPFTLAGGVASIWRTARVIAPLV *******:*:**********:**:*********:*************************  SerotypeD SerotypeA SerotypeB  VGFVVALPLFVFWELKVARHPMLPFRILKDRQVLASLFIANLLNTAWYTQGDYLYYTLLV VGFVVALPLFVIWELKFARHPMLPFRILKDRQVLASLFIANLLNTAwYTQGDYLYYTLLV VGFVVALPLFVIWELKVARHPMLPFKILKDRQVLASLFIAMLLNTAwYTQGDYLYYTLLV  Serotypeo SerotypeA SerotypeB  AFDRDIISATRVQNIYSFTSVVIGVCLGLIIRKVRRLKwFIVAGTLLFVLAFGLLIRYRG AFDRDI ISATRVQNIYSFTSVVIGVCLGLI IRKVRRLKWFIVAGTLLFVLAFGLLIRYRG AFDRDI ISATRVQNIYSFTSVVIGVCLGFI IRKVRRLKWFIVAGTLLFVLAFGLLIRYRG  SerotypeD SerotypeA SerotypeB  GYS I SDFAGLVAAEVVLGIAGGLFPYPTQVMIQSAVQHERTAVVTSLYLASYSVGSALGN GYSVSDFAGLVAAEVVLGIAGGLFPYPTQVMIQSAVQHERTAVVTSLYLASYSVGSALGN GYSVSDFAGLVGAEVVLGIAGGLFPYPTQVMIQSAVQBERTAVVTSLYLASYSVGSALGN ***:*******************************************************  SerotypeD SerotypeA SerotypeB  TIAAAIWTNTMPSHLYNDFIRAGLSTTDASTLQALAYASPLQFI IDYPPGTPEREAVGSA TIAAAIWTNTMPSBLYNDLLRAGLSTVDASTLQALAYASPLQFIAEYAPGTPEREAvGSA TIAAAIWTNTMPSHLYNDFLRAGLSAADASTLQALAYASPLEF IVEYPPGTPEREAVGSA ******************: :*****: .**************:**  :*.************  SerotypeD SerotypeA  YREVQRYLTITGICI STVIVFMALSLRNPRLGDEQSLPDAEKLEKVPSEMSGATEETTET YREVQRYLTITGICLSTVIVFVSLTLRNPRLGDEQSLPDAEKLEKVASEVSGTTEETTKT  Serotypes  YREVQRYLTITGICI SSVIVLVALTLRNPRLGDEQSLPEAEKLEQVPSKMSGANNEGTEE **************:*:***:..*:*************:*****:**..**::*  SerotypeD SerotypeA SerotypeB  *:  KQKN KHEN TKQKN  Figure 13: Amino acid alignment of the Sitip protein from serotypes A, B and D strains of C. neoformans Alignments performed with Clustal W http://www.ddbj .nig.ac.jp/searcb/clustalw-e.html A. Serotype D strain B3501A Sitip (604 amino acids) (http://www-seciuence.stanford.edulgroup/C.neoformans/index.html) B. Serotype A strain 1199 Sitlp (604 amino acids) (http://www.dumru.mc.duke.edu/) C. Serotype B strain W1v1276 Sitip (605 amino acids) (www.bcgsc.bc.ca The additional glutamic acid residue (E) in the C-terminus of serotype B Sitip has been bolded.  107  SerotypeD SerotypeA Se’rotyp eB  Figure 14: Similarity tree of the Sitip protein from serotypes A, B and D strains of C. neoformans Similarity tree produced from alignment (Figure 13) in Clustal W. http://www.ddbj .nig.ac.jp/searchlclustalw-e.html  108  40  50  60  80  0  Gene Feature  Exon Intron Exon Intron Exon Intron Exon Intron Exon Intron Exon 1 1 2 2 3 3 4 4 5 5 6  --  Serotype A vs D •SerotypeAvsB LlSerotypeBvsD  Exons and introns were manually determined in the nucleotide sequence by comparison to the S. cerevisiae homolog SIT1p 1/Arn3p protein sequence and identification of C. neoformans exonlintron consensus sequences donor 5’-GT(XX)GY-3’ and acceptor 5’-YAG-3’ (See sequences in Appendix IVAa). Nucleotide similarity was determined by comparing sequences using the BLAST 2 sequences algorithm at NCBI (http ://www.nchi.nlm.nih.gov/hlast/bl2seq/wblast2.cgi). Instances where no bar is apparent indicate that there was no significant similarity in the sequences compared (Intron 1, 4 and 5).  Figure 15: Comparison of exon and intron nucleotide sequence similarities for the SIT1 gene in serotypes A, B and D strains of C. neoformans  Z  30 a, 20  C..)  a,  :  90  100  b. Identification of siderophore related genes in C. neoformans. The completion of total genomic sequencing projects for strains representing three serotypes of C. neoformans (serotypes A, B and D) in conjunction with the growing database resources in other fungi, allowed an in depth initial in silico analysis to search for siderophore related genes in C. neoformans. A review by Haas (2003) summarized siderophore synthesis and transport in fungi. The review included a table of fungi, detailing the presence or absence of siderophore related genes.  These included structural genes for siderophore transport and  synthesis (Ornithine N -monooxygenase and non-ribosomal peptide synthase) as well as 5 regulatory genes of siderophore systems in fungi (transcriptional activators and repressors). Using this table as a guide, the C. neoformans databases were searched using all fully characterized fungal homologs involved in siderophore synthesis, transport and regulation. C. neoformans databases were searched for serotype A at Duke: (http://www.dumru.mc.duke.edufj, D  Stanford-(http://www-seguence. stanford.edulgroup/C.neoformans/index.html)  or  TIGR:  (http://www.tigr.org/tdb/e2k1/cnal/) and serotype B at MSGSC (www.bcgsc.bc.ca). Results of these searches are summarized in Table 15. Through this analysis, multiple siderophore transporter homologs were identified in C. neoformans: SIT]/ARN3 of S. cerevisiae (characterized in this study and found on chromosome 1 of B3501A, JEC21 and H99) and ARN4/ENB] of S. cerevisiae and MIRB of A. nidulans. ARN4/ENB] and MJRB may be equivalent homologs resulting in one gene because they hit the same genomic location in C. neoformans, where MIRB is the closest homolog (chromosome 6 of B3501A, 7 of JEC21 and 8 of H99). There is also a putative homolog of MIRA of A. nidulans in the serotype D strains (chromosome 4 of B3501A and 5 of JEC21) and MJRC in JEC21 (chromosome 3).  Other transporters do register significant hits, however the homolog  assignment has been given to the gene that has the lowest e-value for that genomic location where more than one transporter hits a given location. The serotype A and D strains do not have putative homologs for the ornithine monooxygenase enzyme involved in siderophore synthesis. They do have putative homologs for a non-ribosomal peptide synthase, however the e-values are fairly high and the top annotated hit is for an aminoadipate-semialdehyde dehydrogenase. The putative homologs are found on the same chromosome as the MIRB homologs (chromosome 6 in B3501A, 7 in JEC21 and 8 in H99).  110  In contrast the serotype B strain WM276 (C. gattii) does have putative homologs for both enzymes necessary for siderophore utilization. The assembly and annotation of the serotype B strain WM276 has not been assigned chromosome numbers at this time, therefore gene locations and the number of homologs can not be accurately determined. Although serotype A and D strains of C. neoformans do possess putative siderophore transporters, they do not appear to have homologs for both of the enzymes necessary to synthesize siderophores, with the exception of the serotype B of C. gattii.  The same  phenomenon is noted in the fungi S. cerevisiae and C. albicans (Table 15) where transporters but not synthesis enzymes are present.  It is likely that these fungi capitalize on siderophore  production by nutritional competitors in their environment by possessing these transporters. These results are interesting because the more evolutionarily similar basidiomycete Ustilago maydis can synthesize siderophores. In contrast to their similarities in siderophore related structural genes, C. neoformans does not have a homolog of the well characterized S. cerevisiae transcriptional activator AFT]. It does however have a putative homolog of the transcriptional repressor GAF2/FEP] of Schizosaccaromyces pombe (chromosome 1 of B3501A, 10 of JEC21 and 10 of H99). The homolog to this gene has been named CIR] (Cryptococcus Iron Regulation) and is being characterized by post-doctoral fellow, Wonhee Jung, in the Kronstad laboratory. A second putative repressor that is a homolog of Penicillium chysogenum SREP is present in B3501A chromosome 9 and JEC2 1 chromosome 1. An additional repressor homolog is also present on chromosome 1 of H99, however it is still most closely related to GAF2/FEP1 of S. pombe and therefore may represent a second homolog of that gene. Like C. neoformans, U maydis has a homolog for the transcriptional repressor called URBSJ, but not a homolog of the yeast transcriptional activator AFT].  111  Table 15: Siderophore-related genes in C. neoformans. CHR= chromosome followed by e-value for the related database: tBLASTx for serotype A and D; BLASTn for serotype B  Str,tetnral Gnneo  Organism  Gene  Serotypn DC. nnofonoans Scrotype Ahomolog (a) Stanford or TIGR DUKE DUMRC  Seqoenee >YHL04OC CIte 8  ,ltorn Transporter 1 Sidnro  acsbara,,,vco.vwac,Waiae 1 , .  .S’chiaoaaccarootycc.opo,,,ho  ARNI ENBt/ARN4  ‘YOLI58C Ctn tS  SITI/ARN.1  >YEL065W Chr 5  TAFt/ARN2  ‘YHLO47C Chr 8 9.So-93  CHR Staoford ln-60 CHR I TIGR 2,6r-106 CHR 6 Stanford lc-30 CHR 7 TIOR 7.Oe-51? CUR I Stanford 3n-89 CUR t TIGR 4.2n-t 15 dR t Stanford e-75 CUR t TIGR 5n-37  Serolyite B @BCGSC  BLASTn only  CUR I 3n-77  No Hit  CHR8 30-3 t CUR I 4n-89  No Hit No Hit/cootig_284 with onrotype D StTt IBLASTo  CHR t 2n.35  No Hit  ALO3 1534 AL033t27  Zt/13 12 C0ARN I/CaSt Caodeda a/b/stow  TA  Aaposgi/bcroidu/aaa  airA  ‘gi117062tl84lgblAY027565.tI  ojrB  sgil3O/158789]gblAYI3I33O.tp Sgil3(6/5879lIgbIAYI35 152. t(  ntird Aopcrg/llo.eJ’ooo’gatoo  Ornithine N’-monoooygenase  *  Uall/ago,oavdio  sidl  .Vchizasaccaroorvceapo,,shs-  CURS te-tI  No Hit  CHRS o.IOb  eontig_218 t.le—113  CHR S 3e-22  No He?  No Hit  No Hit  eonlig_Itt tOe-Sb  *  Nouraspora eras at  Socohanooycea cerevislac  CHR4 Stanford 30-tO CHR S TICS 2.5r—I9 CHR6 Stanford 4n-121 CHR 7 TIOR 5.30-133 CHR I Stooford Io-26 CHR 3 TICS 1,4n-40  >gilt70588lgblM98520.Ij  -  AL 138854  Caw/ido o/hicaa.r  Nonribonomtd topside synthrino.  .-lapergellus ole/it/oats  stdA  ‘gil327093921gb1AY2235t1 I >gil227792371dbjlAB07 1287.  No Hit  No Hit  .tsporgil/eaa srsoac  dITA  I  No Hit  No Hit  .4spcrge//oofo,olgalu.r  *  >gi127316321gb(U62738.tI  CHR 6 Staoford 5n-06 CUR? TIGR 8.20-18 atoinaodipate-ootoiatdnlo’de  CHt?53e-/t6  cootig III 7.7r-t0  detlydmgonasr  CI-tR 83e-05  No Hit  promotnr  No Hit  No Ho  No Hit  >gi17034671gb1L29t?5t.tI  CHR t Stanford Is-IS CHR tO TIGR I.4e-22 CHR I Stanford 2e-1l CUR tO TIOR I 2r-21 dO Stanford 7n-10 CUR I TIGR 9.tn-06  attd CHR I tn-t6 CHR tO3e-tJ and CUR t 2n-t2 CHR lIt Sn-Itt md CUR I Sn-9  Nesrospora crania  *  ..1.ipsrgi//o.s pot/a/sos  1.1859119  Ust,lagscoasat/s  oid2  Saccharo,oyccx cerews,00.Schtaaaaccaesoec-.spaohc. Cain?lila a/bleats,  No Hit COotig_t  It 5.2r.12  -  ALt38ttS4 -  CHR 7 TICS 5.3E-21 antinoadipatn-tennatdnttyde .4apergi/ttceoldu/aoa  sidC  .itapo-rgillusJiso/gatos  +  Neoroapora crasaa  *  .‘lspor/T//oopa//o/aoa  1.185909  Use lags s,avd/s  ob,ot  >gip327093941gb1AY2238l2.II  Regulator-encoding genes Iron-reaponsir’r tronooriplionat repreonor  agil t290691gbJL5-t05O.I  Saccharo,ovcoa cerceioiae &hlaoaeesresro’ceapaoche  -  GAF2/FEPI ‘gi12t74532/NSbIAF52t/9?3,tl  Caodola a/b/oars  SFUI  .‘tspegl his ,bdo/sos  amA  Aapergilhsofa,oigatua  *  Neut-oapca-aeraara  arc  >gi45852l2gbAFO95898.I  CHR 9 Stanford c 90-13  trait reoponoivn transcriptional activator  CHR tO7n-t8  No Hit ooobg 45 2.2e.6  enolig 214 90-8  cgii3552tl27gbAFO87I30,IJ  CHR tO TIGR 2n-08  CHR tO to-t3 andCHR I 2e-t18  sootig_214 tin-IS  i-gi1l5179t51gb1U484t4.II  CHR 9 Stanford 4e-I5 CHR Itt TIOR l.8e-07  CUR It) 6r-l3 and CHR I 4r-8  No Hit  Pc-old//la,,, chysagc-aost  amP  &cryliaciarc-c.a  birt  >giIt3620I72IembA3309O5l. II partiat  CHR 9 Stanford 4n-t5 CUR I/I TICR 1.8e-tS  and CHR I 4e-9  Saccharo,,,cces co-,oria’iao  AFTI  >YCLO7IW Cltr 7  No Hit  No Hit  No Hit  Saeeharo,ovco.,cc.,-cclslae  AFr2  ‘YPL2O2C Chr 16  No Hit  No Hit  No Hit  Candle/a a/b/coos  *  .1sjorg,l/a.a sic/a/any .4ope,iltccofcaoigotoa Neoraapara crassa  -  -  -  112  CHR  to 3e-t4  No Hit  c. Comparison of Sitip to fungal homologs. C. neoformans Sitlp  was compared to orthologs in the other fungi by amino acid  sequence alignment and similarity trees (Figure 16 and 17). Interestingly, we found that the C. neoformans homolog clustered with the ascomycetes S. cerevisiae and C. albicans and not the more evolutionarily similar basidiomycete U maydis. These results are concordant with the findings from homolog searches of siderophore related genes (Table 15), where C. neoformans is more similar to S. cerevisiae and C. albicans than U maydis with respect to the presence or absence of siderophore synthesis genes. The fungi clustered in a pattern that relates to whether they appear to produce and take up siderophores, as is the case with U maydis and Aspergillus nidulans (Haas, 2003), or whether they only have homologs for siderophore transporters but not synthesis such as C. neoformans, S. cerevisiae and C. albicans (Haas 2003).  The closest  homolog, Arn3p/ Sitip of S. cerevisiae has 39 % identity and 55 % positive amino acid matches to C. neoformans Sitip (resulting in an e value of-.l 12).  113  Figure 16: Amino acid alignment of the Sitip protein from the serotypes D strain B3501A of C neoformans with fungal homologs. Alignments performed with Clustal W. http ://www.ddbj .nig.ac.ip/search!clustalw-e.html Fungal homolog sequences and GenBank Accession numbers can be found in Appendix IVAb. C. neoformans SIT] sequence was from the Serotype D strain B3501A (http:I/www seguence.stanford.edu/group/C.neoformans/index.html) Coding sequence was manually determined. (Appendix IV-Aa). An-Aspergillus nidulans Ca-Candida albicans Cn-Cryptococcus neoformans Sc-Saccharomyces cerevisiae Urn- Ustilago maydis  CLUSTAI W (1.83) Multiple Sequence Aliqnments Sequence type explicitly set to Protein Sequence format is Pearson Sequence 1: CnSIT1p 604 aa Sequence 2: ScSITlp/ARN3 628 ae Sequence 3: ScARN1 627 aa Sequence 4: ScARN2/TAF1 637 aa Sequence 5: ScARN4/ENB1 606 aa Sequence 6: CaSITA/CaARN1 604 aa Sequence 7: AnmirA 609 aa Sequence 8: AnmirB 604 aa Sequence 9: AnmirC 607 aa Sequence 10: UmSitl 583 aa  CLUSTAL—Alignment file created [clustalw.aln] ScARN1 MESVHSRDPVKEEKKHVFMGMEHELNPETHNDSNSDSYGLPQL.nA V1flJJfl ScARN2 /TAF 1 MIEVPEDNRSSQTKRKNTEKNCNELMVDEKMDDDSSPR DEMKDKLKG CaSITA/CaARN1 MTSYQSSNNHSSEEDKHLSGDEKTFSPSDIVEKAIVE ScSIT lp/ARN3 MDPGIANHTLPEEFEEVVVPEMLEKEVGAKVDVKPTLTTSSPAPSYIE CnSIT1p MPDQLYPEYEIJQRAISNTKNADDSTAFG ScARN4/ENB1 MLETDHSRNDNLDDKSTVCYSEKTDSNVEKSTTSGLRRID AnmirC MPLLEPSATAYGTFGDMRPDTEDEGERLLTDGYVSDDDCSAVTSVD UmSitl MSRPFDAENVEHRDTSHDSMDMTDQLA AnmirA MALDDISAVPKGALDTDPAVERPPPLLDAORSDSE Anmirs MTIGSKFSLLAGTRKTDGPTEI SASSPPDVETPSAEKTATASAGNKEVGINDNSSDEALP  ScARN1 ScARN2 /TAF1 CaSITA/CaARN1 ScS IT lp/ARN3 CnSIT lp ScARN4 /ENB1 AnmirC UmSit 1 AnmirA AnmirB  NRSLI IQQTEI IG-SAYNKWYLQAILLLSAFICGYGYGLDGNIRYIYTGYATSSYSEHSL TKSLIIRKSELMA-KKYDTWQLKAIFLFSAFICTFAYGLDSSIRGTYMTYAMNSYSAHSL EKSICVEKA3ILANQWKHTFWFKJLLGFSAFLCGYAYGLDSQTRYVYTAYATASWSEM5L LIDPGVHNIEIYA-EMYNRPIYRVALFFSLFLIAYAYGLDGNIRYTFQAYATSSYSQHSL EKSPGVRRIEI IA--ASFTTWHRWVLFI SVFFMSCNYGLDGSVRYTYQAEALSELGTSAQ AVNKVLSDYSSFTAFGVTFSSLKTALLVALFLQGYCTGLGGQISQSIQTYAANSFCKHSQ SVQEGVRKIEAIN--ITWTTRSLVIAYI SIFLMAFCTSLEGQTIMSLSAYATSAFSKHSL DKQICVVAAEAGR---TVADWTLWMGILGIALVAYLYGLDNNTMWAWQTYATTSFNDYPA RLQPCVKRAEMLR--KGWTRQGLI IAFTGLFLATLSINFGDYSTQVYVPYATSAFKQMSA SQHVQTGVQKIQAVTLVWSKWSLVAVFCLLWLVTLANGFRQS ILYSLTPYATS SFQSMSL  114  SCARN1 ScARN2 /TAF1 CaSITA/CaARN1 ScSIT1p/ARN3 CnSIT1p ScARN4 /ENB1 AnmirC UmSit 1 AnmirA AnmirB  LSTINVINAVVSAASQI IYARLSDVFGRLYLFISAVILYVVGTI IQSQAYDVQRYAAGAI ISTVSVIVLMISAVSQVIFGGLSDIFGRLTLFLVSIVIJYIVGTIIQSQAYDVQRYAAGAV LTTVNAITGVVAAASQPVYARLSDVLGRLELFIVAVLFYVVGTI IECQSPTINAYVAGAV LSTVNCIKTVIAAVGQIFFARLSDIFCRFSIMIVSIIFYSNGTIIESQAVNITRFAVGGC VSTVTVVRSIVAAAAQPAFAKVSDYFGRVSILI ISVIIYVVGTIVTATSTNLAAFCGGSV VGSINTVKSIVASVVAVPYARISDRFGRIECWIFALVIJYTIGEII SAATPTFSGLFAGIV I STVLVVQNVVNAVIKPPMAKIADVFGRFEAFCVSILIYVLGYIQMAASTWVQTYASAQI YTAVSVVQAVI IAVGKFPIAKLADVFGRAQAYALSVFLWVIGFVIIAIAQNTRYVAGGTV MSAARVVGNITRIAAYPI IAKLGDVFGRAENFILS IVFQAVGYAIYAGCKNVGQYIAGGI LTVINIVSS Y 1 IPVAKVVDVWGRAEGWLVMVGLSTLGLIMMAASKNLETYCAADV AFIVSAI *  SCARN1 SCARN2 /TAF1 CaSITA/CaARN1 SCSIT1p/ARN3 CnSIT1p ScARN4/ENB1 AnmirC UmSit 1 AnmirA AnmirB  *  *  *  *  WMLDVVGVLLMGCSLCCILVPLTLAG-GVKTTWNDSRLIGPFVLGFVLIP--ILWIWEYR WKLDVVGVLLFTAGVGCILVPLTLAG-GVSTNWRNSKIIGPFVLGFVLVP--GFIYWESR WRLDVIGLLLLTVSLCCLLVPLTLAG-GIRETWKKAHI IVPIVIGGVLIP--VFLLWEGY WKLDI IGMLLITVFFGCVLVPFTLAG-GLKEEWKTAHIIVPEVIGWVVVLP-LYMLWEII( WQIDLVGLLLLAAVLALILLPFTLAG-GVASIWRTARVIAPLvvGFvvALP-LFvFWELK DDINLIGVILFTAFLVLVLLPLTIAG-GATSKWREGHIIAMIvvGGCLGF--IFLIWELK YDLDIFGLALLSAAVTLILVPLTLAA-NTIQJGWK5NSIVAMIVIQVVCLI LLPFWETSIcI( IDIDALGLFLICVGFLLILLPvNLAK-LQPNGWSTGWIIAMLVICGVMLIS---FCVWEC VQLDAFGAILLLLGL5LFLVPL5LTGSGN5DDWBRGSFIAJ4LVLCVVIFVA--FLAWD’pW FAFDIPGVILLAGCLTVFLLPflLAT-RAPNGWK5DYI IANIVTGFVVMVL--FVLYQAY *  ScARN 1 ScARN2 /TAF 1 CaSITA/CaARN1 ScSITlp/ARN3 CnSIT1p ScARN4/ENB1 AnmirC UmSit 1 AnmirA AnairB  *  SWDVGMWAFIFPLSCVPIVLCMLHMQWRARKTPEWHALKGQKSY--YQEIJGFIKILRQLF SWNIANWAFIFPLCCIPLILCMLHMRWKVRNDVEWKELQDEKSY--YQTHGLVQMLVQLF KWGIGMWAFILPLSCIPLVCCMIHMRWLAGKTEEWRVFRQRKTK--FQELGVAGFSKYLF KWGIGMWAFILPLACIPLGICMLHMRYLARKHAKDRLKPEFEAL--NKLIcWKSFCIDIAF GWGIGMWAI IFPVTVIPLLFSLIQAEWRABRKGLLREIP---SP--LRTFGDRHMWADIF RWGYGIFCIIVPISTLILVIJPYVYAQYISWRSGKLPPLKLK EKGQTLRQTLWKFA RWGYGMWSI IIJPASFLPIJAIJSLLLNQRKAKRLNLIKERP HHRRGFVAAVRRTW RWGPGFFSICAPVAAFSI IFALAINQRRARAMGIjVPSRP YKHMSFLAAVYNFL RWGFGNWAI IEPVC SVIJLVCTMLYYQKRARKDPSPAEFASEPTERNVDDGWWKRIYNLVW RWGFGAFAI IFPFVASPVYFVLKVGLNRAEKQGIIQPRIJR SGRTLSQNFKYYF •  ScARN1 ScARN2 /TAF1 CaSITA/CaARN1 ScSIT1p/ARN3 CnSITlp SCARN4 /ENB1 Anmirc UmSitl AnmirA Anmirs  :*  FYNAGYVGVILILLI ILSDFSSLKWRLLYQFVPTWPFIINTWIAGNITSRANP---VVNW FYYVGLVGVk4LQVVLMLSDNSSLKWRLFYTLIPSWPSIITTWVSGSVVEAANP---LENW LFQIGYSGI I IMLLFITJSDFSSLRWRLFFTLCPSFPFIINTWISGNVTAAVG TRW FYQLGLTGI ILILEVIASDFSNLNWRLLALFIPALPFIINTWISCNVTSAIfl tYQFGYTGVQLLVEVLIADVTSLRSRLLFSYIPATPFLINAWISGNVASAVLT---HSTW IQQFGYSGFRIJLATALTGDLSGLRDRTFAMNIFLIPVIINTWVSGNIVSSVAGNVAPYKW LPELPFLVTVWIGPTIADVVLE---NSSW 1 FYAAGSTGLQILQQVFIADSSSLLNRALLAI IYAFGNTGVQIMQQIVLADYI STKWRGAAIGLVSLPYVINFWASAQIYPKIVA----VNW FEAIGSTGFGLTQQVFVADVTNIJINRAVWSTLPDSLTVIPALYLGTEIAEAVLE--KNEW FYSVGFAGNNYILCVLAADITNLRNRGIAFAFTSSPYMITAFAGSKAAEJ<FLVN---VNW *  ScABI41 ScARN2 /TAF1 CaSITA/CaARN1 SCSIT1p/ARN3 CnSIT1p SCARN4 /ENB1 Anmirc UmSitl AnmirA Anmirs  **  *:  *:*::  *  •:  *  *  FARDPILPYRLVKDRAVWSSMGI SFLIDFIflNAAD-flflVMIVAVNE5VK5ATRIAfl LALVPFAPFKLLKDRGVWAPLGIMFFICflYQNAAG-YLYTILWAVDE5ASSATRI 11Th GARDPI IPLHLMEDRGIWSVV5I5LFFDFVFAVE5N-FLYTVId4VAVNESQ55ATRIASL YSRHPLTPWDLIQDRGIFFAI,LIAFFINFNWYNQGD-YMYTVIJVVAVHESIKSATRITSL VARHPMLPFRILKDRQVLASLFIANLLNTAWYTQGD-YLYYTLLVAFDRDIISATRvQNI FAZNPFIPRVYLGDPTIYVALLMEFVWRLCLQIELE-YLVTVIJMVAFGESTLSAQRIAQL LAPKPLLSLHLLKQRTALAGCCLAFFYFMAFYFSVQPYLY5yLQVVQGyDVATAGRVTQT VAPKPILNRRWRLI4HDVHFATAIGFFDFFSFYASWVPAYYWSLIVI4G-yDT4TGATyFSNC CAKKPF IPYRMIKNRTVAAACLJflILDFflIYSVFSV-FFTSYLQVAAHHGAQPATRIDN5 WAPQPFLKYEFLTNRTVLGACLIDATYQNSYYCWN5-YFNSFLQVVCNLPVAEAGYVG5T *:  *  115  *  SCARN1 SCARN2 /TAF1 CaSITA/CaARN 1 ScSIT1p/ARN3 CnSIT1p ScARN4 /ENB1 Anmirc UmSiti AnmirA AnmirB  SSFVSTVASPFFAI.LVTRCTRLKPFIMFGCALWMVANGLLYHFRG----GSQSHSGIIGA YSFVTAVVAPFLGLIVTRSSRLKSYIIFGGSLYFITNGLFYRYRS----GQDADGGIIAG SSFVSVVTGFIFGLFVVYFRRLKGFVVFGCANWNVAFGIMYHFRS----QLHANAGI ICC YSFVSVIVGTILGFILIKVRRTKPFIIFGISCWIVSFGLLVHYRG-----DSGAHSGIIGS YSFTSVVIGVCLGLI IRKVRRLKWFIVAGTLLFVLAFGLLIRYRGGY--SI SDFAGLVAA YNFLQSCTNIVVGIMLHFYPHPKVFVVAGSLLGVIGMGLLYKYRV----VYDGISCLIGA FAFTSTIAAFGVS ILIKYTRRYRVYVTLGCVIYMTGLLLMLLYRK----EGSSPLQVLGT QSLALTVFGIAAGFLSLGTKNYKWIMISGACIRLLGIGLMIKYRS----SGSSNVQAVFP LRVAFQVAGI FAAYFNRFTKRSQVWVFTGVPLCVIGMGVISLYLVDMGEGRVGNEAAFVTA FQVVSGVLIJFMVGFAIRKTGYFRWLLFIGVPLYI FAQGLMIHFRQ----PNQYIGYIVMC *  ScARN 1 ScARN2 /TAF1 CaSITA/CaARN1 SCSIT lp/ARN3 CnSIT ip ScARN4 /ENB1 AnmirC UmSit 1 AnmirA AnmirB  LCVWGVGTTLFTYPVTVSVQS-AVSHENMATVTALNYTLYRIGSAVGSAVSGAIWTQTLY MVIWGLSSCLFDYPTIVSIQS-VTSHENMATVTALNYTVFRIGGAVAAAISCAIWTQSLY MCLMGFGTGFFSYPINVSAQS-CVSHEHMAVISSALYTTYRIGYAVGSSVAGAIWSQMLY LCLLGFGAGSFTYVTQASIQASAKTHARMAVVTSLYLATYNIGSAFGSSVSGAVWTNILP EVVLGIAGGLFPYPTQVMIQS-AVQHERTAVVTSLYLASYSVGSALGNTIAAAIWTNTMP EIVVCIAGGMIRFPMWTLVHA-STTHNEMATVTGLLMSVYQIGDAVGASIAGAIWTQRLA QVIVGNGGGLLNVPVQLGVQA-SASHQEVAAATAMFLTSMEMGGAVGAAI SGAVWTHNIP QVLQGMGGGFLGITLQVAAQV-SVRHQOVATVTAYFLLLTEMGCACGNALVGAVQTNVLP KSLIGIGRGFYQTASQVSVQA-KVSRGEVSVVTAVFFAAMS IGGAIGTSVAGAIWRSTIJP EIFISIGGSIFVLLQQLAVLV-AVDHQYVAAALAVLFISGGIGGAVGNAISGAIWTNTFL .*  SCARN1 SCARN2/TAF1 CaSITA/CaARN1 ScSIT1p/ARN3 CnSIT1p ScARN4 /ENB1 AnmirC UmSiti AnmirA AnmirS  KQILKRNG DVAIJATTAYESPYTFIETYTWGTPQRNALNNAYKYVQRLETIVA PKLLHYMG DADLATAAYGSPLTFILSNPWGTPVRSANVEAYRHVQKYEVIVA SRLVKYLG DSTLATSVYTDPYTFIAQYVWGTPEREAAVKAYGEVQRVLMSVC KEI SKRI S DPTLAAQAYGSPFTFITTYTWGTPERIALVNSYRYVQKILCI IC SHLYNDFIRAGLSTTDASTLQALAYASPLQFIIDYPPGTPEREAVGSAYREVQRYLTITG KELIQRIJG SSLGMAIYKSPLNYLKKYPIGSEVRVQMIESYSKIQRLLIIVS RKJJNLYLP DEYKSEAGAI FGKLTKALSYEMGTPVRSAINRSYQETMNKLLVLA GYYEKYLP MLNATQRAAIYASPFTAVSQYPIGTPERTGMINAYNDYMRILLIVA PKLAQHLP AELKOQAQAX FGS IVVAQKYEVGTPARDAIDMCYRQSQRMLAIAA PALMRNLP AAG 1 ESAKARAVAIYGDLRVQLSYPVNSPERIAIQESYGYAQARMI *  SCARN 1 ScARN2 /TAF 1 CaSITA/CaARN1 ScSITlp/ARN3 CnSITlp SCARN4/ENB1 AnmirC UmSit 1 AnmirA AnmirS  SCARN1 SCARN2 /TAF1 CaSITA/CaARN1 ScSIT1p/ARN3 CnSIT1p SCARN4/ENB1 Anmirc UmSiti AnmirA AnmirB  *  *  LVFCVPLIAFSLCLRDPKLTDTVAVEYIEDGEYVDTKDNDP ILDWFEKLPSKFTFKRE LVFSAPMFLLTFCVRDPRLTEDFAQKLP-DREYVQTKEDDPINDwIAKRFAKALGGHKKD IAFVAPMIVSALFMRDHKLTNEQSLEDVEKQEEKDSLANFFGNFKKKRVAV LVFCFPLLGCAFNLRNHKLTDSIALEGNDULESKNTFEIEEKEESFLKNKFFTHFTSSKD ICISTVIVFMALSLRNPRLGDEQSLPDAEKLEKVPSEMSCATEETTETKQKN ISFAAFNAVLCFFLRGFTVNKKQSLSAEEREKEKLKIKQQSWLRRVIGY LLATLPLIPLSLLMSNYKLOKMSESSDHDDASPRNGLGPCERAKRT IVLAVPPI ILGLVVNDRKLNDNQNCVSNELACMRRMSSENESTDNK LAALAPMLIIMFFLENVPLTDETflIELHGNREAVKKNS5GQEGKEAR5 TGLMAIMFIWMFMVKNYNVKNM5QTKGMVF --  LQNPNRICVRKNDL RIO  116  Figure 17: Similarity tree of the Sitlp protein from the serotypes D strain B3SO1A of C. neoformans with fungal homologs Similarity tree produced from alignment http://www.ddbj .nig.ac.jp/searchlclustalw-e.html An-Aspergillus nidulans Ca-Candida albicans Cn-Cryptococcus neoformans Sc-Saccharomyces cerevisiae Urn- Ustilago maydis  117  (Fig.  16.)  in  Clustal  W.  ScARN1 ScARN2/TAF I  Ca,SITA/CaARN I SoSITI p/ARN3 CnSITI p ScARN4/ENB I A mm’irA AnmirB AnmirC UmSiti Figure 17: Similarity tree of the Sitip protein from the serotypes D strain B3501A of C. neoformans with fungal homologs Similarity tree produced from alignment (Figure 16) in Clustal W. http:/Iwww. ddbj .nig.ac.jp/search!clustalw-e.html An-A spergillus nidulans Ca-Candida albicans Cn-Cryptococcus neoformans Sc-Saccharomyces cerevisiae Urn- Ustilago maydis  117  d. Expression of SIT] in low iron and iron replete conditions. The SIT] gene was identified by SAGE as having an elevated transcript in cells grown in low iron vs. iron replete conditions (Lian et a!., 2005). The SIT] transcript was detected by Northern blot during growth of cells in low iron conditions but absent during growth conditions of high iron in all strains (Fig. 18). These results suggest that the Sitip may be important in the acquisition of iron when C. neoformans encounters a low iron environment. These results were consistent with SAGE analysis in B3501A (Lian et a!. 2005) where a 3.33 fold increase of the SIT] transcript was noted from cells grown in LIM vs. LIM+Fe.  B3501A +  JEC2I +  H99 +  A SITI  B  rRNA  Figure 18: Transcript levels for the SIT1 gene in culture media with low or high iron levels for strains JEC21, B3501A and 1199. Cells were grown in low iron media, ElM (designated -) or iron replete media LIM+Fe (designated +). The Northern blot was prepared from total RNA isolated from cells grown in ElM or LIM+Fe medium at 37°C for 6 hours. Blots were hybridized with a DNA fragment from Exon 6 of the SIT] gene of C. neoformans. A. SIT] transcript levels B. rRNA (18S and 28S) bands as a loading control  118  B. Construction of Siderophore Transporter Mutants. a. SIT] gene and Sitip protein structure. SIT] is 2144 nucleotide bp in length in serotype D and serotype A and the predicted Sitip polypeptide 604 amino acids in length. The gene contains six exons and five introns (Figure 1 9A), and the encoded polypeptide has 13 predicted transmembrane regions by TmHMM (http://www.cbs.dtu.dklservices/TMHMMJ). The predicted structure includes an extracytosolic N-terminus, 6 regions in the cytosolic face, 6 regions in the extracytosolic face and a cytosolic Cterminus (Figure 19B). There is an unusually large extracytosolic loop at the C-terminus (58 amino acids). The Am family of protein in S. cerevisiae (of which Sitlp is a homolog) possess a large loop at the C-terminus (44 amino acids) that has been shown to be involved with the localization of the protein, more specifically the cycling of the protein from the plasma membrane to endosomes (Kim et al., 2005).  The loop in C. neoformans Sitlp therefore  represents a strong candidate for site-directed mutagenesis in the future to investigate its potential role in the localization of the protein.  119  pJ C  48bp  B  197bp  62bp  469bp  I7j  84bp  494bp  TA(AIG)  face C-terminus  Ic___  Extracytosolic face  230bp  488-546  74bp  445-464 393-398 227-262 Predicted Transmembrane Regions 604 amino acids (aa)  314-332  351 -369  EExon Ilntron 2144 nucleotide bp  240bp  1131  6lbp  Figure 19: Structure of the SIT1 gene and Sitip protein A. SIT1 gene structure B. Predicted topology of Sitip  66-110  160-1 71  195-203 134-136  linus  185 bp  A  b. Construction and confirmation of null mutants. Null mutants were constructed in three strain backgrounds to investigate the function of SIT]: serotype D strains JEC21 and B3501A and serotype A strain H99. The entire coding region (6 exons; 5 introns) was deleted by using a knock out cassette constructed by PCR overlap (Davidson et al., 2002). A URA5 marker was used for the serotype D strains (Varma et a?., 1992). A modified overlap strategy was used (Yu et a?., 2004) with a neomycin marker for the serotype A strain. The structure of the knock-out constructs for serotypes A and D including primer locations is detailed in Figure 20.  One homologous integrant was characterized for  JEC21 and two independent homologous integrants were obtained for both B3501A and H99. Mutants were confirmed by colony PCR (Appendix IVC) and Southern analysis (Figure 21 and 22). PCR confirmation showed homologous integration by using one primer upstream of the integration site and one primer internal to the marker (Appendix IVC).  Southern analysis  unequivocally determined that a single homologous integration event had occurred in the strains, ensuring that no additional ectopic integrations resulted from the transformation (Figure 21 and 22). For serotype D, three enzyme combinations were used to differentiate wild-type and mutant alleles: EcoRl/NcoI, Seal and StuI. A schematic diagram of restriction enzyme sites in the knock-out construct and the wild type gene, as well as images of Southern blots, can be found in Figure 21. For serotype A, FspI was used to distinguish the wild-type and mutant alleles (Figure 22). sit] mutants were reconstituted with the wild-type SIT] gene cloned into a vector containing a marker for resistance to nourseothricin (NAT).  121  853 bp  1151 bp  SITA3  SITA2  5’ Flank  Colony PCR Screen 1310 bp  -  SITAI  SITNF  3355 bp  URA5 1760 bp  Marker  4189 bp  Neomycin 1876 bp 1-  6328  1—  SITIC  Colony PCR Screen 1411 bp  -  SITIA  SITIB  -  SITA4  SITA5  -  SITID  SITIE  Primers for knockout construction and colony PCR screening are denoted with black arrows. A. serotypeD and B. serotypeA  Figure 20: Structure of the Essitl knock out constructs  S ITAUP  B  SITIUP  A  3’ Flank  SITNR SITA6  1-  1162 bp  742 bp  1-  SITIF  Asiti Construct EcoRl  A. Stul  ScaT  5’742bp  Stul Seal  NcoI  ‘r4r  4r  URA5176Obp  3’ 853bp  Probe  B.  SIT1 Genomic Sequence EcoRI Stul  Seal  5’ 742 bp  Seal  Ncol  Stul  4r  ‘4r  ‘4r  SIT] 2290 bp  3’853bp  Probe  C.  D. -  L)  —  Lfl  m —  4571 bp  ,-  —  iD  rJ)  •  I  Ii)  )  .-  .-  C  N  c/)  Z1  I  N  I  4001  0—  2723 bp 2218 bp  E.  N  c-)  C -  ci  _e  c  Figure 21: Confirmation of the Asitl knock out in the serotype D strains B3501A and JEC21.  r1  -  Southern analysis to confirm homologous integration of the tXsit] knock out construct. The Southern blot was hybridized with the 5’ of the Asit] knock out construct. A. Enzyme recognition sites in Asit] construct. B. Enzyme recognition sites in the SIT] gene. C. Southern blot of genomic DNA digested with: C. EcoRl/Ncol D. Seal and E. StuI and probed with SIT].  arm  >6000 bp  ‘i bp 123  iXsitl Construct A. FspI  FspI  ¶4,  r’  Neomycinl876bp  3’ 1162bp  Probe  B.  SIT1 Genomic Sequence FspI  FspI  5’ 1151 bp  SIT] 2258 bp  3’ 1162bp  Probe  C.  7000 bp  •* ___.  Undigested DNA  2000 bp  Figure 22: Confirmation of the 1!isitl knock out in the serotype A strain H99.  Southern analysis to confirm homologous integration of the iXsit] knock out construct. The Southern blot was hybridized with the 5’ arm of the Asit] knock out construct. A. FspI enzyme recognition sites in Asit] construct. B. FspI enzyme recognition sites in the SIT] gene. C. Southern blot of genomic DNA digested with FspI and probed with SIT].  124  C. The role of SIT] in Iron Acquisition. a. Growth in various iron conditions. The disruption of SIT] resulted in greatly reduced growth of C. neoformans under low iron conditions (LIM), or when a siderophore was provided as the sole iron source (LIM containing the chelator bathophenanthroline disulfonic acid [BPDA] supplemented with the siderophore deferoxamine) for strains B3501A (serotype D) and H99 (serotype A) (Figure 23 A,B,C,D). No strains are able to grow in LIM and the chelator BPDA alone. Interestingly, the JEC2 1 sit] mutant did not show reduced growth in comparison to the wt strain in these growth assays (Figure 23 A,C). All strains showed similar growth patterns in LIM+Fe, although growth was slightly delayed for the sit] mutants. These strains eventually reached wt levels by 36 hours (Figure 23. E,F). These results indicated that Sitip plays an important role in iron acquisition in the low iron environment and in the use of a siderophore as a sole source for iron acquisition for strains B3501A and H99, but not JEC21. It is possible that JEC21 has additional or redundant mechanisms for acquisition of iron. Another interesting observation is that the serotype D strains attained more robust growth than serotype A in LIM and deferoxamine (Figure 23 A,B,C,D). The strains were tested to determine whether loss of Sitip resulted in a more generalized uptake defect by growing the cells in minimal media with urea as the sole nitrogen source. sit] mutants in the serotype D strains JEC21 and B3501A but not the serotype A H99 strain had retarded growth in this media at 24 hours (0D600 of 8 vs. 12). However the OD of the culture was equivalent for all strains by 48 hours (0D600 of 12) (data not shown). This result may suggest that a defect in Sitip has a general influence on membrane transporter function in the serotype D background.  125  Figure 23. Disruption of the SIT1 gene significantly affects growth in LIM and LIM+BPDA+Deferoxamine in B3501A and H99, but not JEC21. All strains have comparable growth in LIM+Fe. A comparison is shown for serotype D strains: JEC21 wt, the SIT]:: URA5 disruption mutant (je-si), reconstituted SIT]:: URA5 + SIT] (je siRl), B3501 wt, two SITJ:. URA5 disruption mutants (b-slO and b-s42) and reconstituted strains SIT]:: URA5 + SIT] (b-s42R1 and b-s42R2) in LIM (A), LIM+BPDA+Deferoxamine (C) and LIM+Fe (E). A comparison is also shown of serotype A strains H99 wt, two SIT]::NEO disruption mutants (h-si and h-s2) and the reconstituted strain SIT]::NEO + SITJ (h-slRl) in LIM (B), LIM+BPDA+Deferoxamine (D) and LIM+Fe (F). These growth experiments represent the average of three trials. Raw growth curve data can be found in Appendix IVE.  126  N)  -  A  8 —  —  0  1  2  3  05  0  -  D6-  0  E  9  10  11  12  13  6  12  18  24 30 Hours  —b-s42R2 (sitl::URJk5 + SIT1::NAT)  —+-—b-s42R1 (sitl::UR.A5 + SIT1::NAT)  —-—b-s42 (sitl::URAS)  *—b-slC (sitl::URAS)  —f—B35O1A (wt)  —-—je-s1R1 (sitl::UPA5 + SIT1::NAT)  —R—je-sl (sitl::URA5)  —+—JEC21(wt)  36  42  48  54  cc  C UI L.  0 (N  z I— U’) +  cc  0 00 UJ  0”  Ui  ;-; 4-’  ;‘;  Cfl .—  —  -  r.4  4-’ (it  r  ‘to %—  ,-4 -  0  C f  (N  -I  -l  -  v-I  o  cc  N  WU  If)  ‘.0  009  ao  128  (N  C  CD  -  C  7  8  0  051  1.0  0  o  E  9  10  6  12  +  +  Hours  30  SIT1::NAT)  SIT1::NAT)  1824  —b-s42R2 (sitl::URA5  —+—b-s42R1 (sitl::URA5  —s—— b-s42 (siti: : URA5)  —*—- b-slO (siti: :URA5)  —E—B35O1A(wt)  —*—--je-slP.1 (sitl::URAS+SIT1::NAT)  11 -  ———je-s1 (sitl::URAS)  —+—JEC21 (we)  12  13  36  42  48  54  0  D  8  9  0  01  1-  2  3  4  05  O6.  7.  E  1  11  12  13  H99  6  12  18  h-slRi (siti;:NEO + SIT1::NAT)  X  F  24 30 Hours  —  h-s2 (sitl::NEO)  h-si (sfti::NEO)  I A  36  42  48  54  -  C)  -  E  0  0  a  E  0  1  2  3  0  4  8  I  10 1  11  12  13  i  b-s42 (sitl::URA5)  •  6 12  *  18  T  24 30 Hours  b-s42R2 (sitl::URA5 + SIT1::NAT)  b-s42R1 (sitl::URA5 + SIT1::NAT)  (sitl::URA5)  b-sb  X  je-siRl (sitl::URA5 + STT1::NAT)  A B3501A(wt)  je-si (sitl::URA5)  • X  JEC21 (wt)  •  36  42  48  54  F  0  1  2  0  3-  4  05  8  9  10  11  12  13  6  12  /  //  18  24 30 Hours  SIT1::NAT)  /  +  //  ———h-s1R1 (sftl::NEO  —*-—h-s2 (sitl::NEO)  ———h-sl (sitl::NEO)  —+—H99  36  42  48  54  b. Siderophore utilization. Siderophore utilization assays were performed on iron free plates with deferoxamine to further determine whether a siderophore could be used by C. neoformans to acquire iron when no other source was available. Zones of growth were apparent for all wt and reconstituted strains but no zones of growth were present for the sit] mutants (Figure 24. A,B,C). These siderophore utilization assays showed that sit] mutants in all strains were unable to use deferoxamine as a sole source of iron.  There is a notable difference in the JEC2 1 results in the siderophore  utilization assay vs. the liquid growth curves where no growth was noted on the plate assay and wild-type growth was present in the liquid medium. Growth of the JEC2 1 sit] mutant was noted on the plates after 7 days incubation indicating again that this strain may have additional mechanisms to acquire iron from a siderophore that are not expressed on agar plates. The loss of the high affinity permease, FTR], in serotypes A (H99) or D (ATCC 24067) was also investigated to determine if it led to a change in siderophore utilization. Wild type, null and reconstituted strains were tested in the assay and it was found that all strains could use siderophore-bound iron as a sole source (data not shown). All experiments were repeated three times to ensure consistency in the results.  133  wt  Asiti  Asitl  +  SITI  A  B  C  Figure 24: siti mutants are unable to use a siderophore to acquire iron in all strains as shown by siderophore utilization assays. To test for siderophore utilization, a culture of 200 iL of 1 o CFU/mL cells were spread on LIM plates containing 550 tM BPDA. A disk containing 10 iL of 100 tM deferoxamine was placed in the center of each plate and the cultures were incubated two days at 30°C. A. JEC21 B. B3501A and C. 1199.  134  D. Involvement of the cAMP Pathway in Siderophore Utilization. In S. cerevisiae, the cAMP pathway has been implicated in the regulation of iron uptake genes (Robertson et al., 2000). It was shown that one of three PKA catalytic subunits, Tpk2p, negatively regulated a number of genes involved in iron uptake including the ARN3/SIT] gene. We were therefore interested in the affect of the cAMP pathway on siderophore utilization in C. neoformans. Serotype A and serotype D cAMP mutants (defective in the catalytic subunit of PKA (PKAJ) or the regulatory subunit (PKRJ) were tested in the siderophore utilization assay to determine if specific components were necessary for the utilization of deferoxamine as a sole source of iron. The results suggest that FKA 1 was necessary for siderophore utilization in the serotype A H99 background (Fig. 25). That is, no growth with deferoxamine was present for the pica] mutant (catalytic subunit knockout of PKA) and growth was enhanced in the pkr] mutant (regulatory subunit knockout of PKA-constitutively active) indicating that siderophore use is positively regulated by the cAMP pathway in this background. This relationship is opposite to that observed in S. cerevisiae (Robertson et al., 2000). No difference in growth was noted for the pka2, pica]  +  PKA1 (data not shown) mutants in comparison to wild type. The gpal mutant  had slightly reduced growth in comparison to the wt type strain (data not shown). GPA] encodes the alpha subunit of a heterotrimeric GTP binding protein that is involved in the induction of the cAMP pathway and is epistatic to PKAJ (Aispaugh et al., 1997). A reduction in growth would be expected in the gpal mutant where PKA 1 is necessary for siderophore utilization. In striking contrast to the serotype A results, no significant difference in growth compared to wild-type was noted for mutants of the cAMP pathway in the serotype D, strain JEC2 1 background for each of mutants (pica], pka2, pkalpka2 [data not showni or pkr]) (Fig. 25). These results suggest that cAMP pathway is involved in siderophore utilization in serotype A but not serotype D, at least for the representative strains tested here. The cAMP mutants were checked for presence of the appropriate marker and a subset of published phenotypes to confirm that they retained their expected defects in cAMP signaling (Alspaugh et a?., 1997; D’Souza et a?., 2001; Hicks et a?., 2004). Specifically, the strains were tested for melanin and capsule production where in serotype A, melanization and capsule was reduced for pica], unchanged for pka2 and increased for pkrl in comparison to the wt strain. In serotype D, melanization was unchanged for pka], reduced for pka2 and slightly increased for pkr] in comparison to the wt strain. Capsule size was similar in  135  all the serotype D strains tested. These results are concordant with publish ed phenotypes for these strains. All experiments were repeated three times to ensure consistency in the results.  wt  Apkal  Apka2  Apkrl  A  B  Figure 25: The cAMP pathway is involved in siderophore utilizat ion in serotype A but not serotype B strains. To test for siderophore utilization, a culture of 200 iL of 1 CFU/m L cells were spread on LIM plates containing 550 iM BPDA. A disk containing 10 L of 100 p.M deferoxamine was placed in the center of each plate and the cultures were incubated two days at 30°C. A. JEC21 B. H99.  136  E. SIT] and Melanization. a. Production of DOPA melanin. Production of melanin by the enzyme laccase is important for virulence in C. neoformans and a variation in production or deposition may alter the pathogenicity of the fungus. Laccase expression is activated through the cAMP pathway, the same pathway that is thought to be activated in low iron conditions. Therefore, the sit] mutants were tested for melanin production and a striking phenotype was noted in the serotype D strains JEC21 and B3501A. Specifically, evaluation of melanin production on DOPA medium plates revealed a variation in melanization between wt and sit] for strains JEC21 and B3501A (Fig. 26A). In JEC21, the sit] mutant was significantly more melanized than wt. In B3501A, the sit] mutant produced melanin more rapidly than wt and the surface of the colony appeared coarser with a higher sheen in comparison to wt (Fig. 26A). B3501A wt cells were able to invade the agar whereas the sit] mutant could not (Table 16). No defect in melanization was noted for the H99 sit] mutants (Fig 26A). All experiments were repeated three times to ensure consistency within the results.  b. Presence of extracellular melanin-like granules. A closer inspection of cells grown in DOPA by DIC microscopy (1000X) revealed an accumulation of extracellular granules in the sit] mutants that may be related to melanin polymers (Fig. 27A and B). Cells grown in low glucose asparagine medium (LGA) lacking the DOPA substrate did not produce granules. If these granules are related to melanin, this result indicated that SIT] may play a role in melanogenesis or melanin placement in serotype D strains. Further, this may represent a mislocalization of melanin from the cell walls and may indicate that Sitip plays a role in membrane transport. Interestingly, no difference was noted in the serotype A strain H99 for either melanin production or the accumulation of extracellular granules (Fig. 26A;27C). This illustrates another difference between serotypes. All experiments were repeated three times to ensure consistency within the results.  137  00  C  B  A  N 12  C  cr  ri  N  To test for melanin production, a culture of 10 tL of i0 CFU/mL cells were spotted onto 0.1% glucose plates containing A. 1mM DOPA B. 4mM DOPA and C. 4mM DOPA + 1.0% glucose. Plates were incubated two days at 30°C.  Fig 26: Melanin Production on DOPA Medium.  I  (‘3 CD  -  Asiti Granules in Focus  To investigate the melanin defect of siti mutants, a culture of 10 tL of i0 CFU/mL cells were spotted onto 0.1% glucose plates containing 1mM DOPA. Plates were incubated four days at 30°C. Cells were scraped from plates and resuspended in sdH2O. 10 pL of the suspension was viewed at 1000X in oil immersion. Two images are represented for each strain with cells in the field of focus and then the extracellular granules in focus. Inset frames show a magnffied image of the cells or granules. A. JEC21 B. B3501A C. H99.  Fig 27: DIC microscopy of melanized cells at four days growth on DOPA media.  C  B  A  Cells in Focus  c. Effects of other factors on melanization. A surprising result in these studies was the alteration of melanin deposition that was noted in SIT] mutants for the serotype D strains JEC21 and B3501A. There have been previous reports of melanin existing in a granular form (both intracellular and extracellular) in association with C. neoformans (Eisenman et al., 2005, Chaskes and Tyndall, 1975). Numerous parameters were tested to try to further determine what role SIT] may be playing with respect to melanogenesis because melanin has been implicated in virulence.  Specifically, additional  substrate compounds and conditions were tested for effects on the melanin phenotype of sit] and wt C. neoformans by plate assay and investigation by DIC microscopy to monitor the accumulation of the extracellular granules. A number of physiological effects were investigated. These included the effects on melanin levels and deposition by: excess substrate, high glucose, high pH, exogenous ferric vs. ferrous iron and ferric and ferrous iron chelators and a variety of diphenolic substrates. The results are summarized in Table 16. The strains were tested at a higher substrate concentration (Fig. 26B) to determine if wt melanin levels would be restored to mirror the sit] mutant phenotype. If the increased level of melanin noted in the sit] mutants was a result of additional substrate available to laccase at the cell surface due to lack of Sitlp, then excess substrate should allow wild-type melanin levels to equal those of the mutants. A restoration of melanin levels by high substrate could indicate that the aberrant melanization noted between wt and mutant was the result of higher substrate availability to laccase at the cell surface caused by lack of DOPA/melanin influx by Sitip. The higher DOPA concentration restored the phenotype for B3501A but not JEC21. These results suggest that substrate concentration may be a factor of the aberrant melanization in B3 501 A but not JEC21. Melanization in C. neoformans is partially controlled by the cAMP pathway (Alspaugh et al., 2002). In conditions of low glucose, the cAMP pathway is believed to be activated leading to expression of laccase and production of melanin. Melanization is repressed by high glucose. Strains were tested for glucose repression using 4X [DOPA] and 1.0 % glucose (Fig.26C). We found that glucose could repress melanization in all strains indicating that the SIT] mutation did not affect the regulatory influence of glucose on melanization. Laccase requires an acidic environment for activity, so not surprisingly, high pH repressed melanization in all serotype D strains.  140  Melanin is able to both reduce and oxidize iron (Jacobson, E.S. 2000). It was therefore tested whether different redox conditions would influence the melanin phenotype when an iron transporter (Sit 1 p) was absent. Iron sources with different oxidation states Fell or Feill were added to DOPA medium. DOPA with chelators of both Fell (BPDA) or FelIl (deferoxamine) were also tested. The addition of ferric iron (FeEDTA) did not alter the melanin phenotypes of wt or sit] mutants.  The Fell chelator BPDA led to albino phenotypes, however addition of  excess copper restored the melanization in all strains indicating that loss of melanization by BPDA addition was likely a result of copper not iron chelation. Four molecules of copper are necessary for a functional laccase enzyme (Williamson, 1997). In contrast, the addition of ferrous iron (Fell  +  the antioxidant ascorbic acid) or a ferric chelator (Felil  +  deferoxamine)  caused the loss of the extracellular granules in sit] mutants. This may indicate that the oxidation state of iron can contribute to the placement or solubility of melanin. This result was not surprising as many redox interactions are possible between iron and melanin (Jacobson and Hong, 1997). Past research has shown that structurally varied diphenolic substrates can lead to different solubilities and placement of melanin resulting in either extracellular and/or intracellular localization (Chaskes and Tyndall, 1975). These authors noted that para substrates often lead to extracellular melanin, while ortho compounds (like DOPA-3,4, dihydroxyphenylalanine) normally lead to intracellular melanin. Since these phenotypes appeared to relate to observations for the melanin phenotype of serotype D sit] mutants the effects of a variety of diphenolic substrates were tested in place of DOPA. Specifically the para compounds 2,5 dihydroxybenzene diacetic acid and hydroxyquinone (1,4) and the ortho compound 3,4 dihydroxybenzoic acid were tested. A general reduction in pigmentation with these diphenolic compounds was noted and a reduction in the presence of extracellular granules was found for all substrates (Table 16). The expected increase in extracellular granules with para substrates was not observed. These results indicated that the melanin phenotype that is observed in the sit] mutants is not affected in the presence of other diphenolic substrates and the results contradict the findings of Chaskes and Tyndall (1975) at least in the strain backgrounds tested. All experiments were repeated three times to ensure consistency within the results.  141  Table 16: Summary of melanin production for sit] mutants in serotype D strains.  Characteristic Melanization ExtracellularGranulesb Agar Invasion 4X (DOPAI Melanization ExtracelIularGranuIes Agar Invasion 4X IDOPAI + Glucose MeIanization Extracellular Granules” Agar Invasion DOPA pH9 MeIanization Extracellular Granules” Agar Invasion MeIanization DOPA + Fell Ascorbic Acid Extracellular Granulesb Agar Invasion DOPA + BPDA MeIanization Extraceflulartjranules” Agar Invasion DOPA + BPDA + Cu(SQ), MeIanization Extracellular Granulesb Agar Invasion DOPA + FCEDTA Melanizalion Extracellular Granulesb Agar Invasion DOPA + Deferoxamine Melanization’ Extracellular Granules” Agar Invasion 2,5 Dihydroxybenzene diacetic acid Melanization Extracellular Granulesb Agar Invasion 3,4 dihydroxybenzoic acid Melanization Extracellular Granulesb Agar Invasion hydroxyquinone(1,4 pars) Melanization’ Extracellular Granulesh Agar Invasion DOPA  Symbols For Melanization  -  *sgg Fig. 26 for visual  +  assessment of pigmentation  ++ +++  JEC2I  je-s1  je-siRl  B3501A  b-slO  b-s42  b-s42R1  b-s42R2  ++  +++  ++  +++  +++  -1-++  +++  +++  +  +++  +  +++  -i--t-+  +  +  -  -  -  + +  -  -  +  +  ++ ++  -H-+ ++  ++ ++  +++ ++  +-f-+ ++  +++ ++  ++4++  +++ ++  -  -  -  +  -  -  +  +  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  ++  +++  ++  +++  +++  +++  +++  +++  -  -  -  -  -  -  +  -  -  -  +  -  -  +  +  -  -  -  -  +  -  -  -  -  -  +  +  +  +  +  +  -  -  -  -  -  -  -  -  +++  +++  +++  +++  ++-f-  +++  +++  +++  -  -  -  -  -  -  -  -  -  -  -  +  +  ++  -t-++  ++  +++  +++  +++  +  -  -  +++  +4—I-  ++  ++  -  -  +  -  -  +  +  ++  +++  +++  +++  +++  +++  -  -  +  +  -  -  -  -  -  ++  -H-f-  -  -  -  -  -  +  -  -  +  -  -  -  +  +  +  +  +  +  +  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -t-+  ++  ++  ++  ++ +  -  -  ++  -  -  +  +  +  +  +  +  +  -  -  -  +  +  +  +  +  -  +-f  -  ++  ++  ++  ++  ++  —  —  —  -  -  -  albino minimal pigment formation medium pigment formation excessive pigment formation  142  *  —  -  —  —  —  +  +  +  +  +  5 S ymbols for Extracellular Granules see Fig. 27 for visual assessment of extracellular granules  -  no extracellular granules  +  minimal extracellular  ++  granules medium extracellular granules  +++  excessive extracellular  granules  F. Influences of SIT] on Cell Wall Integrity. a. Temperature sensitivity and cell wall integrity. Because an accumulation of extracellular granules may be a result of blocked incorporation or displacement of melanin from within the cell wall, alteration to the structure of the cell wall was investigated.  Cells were tested for temperature sensitivity (a phenotype  associated with changes in cell wall intrgrity) by spotting serial dilutions of cells on LGA (Figure 28A) or DOPA (Figure 28B) media and incubating plates at 30°C or 37°C. The sit] mutants showed slightly less growth than wt at 30°C and significantly less growth at 37°C for strains JEC21 and B3501A (Fig. 28. A,B). The temperature sensitivity is particularly severe in the B3501A background. No difference was noted for 1199 (Fig. 28.A,B). The addition of DOPA to the medium to allow melanin production did not change the results (Fig. 28B). The compounds calcofluor white, sodium dodecyl sulphate (SDS) and congo red were also tested in low glucose or DOPA plates to examine cell wall integrity. Cells with cell wall integrity problems are generally more sensitive to these compounds. Only a slight increase in sensitivity was observed for wt cells vs. sit] mutants to these compounds in JEC21 and B3501A and no difference was noted for H99 (Fig. 28 A,B). All experiments were repeated three times to ensure consistency within the results.  143  -  -  ID  A  0  Oi U,  -,  w  0  C4  I  a,  C)  0  w  C4  37°C  37°C 300 igImL CR  37°C 30 ig/mL CW  To test for temperature sensitivity and cell wall integrity, a culture of 5 L of serial dilutions of cells were spotted onto A. 0.1% glucose orB. 0.1% glucose + 1mM DOPA plates. Serial dilutions were 107-102 CFU/mL. Plates were incubated for two days at 30°C or 3 7°C. SDS-sodiuni dodecyl sulfate; CR-congo red; CW-calcofluor white  1  I  30°C  37°C 0.015 % SDS  b. Alteration of cell wall structure. Because the cell wall may be compromised by the loss of SIT], a closer examination of the cell wall of sit] mutants vs. wt for JEC21 and B3501 was initiated to determine if the structure had been changed in the sit] mutants. Transmission electron microscopy (TEM) of melanized cells (grown for four days on DOPA) and non-melanized cells (grown four days on LGA) were used to further elucidate any changes to the cell wall. There was a striking reduction in cell wall organization between the sit] mutants and wt cells for JEC21 (Figure 29A) and B3501 (Figure 29B) and H99 (Figure 29C). For wild-type and mutant cells, the figure shows two magnifications of the same cell for each strain background: 30,000X magnification of the full cell and 150,000X magnification of the cell wall. A 150,000X magnification of the cell wall of an independent mutant cell is shown to illustrate the consistency of the microscopy findings. One image of 1 50,000X magnification is shown for a reconstituted strain in each background. In wild-type and reconstituted cells, there are distinct dense concentric rings in the cell walls and ordered polysaccharide fibrils are apparent, this is consistent to what was reported by Eisenman et al., 2005. In contrast, the mutant cell walls were much less dense, varied in thickness and had unordered truncated fibrils. The cell wall appeared to be porous in comparison to wild-type suggesting a loss of integrity to the cell wall structure. The mutant cells were also far more distorted by the TEM processing. For 40 cells documented in each strain, 100 % of JEC21 sit] mutants were significantly distorted (not spherical) compared to 12.5 % of wt. In B35O1A 89 % of sit] mutants were distorted vs. 7.1 % of wt (data not shown). An independent preparation for TEM with a prolonged dehydration steps reduced the distortion in the mutant cells, however the defect in the cell wall was still apparent (data not shown). These results suggest that the loss of sit] caused an altered organization of the cell wall for strains JEC21 and B3501A. H99 was not initially tested in TEM because there was no indication of aberrant melanization, temperature or cell wall sensitivity in the H99 sit] mutant. However, the H99 sit] mutant was later tested and a defect in the cell wall was apparent in this background as well (Fig 29C). The cell walls of the wild type strains increase in thickness in the order of JEC21, B3501A then H99. It is possible that the greater thickness in the H99 cell wall may have made it more resistant to temperature stress and the cell wall integrity compounds even though a defect in the cell wall was present as noted by TEM. Resin infiltration in the wt H99 cells was also less effective than JEC2 1 or B3501A, further suggesting that more rigidity may be present in the cell wall of this strain.  145  Finally, the defect in the cell wall noted in the sit] mutants was apparent even in non-melanized cells that were grown on LGA. The non-melanized cells were more distorted by the TEM processing, and the wild type had less density in their cell walls compared to the melanized cells, which may indicate some contribution to rigidity of the cell wall by the incorporation of melanin. However, taken as a whole, these results may indicate that the cell wall defect is not a direct or complete result of loss of melanin from the cell wall. A summary of cell wall defects noted by TEM can be found in Table 17.  Table 17: Summary of cell wall defects for sit] mutants noted by TEM.  JEC21 je-si je-siRi B35O1A b-s42 b-s42Ri H99 h-si h-siRl  +DOPA NO YES NO NO YES NO NO YES NO  146  -DOPA NO YES NO NO YES NO NO YES NO  C  B  A  1 50,000X  1 50,000X  1 50,000X  To prepare cells for TEM, a culture of iO CFU/mL cells were spotted onto 0.1% glucose plates containing 1mM DOPA. Plates were incubated four days at 3 0°C. Cells were glutaraidehyde and osmium fixed and stained with uranyl acetate and lead citrate. A. JEC21, B. B3501A and C. H99. Images show a cross section of the cell wall, cytoplasm and polysaccharide capsule.  Figure 29: Transmission electron microscopy (TEM) of melanized cells for wild type and siti mutant strains.  30,000X  Ce112  G. Summary of Additional Phenotypic Analyses of sit] Mutants. Overall, this work has suggested possible connections between SIT] and the cAMP pathway and has shown that the SIT] gene plays a role in iron acquisition during low iron conditions. Therefore phenotypes were tested that are affected by cAMP signaling or the low iron nutritional cue. Specifically, the mutants were assessed for the elaboration of the polysaccharide capsule in low iron and iron replete conditions. There are two obvious possible connections here. First, iron sensing may be affected by the loss of SIT], thereby altering the induction of the capsule. Second, a melanin phenotype is observed in the serotype D sit] mutants. Melanin production through the regulation of laccase is also controlled by the cAMP pathway and capsule formation may also be affected if SIT] has a generalized effect on the cAMP pathway. The wild-type, mutant and reconstituted cells were tested in all three strain backgrounds (JEC21, B3501A and H99). Cells were grown in the same manner as for the Northern analysis in LIM or JR media and harvested at 6 hours. The cells were stained in India ink and viewed at 1000X under oil immersion. No difference in capsule size or induction was noted between wt or mutant cells in any background. All strains produced capsule in low iron and repressed capsule in iron replete conditions.  These results suggest that SIT] is not required for iron sensing that leads to  elaboration of the polysaccharide capsule. To further investigate a generalized cAMP relationship to SIT], mating was tested for wt, mutant and reconstituted cells with the congenic MATa mating strain JEC2O (serotype D) or Bt63 (serotype A). Mating is also partly controlled by the cAMP pathway in C. neoformans (Casadevall and Perfect, 1998; Kronstad et al., 1998; D’Souza et al., 2001; Aispaugh et al., 2002). The assays were performed by spotting 10 iL of 1O CFU/mL of the a and a strain on V8 8 agar