Joseph Heitman, PhD,MD - US grants

DESCRIPTION (Adapted from the applicant's abstract): The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin block signaling events required for T-cell activation. CsA and FK506 prevent responses to antigen; rapamycin inhibits responses to IL-2. These three natural products also have antifungal activities, and previous studies reveal a remarkable conservation of action from microorganisms to vertebrates in which the drugs diffuse into the cell, associate with an immunophilin protein, cyclophilin A for CsA and FKBP12 for FK506/rapamycin, and are toxic to other proteins. A conserved calcium-regulated phosphatase, calcineurin, is the target of the cyclophilin A - CsA and FKBP12-FK506 complexes. The targets of rapamycin, the TOR proteins, are conserved proteins likely to transduce growth-promoting signals. Although touted as potential antifungal agents, little is known about the actions or targets of these agents in pathogenic organisms. The investigator proposes to study the antifungal properties of immunosuppressants in the pathogenic basidiomycete Cryptococcus neoformans, whose incidence has risen, especially in HIV infection, to become the leading cause of fungal meningitis. The investigator discovered that C. neoformans is resistant to CsA or FK506 at 22C but sensitive to both drugs at 37C. Genetic studies suggest immunophilin-drug complexes inhibit calcineurin to prevent growth at 37C. Because growth at 37C is required for infection, the Investigator s hypothesis is that calcineurin is required for pathogenicity. The C. neoformans calcineurin A gene was cloned, sequenced, and disrupted by homologous recombination. As predicted, calcineurin mutants are viable at 24C but not at 37C. Two calcineurin mutants are nonpathogenic in an animal model. Reintroduction of the calcineurin A gene restored growth at 37C, and the Investigator will test whether virulence is also restored. A nonimmunosuppressive antifungal FK506 analog was identified. The Investigator will analyze drug-resistant C. neoformans mutants and clone C. neoformans FKBP12, cyclophilin, and TOR genes. The Investigator s goals are to identify immunophilins, calcineurin, and their functions in C. neoformans to delineate a signal transduction pathway regulating pathogenesis and to define novel antifungal drug targets.

Fungal infections are increasingly common as a result of AIDS, transplantation, high dose chemotherapy, steroid treatment, antibiotic treatment, and invasive procedures. However, existing antifungal agents are limited to amphotericin B, flucytosine, and azoles, and drug resistant strains are emerging. We propose to elucidate signal transduction cascades regulating fungal growth and virulence as targets for antifungal drug development. We propose studies on both Candida albicans, the most common human fungal pathogen, and Cryptococcus neoformans, the leading cause of fungal meningitis and an important opportunistic fungal pathogen. We have discovered that the immunosuppressive drugs rapamycin, cyclosporin A, and FK506 have potent antifungal activity. In studies supported by this award, we have identified the fungal drug target proteins, including FKBP12, the Tor1 kinase, and two cyclophilin A homologs. Our studies demonstrate that the antifungal effects of rapamycin are mediated via a complex with FKBP12 that inhibits the fungal Tor1 kinase homolog. We have identified nonimmunosuppressive analogs of each of these drugs that retain antifungal activity. By genetic and biochemical approaches, we show that these analogs take advantage of structural differences between host and fungal enzymes, sparing immune function while impairing fungal cell growth. Finally, we have identified examples of potent synergistic drug interactions. For example, the calcineurin inhibitors cyclosporin A and FK506 are potently synergistic with azoles in Candida albicans. Here we propose to establish the cellular functions and targets of the rapamycin target protein Tor1. The TOR1 gene will be disrupted in diploid strains of C. neoformans to test if it is essential. Targets of the Tor kinase will be identified by genetic and two hybrid screens, and by analyzing gene expression with genome arrays. We will also identify the targets of the C. neoformans cyclosporin A target proteins, the Cpa1 and Cpa2 cyclophilins, which are important for cell growth and virulence. We will determine the molecular basis of synergistic drug interactions. First, we will identify a novel target of the FKBP12-FK506 complex that is synergistic with proton pump inhibitors in C. neoformans. Second, we will focus on the roles of FKBP12 and calcineurin in azole action in C. albicans, testing the hypothesis that calcineurin is either essential or becomes essential during cell membrane stress as a result of azole treatment. Finally, we will test rapamycin and nonimmunosuppressive rapamycin analogs, and the synergistic combination of calcineurin inhibitors and azoles, in animal models of cryptococcosis and candidiasis. Our long term goal is to identify unique targets and develop novel antifungal drug therapies.

DESCRIPTION (Adapted from applicant's abstract): Fungal infections are increasing as a result of AIDS, transplantation, and high dose chemotherapy. Yet the armamentarium of antifungal agents is scare--amphotericin B, azoles, flucytosine--drug resistant isolates are emerging, and a molecular understanding of fungal virulence is limited. Dr. Heitman and his group propose to elucidate signal transduction cascades regulating virulence of Cryptococcus neoformans, the leading cause of fungal meningitis and a common opportunistic infection in AIDS patients. Several features make C. neoformans ideal for studies of fungal pathogenesis. The organism exists as a stable haploid with a defined sexual life cycle. Gene disruption by homologous recombination is now possible in this organism, and animal models have been established to test both mutant strains and candidate drugs in a viurlence setting. These advances provide tools to establish the molecular basis of traits associated with virulence: including growth at elevated temperature, capsule, melalin, and urease production, and mating type. This group recently identified a molecular factor regulating C. neoformans virulence as calcineurin, a conserved Ca++-calmodulin activated protein phosphatase. They cloned the C. neoformans gene encoding the enzymatic domain (A) of calcineurin, disrupted the gene, and found that calcineurin mutant strains are viable but sensitive to conditions in the infected host: elevated temperature, alkaline pH and 5% CO2. As a consequence, strains lacking calcineurin are nonpathogenic in animals. Calcineurin is the target of the immunosuppressants cyclosporin A (CsA) and FK506, which inhibit |T cells and revolutionalized therapy of transplant patients. Much is known on calcineurin biochemistry and structure, including the crystal structures of calcineurin alone and bound to FK506,a multitude of CsA and FK506 analogs have been developed, and simple in vitro assays have become accessible techniques. Based on known signalling roles in other organisms, the investigator hypothesizes that calcineurin regulates C. neoformans virulence by dephosphorylating proteins required for temperature-, pH- and CO2- resistant growth. The group proposes to delineate this signalling cascade to understand the regulation of pathogenesisis and to provide targets for therapeutic intervention. One of several strong features of this submission is the solid track record of this Howard Hughes Assistant Investigator (Dr. Heitman) in uncovering calcineurin-dependent phenotypic changes in S. cerevisiae in response to immunosuppressants, and exposing the catalytic subunit of this Ca++ and calmodulin dependent protein phosphatase in C. neoformans as a key player in temperature, pH and CO2 resistant growth and a virulence determinant. The focus of this proposal rivets on calcineurin as the investigator seeks first to identify and further characterize the molecular components of calcineurin by cloning the regulatory (B) component of the calcineurin heterodimer, and then shifts to identify the substrates for calcineurin phosphatase by a combined approaches using affinity chromatography coupled with the two hybrid system to identify proteins that bind calcineurin. These efforts will be supported by classical genetic suppressor analysis to identify multicopy suppressors of calcineurin mutants and thus identify the genes that function in signal transduction downstream of the Ca++/CaM protein phosphatase. Earlier events prior to calcineurin activation will also be examined in this proposal, as the C neoformans calmodulin gene will be cloned, mutated in several domains (including the Ca++ binding domains, and the calcineurin interaction site) to generate clones with altered interaction or activation capabilities specifically with calcineurin and potentially with other Ca++/CaM dependent proteins involved in growth phenotype and virulence. In addition, the group will examine the role of extracellular Ca++ on calcineurin dependent growth, and Dr. Heitman plans to establish aquarian as a reporter protein for cellular conditions in which intracellular Ca++ levels fluctuate in C. neoformans. The soundness of the molecular biology of this proposal, the pivotal position of Dr. Heitman as an investigator into the biology of calcineurin, the strong line-up of co-investigators (Dr. Maria Cardenas; biochemical and protein purification support and Dr. John Perfect; transformation of C. neoformans and virulence analysis) combine for a highly enthusiastic rating of this application.

Fungal infections are increasing as a result of AIDS, transplantation, and high dose chemotherapy. However, existing antifungal agents are limited to amphotericin B, azoles, and flucytosine, and drug resistant isolates are emerging. We propose to elucidate signal transduction cascades regulating virulence of Cryptococcus neoformans, the leading cause of fungal meningitis and a common opportunistic pathogen. Several features make C. neoformans an ideal model fungal pathogen. The organism exists as haploid cells with a defined sexual cycle. Gene disruption by homologous recombination is now possible, and established animal models permit analyses of mutant strains and candidate drugs in a virulence setting. These advances provide tools to elucidate signaling cascades regulating virulence traits of this organism, including capsule and melanin production in response to host signals, and to explore the association between virulence and MATalpha mating type. We have discovered that the G protein Gpa1 is required for mating and capsule and melanin production in C. neoformans. gpa 1 mutant strains are avirulent in an animal mode of cryptococcal meningitis. The phenotypes of gpa 1 mutant cells are suppressed by cAMP, suggesting Gpa1 regulates an adenylyl cyclase/cAMP/protein kinase A cascade. We have cloned C. neoformans homologs of Ras1, the PKA catalytic subunit and the Sch9 kinase, which are known to regulate an analogous G protein/cAMP dependent signaling pathway that regulates pseudohyphal differentiation in S. cerevisiae. In both S. cerevisiae and C. albicans, a MAP kinase signaling pathway also regulates filamentation and virulence. We and others have identified components of a homologous MAP kinase cascade in C. neoformans. Our studies reveal mutants lacking the G beta protein Gpb1, the MAP kinase Cpk1, or the STE12 transcription factor homolog have mating defects and virulence studies with these mutants are in progress. We propose to delineate the MAP kinase and G protein/cAMP dependent signaling cascades regulating C. neoformans virulence to understand regulation of pathogenesis and to identify novel antifungal drug targets.

DESCRIPTION (Adapted from applicant's abstract): Fungal infections are increasing as a result of AIDS, transplantation, and high dose chemotherapy. Yet the armamentarium of antifungal agents is scare--amphotericin B, azoles, flucytosine--drug resistant isolates are emerging, and a molecular understanding of fungal virulence is limited. Dr. Heitman and his group propose to elucidate signal transduction cascades regulating virulence of Cryptococcus neoformans, the leading cause of fungal meningitis and a common opportunistic infection in AIDS patients. Several features make C. neoformans ideal for studies of fungal pathogenesis. The organism exists as a stable haploid with a defined sexual life cycle. Gene disruption by homologous recombination is now possible in this organism, and animal models have been established to test both mutant strains and candidate drugs in a viurlence setting. These advances provide tools to establish the molecular basis of traits associated with virulence: including growth at elevated temperature, capsule, melalin, and urease production, and mating type. This group recently identified a molecular factor regulating C. neoformans virulence as calcineurin, a conserved Ca++-calmodulin activated protein phosphatase. They cloned the C. neoformans gene encoding the enzymatic domain (A) of calcineurin, disrupted the gene, and found that calcineurin mutant strains are viable but sensitive to conditions in the infected host: elevated temperature, alkaline pH and 5% CO2. As a consequence, strains lacking calcineurin are nonpathogenic in animals. Calcineurin is the target of the immunosuppressants cyclosporin A (CsA) and FK506, which inhibit |T cells and revolutionalized therapy of transplant patients. Much is known on calcineurin biochemistry and structure, including the crystal structures of calcineurin alone and bound to FK506,a multitude of CsA and FK506 analogs have been developed, and simple in vitro assays have become accessible techniques. Based on known signalling roles in other organisms, the investigator hypothesizes that calcineurin regulates C. neoformans virulence by dephosphorylating proteins required for temperature-, pH- and CO2- resistant growth. The group proposes to delineate this signalling cascade to understand the regulation of pathogenesisis and to provide targets for therapeutic intervention. One of several strong features of this submission is the solid track record of this Howard Hughes Assistant Investigator (Dr. Heitman) in uncovering calcineurin-dependent phenotypic changes in S. cerevisiae in response to immunosuppressants, and exposing the catalytic subunit of this Ca++ and calmodulin dependent protein phosphatase in C. neoformans as a key player in temperature, pH and CO2 resistant growth and a virulence determinant. The focus of this proposal rivets on calcineurin as the investigator seeks first to identify and further characterize the molecular components of calcineurin by cloning the regulatory (B) component of the calcineurin heterodimer, and then shifts to identify the substrates for calcineurin phosphatase by a combined approaches using affinity chromatography coupled with the two hybrid system to identify proteins that bind calcineurin. These efforts will be supported by classical genetic suppressor analysis to identify multicopy suppressors of calcineurin mutants and thus identify the genes that function in signal transduction downstream of the Ca++/CaM protein phosphatase. Earlier events prior to calcineurin activation will also be examined in this proposal, as the C neoformans calmodulin gene will be cloned, mutated in several domains (including the Ca++ binding domains, and the calcineurin interaction site) to generate clones with altered interaction or activation capabilities specifically with calcineurin and potentially with other Ca++/CaM dependent proteins involved in growth phenotype and virulence. In addition, the group will examine the role of extracellular Ca++ on calcineurin dependent growth, and Dr. Heitman plans to establish aquarian as a reporter protein for cellular conditions in which intracellular Ca++ levels fluctuate in C. neoformans. The soundness of the molecular biology of this proposal, the pivotal position of Dr. Heitman as an investigator into the biology of calcineurin, the strong line-up of co-investigators (Dr. Maria Cardenas; biochemical and protein purification support and Dr. John Perfect; transformation of C. neoformans and virulence analysis) combine for a highly enthusiastic rating of this application.

DESCRIPTION (Verbatim from Applicant's Abstract): Our studies define signal transduction cascades regulating physiology and virulence of the pathogen Cryptococcus neoformans. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin block signaling cascades required for T-cell activation. These compounds also have antifungal activities, and we have identified fungal homologs of the mammalian target proteins calcineurin A, calcineurin B, cyclophilin A, FKBP12, TOR1, and TOR2. Our genetic and biochemical studies demonstrate that CsA and FK506 antifungal affects are mediated by cyclophilin and FKBP12 dependent inhibition of the fungal calcineurin phosphatase homologue. In addition, rapamycin action is mediated by FKBP12 dependent inhibition of the fungal TOR kinase homologs. Our studies demonstrate that in C. neoformans, calcineurin is required for growth at 37 C and other stress conditions and, as a consequence, is required for virulence of both serotype A and D strains in two different animal models. Recently, we discovered that calcineurin is required for mating and haploid fruiting in C. neoformans, which may provide a molecular link between signaling pathways regulating the differentiation and virulence of this pathogen. The lifecycle and sexual cycle of C. neoformans have been defined. Interestingly, the MATalpha mating type is more prevalent in the environment and in patients, and MATalpha strains are more virulent than congenic MATa strains. In addition, in response to nitrogen starvation, strains of the MATalpha mating type filament and sporulate (haploid fruiting). Because cryptococcal infection requires inhalation into the alveoli of the lung, the spores produced by mating or haploid fruiting may represent the infectious particle. We have discovered that, in addition to its role in virulence, calcineurin is also required for mating and haploid fruiting. This discovery may link the known or suspected roles of calcineurin, mating type, and haploid fruiting in virulence. In this proposal, we will first establish which steps in mating and fruiting are regulated by calcineurin. Second, we will identify targets of calcineurin that regulate mating and fruiting. Using the two-hybrid assay, we have identified a conserved calcineurin binding protein and will test a possible role in mating and fruiting. Third, we will analyze the functions of cyclophilin A in C. neoformans and test the hypothesis that cyclophilin A regulates calcineurin. We have discovered diploid strains and are developing these tools to study calcineurin roles in mating and essential genes. Finally, we will characterize two transposable elements discovered during these studies, whose transposition may be regulated by calcineurin and which will be used as insertional mutagens to define other components of the calcineurin pathways.

Cryptococcus neoformans is a fungal pathogen that infects the human brain. The organism has a defined sexual cycle involving haploid MATalpha and MATa strains, and the MATalpha locus has been linked to virulence and differentiation. Most environmental and clinical isolates are MATalpha mating type, and MATalpha strains are more virulent than congenic MATa strains. MATalpha strains also haploid fruit and produce filaments and infectious basidiospores when nitrogen limited. Thus, the structure and function of the mating type loci and their links to virulence and ecology are of considerable interest. Moore and Edman used difference cloning to identify part of the MATalpha locus encoding a mating pheromone. Later, Wickes and colleagues identified a Ste12 homolog that is MATalpha-specific but not encoded by the known MATalpha region. STE12alpha is required for virulence of a congenic lab adapted serotype D strain but is dispensable for virulence of a pathogenic serotype A clinical isolate. To establish functions of the mating type loci, we propose to clone, sequence, and mutate the MAT loci from three divergent C. neoformans varieties. MATalpha and MATa strains of each variety were known or identified in our lab and MATalpha and MATa specific genes were cloned. Six BAC libraries were constructed to isolate clones spanning each MAT locus. Sequencing of the serotype A and D MATalpha and MATa loci is in progress. These loci are unusually large (greater than 100 kb) with more than a dozen genes encoding three pheromones and a pheromone receptor, homologs of Ste20, Stel1, Ste12, and a Zn2+ finger protein. Other genes encode a myosin, a favoprotein, and a translation factor. Our studies reveal the MATalpha and MATa loci encode diverged alleles of the same genes and have extensively rearranged during evolution. Mutation of individual MATalpha locus genes confers defects in mating, fruiting, and virulence. A haploid strain lacking a 50 kb region of the MATalpha locus was inviable, indicating one or more genes in the region is essential. A diploid a/delta strain deleted for this part of the MATalpha locus was still self-filamentous like an a/alpha diploid, and a/a and alpha/alpha diploids were not self- filamentous, excluding a ploidy-dependent model. Our preliminary results support a locus-dependent model. We have discovered a novel, linked region of the MATalpha locus encoding a homeodomain homolog (Hdp1alpha) and a candidate transcriptional regulator (Rum1alpha), and we will establish the functions of these genes. We also propose to develop DNA based methods to test if MAT locus alterations occur naturally and affect fertility or virulence. These studies will provide insights into the role of MAT loci and sexual cycles in fungal differentiation and virulence.

fungal proteins; protein structure function; yeasts; cell growth regulation; cell differentiation; biomedical resource;

DESCRIPTION (provided by applicant): The incidence of fungal infection is increasing and yet available antifungal drugs are limited, some are toxic, and drug resistant strains are emerging. We have elucidated a conserved signal transduction cascade that controls virulence of Cryptococcus neoformans, the leading cause of fungal meningitis. The central element of this virulence pathway is the calcium-calmodulin activated protein phosphatase calcineurin, which is the molecular target of the immunosuppressive antifungal drugs cyclosporin A and FK506. C. neoformans mutants lacking either the catalytic A or the regulatory B subunit of calcineurin are inviable at 37 degrees C and other stress conditions and, as a consequence, are avirulent in animal models. In studies supported by this award, we identified: 1) the calcineurin B regulatory subunit and calmodulin, 2) the calcineurin binding protein (Cbpl) that is a conserved regulator or effector and which is the founding member of a protein family conserved from fungi to humans, and 3) the novel C2 domain protein Cts1 that may function as a downstream effector of the calcineurin signaling pathway to promote cell wall biogenesis and growth at 37degrees C. In parallel we discovered that calcineurin is required for virulence of Candida albicans, the most common human fungal pathogen. C. albicans cnb1/cnb1 mutants lacking the calcineurin B regulatory subunit are severely attenuated in animal models. Yet, in contrast to C. neoformans calcineurin is not required for growth of C. albicans at 37 degrees C. Instead, calcineurin is necessary for C. albicans to survive and proliferate in serum. These studies illustrate how a conserved signaling cascade has been co-opted to control virulence of two divergent fungal pathogens by unique molecular mechanisms. Here we propose to delineate this molecular virulence cascade in both C. neoformans and C. albicans. Importantly, this pathway can be targeted for therapeutic intervention using non-immunosuppressive calcineurin inhibitors that retain antifungal activity and synergistic drug combinations that we have discovered.

DESCRIPTION: One of the most exciting advances in fungal biology is the application of genomics approaches. The genomes of four model fungi (S. cerevisiae, S. pombe, N. crassa, A. gossypii) are complete, and many others are in progress. The genome project for the human fungal pathogen Cryptococcus neoformans has provided the complete genome for the serotype D strain (JEC20), generated 10 to 12X assemblies for the related serotype D strain B3501A and the pathogenic serotype A clinical isolate H99, and 6.5X coverage for a divergent serotype B strain (WM276). Our challenge is to capitalize upon these genomic resources to elucidate the molecular basis of virulence, and to devise novel therapies. We propose to broadly apply Insertional mutagenesis to identify genes encoding virulence attributes necessary for infection. C. neoformans is an outstanding model pathogen. The organism is haploid, so recessive mutations can be directly isolated following mutagenesis. The organism has a defined sexual cycle, facilitating genetic analysis. Genes can be disrupted by transformation and homologous recombination, and robust animal models have been developed. These advances make it possible to satisfy Falkow's molecular postulates of virulence for this fungal pathogen. While genes can be disrupted by homologous recombination, targeting requires long regions of homology (about 1000 bp) and efficiency is not optimal. Random insertional mutagenesis provides a powerful complementary approach to identify genes of interest. We have optimized insertional mutagenesis using a dominant genetic marker and agrobacterium as the gene delivery vehicle, developed congenic strains to conduct genetic crosses and establish linkage, and implemented approaches to identify the mutated genes. Here, we will employ signature tagged mutagenesis to conduct a broad scale analysis of the molecular determinants of development and virulence. In aim 1, we will generate banks of mutants using agrobacterium-mediated gene delivery to insert tagged dominant markers to saturate the genome. In aim 2, we will conduct in vitro screens to identify mutants compromised for virulence factors, combined with screens in heterologous hosts and cultured macrophages to identify candidate virulence mutants. Finally, in aim 3, we will conduct studies in murine models to identify mutants from pooled infections that are altered in virulence or tissue-specific patterns of infection. These studies will enable a genome-wide definition of the gene set contributing to virulence of this common human fungal pathogen.

DESCRIPTION (provided by applicant): This proposal seeks continued and increased support for graduate training in genetics and genomics in the Duke University Program in Genetics and Genomics, a unique training program that is degree granting and currently encompasses 71 faculty and 64 students in the Departments of Biochemistry, Cell Biology, Immunology, Medicine, Microbiology and Molecular Genetics (MGM), Pathology, Pediatrics, Pharmacology and Cancer Biology, and Radiation Oncology in the Medical Center and from the Departments of Biology and Chemistry in the School of Arts and Sciences. Research interests span traditional and molecular genetics, model systems (bacteria, yeast, and flies), plant biology, human genetics, developmental biology, population genetics, and genome sciences. Students apply directly to the program for graduate training at Duke, most from undergraduate programs in the biological or physical sciences. Admission is based on GRE scores, GPA, letters of recommendation, research interests and experience, and an interview at Duke. During the first program year, students attend courses, rotate in labs, and meet with the first year advisory committee. At the conclusion of the first year, students choose a lab and thesis advisor, and ultimately receive their degree via the program or host department. Duke has recently established the Institute for Genome Sciences and Policy (IGSP), and we have forged a close link between the genetics program and both the IGSP and its director, Hunt Willard, We are expanding the genetics program to include genome sciences in a joint program to be called the Duke University Program in Genetics and Genomics (UPGG). Thus, we have expanded our seminar series to the full academic year with the IGSP providing one-half of the support. We have launched a distinguished lecturer series to complement the genetics and genomics seminar series. We have also instituted a program retreat organized and conducted by students, which was held at the beach in 2002 and the mountains in 2003 The program is administered by the Director (Joe Heitman), the Co-director (Doug Marchuk), the Director of Graduate Studies (Marcy Speer), and the IGSP Director (Hunt Willard) together with the executive committee. Currently, the NIH provides support for four new students per year, and Duke University via the Deans supports eight new students per year. Here we seek to expand the NIH supported portion of the training program to a total of eight new students per year.

DESCRIPTION (provided by applicant): Our studies have defined signaling cascades regulating Cryptococcus neoformans differentiation and virulence. As one paradigmatic example, the Ca2+ activated phosphatase calcineurin was demonstrated to govern a signaling cascade enabling growth at mammalian body temperature and virulence. In parallel, calcineurin was found to be required for mating and monokaryotic fruiting, differentiation cascades thought to produce the suspected infectious propagules in nature. Our efforts supported by this proposal have resulted in elucidating many pathway components, including the Ca2+ sensor calmodulin, the calcineurin catalytic and regulatory subunits, the conserved calcineurin binding protein and pathway effector Cbp1, the immunophilins cyclophilin A and FKBP12, and the cell wall regulatory component Cts1. In summary, we have achieved the goals of the two previous awards, and contributed a wealth of additional methodological and conceptional advances fueled by this support. As one example, we recently reported that monokaryotic fruiting represents a modified sexual cycle involving partners of the same mating type. These advances enable us to broaden the scope of this competitive renewal from an original focus on calcineurin in mating and fruiting to a genetic and genomic analysis of mating and fruiting in C. neoformans. Previous studies have linked mating type to virulence of C. neoformans. A central conundrum has been the finding that the vast majority of clinical and environmental isolates are of the mating type. Our discovery of same sex mating provides an explanation how diversity can be maintained in a largely unisexual population. Here, we propose to analyze in detail the monokaryotic fruiting sexual cycle and its impact on the organism. We propose three inter-related specific aims. Aim 1 is a physiological, genetic, and genomic analysis of monokaryotic fruiting including whole genome microarray, insertional mutagenesis, and quantitative trait mapping. In aim 2, we will dissect the role of the Cpr2 pheromone receptor homolog in monokaryotic fruiting. Aim 3 is to examine the role of same-sex mating in the origin of the C. gattii Vancouver Island outbreak. These studies will contribute to an understanding of the roles of sexual reproduction in microbial pathogenesis, and provide insights into the life cycle and virulence cycle of C. neoformans and C. gattii applicable to development of treatment approaches, diagnostics, or environmental interventions to reduce exposure to infectious spores.

[unreadable] DESCRIPTION (provided by applicant): Cryptococcus neoformans is a pathogenic basidiomycetous fungus that infects the central nervous system causing meningoencephalitis. This project's objective is to understand signaling mechanisms controlling sexual reproduction and virulence in this model pathogenic fungus. Tremendous advances have been achieved in studies on the role of G protein regulated signal transduction pathways that control cell type and virulence. Two well-conserved cascades involving cAMP and MAP kinase signaling have been previously characterized. But it still remains largely unknown how fungal pathogens sense environmental and host signals that trigger these G protein governed pathways. We have identified a family of G protein-coupled receptors (GPCRs) and discovered that one GPCR, Gpr4, senses amino acids and interacts with the Ga protein Gpa1 to activate cAMP signaling. Our studies further reveal Gpa1 is required to sense multiple signals, which may provide new paradigms to understand how G proteins are activated by both receptor-dependent and receptor-independent mechanisms. The studies proposed here will provide new approaches to understand how ligand-GPCR-G protein interactions control microbial pathogenesis. The split-ubiquitin system will be applied to study the interactions between the family of GPCRs and associated Ga proteins. A yeast heterologous expression system will be developed to screen for potential ligands of individual GPCRs. Gene deletion mutants for each GPCR have been generated and their roles in signaling and pathogenesis will be studied in vitro and in a murine infection model. The specific aims of this project are to: 1) Identify and characterize sensors involved in cAMP signaling, and to test the hypothesis that a multiple sensor system operates in C. neoformans to activate the Ga protein Gpa1; 2) Elucidate interactions between the GPCR family and the Ga proteins Gpa1, Gpa2, and Gpa3; 3) Identify potential GPCR ligands and their roles in development and virulence. Outcomes of this project should fill critical research gaps between extracellular signal sensing and signal transduction cascades controlling sexual reproduction and virulence factor production in C. neoformans. Understanding ligand- GPCR-G protein interactions and regulation of fungal development will provide insights to develop new drug targets, and may provide the means to control C. neoformans and other pathogenic fungi, which have medical significance of considerable importance. Cryptococcus neoformans is a human fungal pathogen that infects the central nervous system and often causes meningitis that is fatal if untreated. Understanding ligand- GPCR-G protein interactions and regulation of fungal development will provide insights to development new drug targets, and may provide the means to control C neoformans and other pathogenic fungi, which have medical significance of considerable importance. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Fungal infections are increasing as a result of AIDS, transplantation, chemotherapy, steroids and antibiotics, and invasive procedures and medical devices. Antifungal agents were limited to amphotericin B, flucytosine, and azoles, but now the candins, second-generation azoles, and lipid based amphotericin formulations have expanded the antifungal drug armamentarium. Yet with difficulties in delivering parenteral agents, consistent efficacy, need for rapid, short courses of therapy, and emerging drug resistance, therapeutic advances remain to be achieved. Our research focuses on signaling cascades as targets for antifungal drugs. Studies are proposed on Candida albicans, the most common human fungal pathogen that remains a major mucosal pathogen in AIDS patients who fail or do not receive HAART, and Cryptococcus neoformans, the leading cause of fungal meningitis in the world due to the AIDS epidemic. [unreadable] [unreadable] Our studies have defined the mechanisms of action and targets for the antifungal immunosuppressants cyclosporin A, FK506, and rapamycin. Fungal homologs of calcineurin, cyclophilin, FKBP12, and Tor1 were identified, providing insight into biological roles and as conserved drug targets. Nonimmunosuppressive analogs that retain antifungal activity were identified. Synergistic fungicidal drug interactions were demonstrated and mechanisms of action elucidated. Calcineurin inhibition by cyclosporin A or FK506 is potently synergistic with azoles against C. albicans and of therapeutic benefit in animal models. Recent studies implicate calcineurin as an Hsp90 client protein, and Hsp90 mutations or inhibitors are also synergistic with azoles. [unreadable] [unreadable] Here we propose to define Tor, calcineurin, and FKBP12 pathways as targets for therapy. First, we will characterize Tor cascade elements and functions and target this pathway with rapamycin and less immunosuppressive rapamycin analogs (rapalogs). Second, we will elucidate relationships between Hsp90 and calcineurin and their inhibitors that render azoles fungicidal and target this pathway with novel Hsp90 inhibitors, azoles, and calcineurin inhibitors. Third, we will focus on FKBP12 control of an amino biosynthetic cascade targeted by known antifungal agents and define synergistic antifungal drug combinations. Finally, drugs, analogs, and combinations will be tested in animal models of cryptococcosis and candidiasis. Our assembled team of collaborators in natural products, medicinal chemistry, enzymology, structural biology, and animal models complements our expertise in signaling and target identification. The goal is to harness signaling cascades to develop novel antifungal therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The related fungal species Cryptococcus neoformans and Cryptococcus gattii frequently infect humans, causing life-threatening lung and brain infections. Although often considered opportunistic pathogens, these organisms can cause disease in hosts with both compromised and normal immunity. More than 1,000,000 infections occur worldwide annually, leading to >620,000 deaths comprising more than one-third of all HIV/AIDS-related deaths. A specialized genomic region, the mating type locus (MAT), governs cell identity, sexual reproduction, infectious spore production, and virulence. We previously discovered that the Cryptococcus MAT locus is a large, complex gene cluster, and we proposed to elucidate how this unusual suite of sex- and virulence-determining genes evolved from a simpler nonpathogenic ancestral state. Our comparative phylogenomic studies provide evidence that 1) the bipolar/unipolar mating system is a shared characteristic of these two Cryptococcus pathogenic species, and 2) the inbreeding bipolar/unipolar system evolved from an ancestral outbreeding tetrapolar system via gene acquisition and translocation-driven fusion. Similar transitions have occurred in other fungal pathogens of plants and animals, suggesting convergent evolution in concert with host adaptation. These studies illustrate general principles of gene cluster evolution and forces by which recombination has forged the genomes of microbial pathogens. Our studies supported by this award defined the structure, function, and evolution of the MAT locus. In the prior award period, we 1) cloned and sequenced the bipolar MAT locus from pathogenic Cryptococcus species; 2) discovered tetrapolar sexual cycles and MAT loci of closely related nonpathogenic species; and 3) found the two MAT loci of the nonpathogen Cryptococcus amylolentus lie on different chromosomes and are both centromere-linked. We hypothesize the tetrapolar-bipolar transition occurred concomitant with pathogen emergence via two steps. First, genes were recruited into two unlinked MAT loci; second, chromosomal translocation fused the MAT loci. Our recent studies reveal novel MAT features allowing us to propose new aims to test these hypotheses. Aim 1 focuses on MAT locus structure and evolution. We will sequence genomes, define centromeres, and test the hypothesis that inter-centromeric recombination drove fusion of unlinked ancestral MAT loci and key chromosomal translocations that punctuate pathogen evolution. Aim 2 focuses on MAT locus functions. We will address 1) mechanisms governing uniparental mitochondrial inheritance that restrict mitochondrial genome recombination and impact mitochondrial dynamics that promote replication inside macrophages and pathogenesis, and 2) roles of essential diverged ribosomal protein paralogs in development and virulence. Not only do these studies advance our understanding of the dynamic evolution of microbial genomes, but given the association with specific gene clusters and virulence, they also have direct implications for infectious disease treatment and prevention.

DESCRIPTION (provided by applicant): Mucor circinelloides and Rhizopus oryzae are the most prevalent causal agents of the deadly fungal infection, mucormycosis, which is an emerging disease in immunocompromised patients and the second most common fungal infection in both hematopoietic malignancy patients and transplant recipients. However, considerably less is known about the virulence of these species compared to other fungal pathogens. This study focuses on the genetics of sexual development and pathogenicity of two zygomycete species complexes. In the studies proposed for M. circinelloides, we will investigate the roles of three genes that encode a triose phosphate transporter homolog, HMG domain protein, and RNA helicase in the syntenic sex locus of three related Mucor sub-species. The expression patterns of these genes during sexual development will be investigated. Disruption mutants of each gene and isogenic strains will be generated to test their roles in sexual development. Our recent studies found evidence that the sex locus may be involved in spore size and virulence of M. circinelloides, in which (-) isolates produce more virulent larger spores when compared to (+) isolates. We will investigate how genes in the syntenic sex locus are involved in spore size determination and virulence in three host models: cultured macrophages, the wax moth Galleria mellonella, and the mouse. In parallel, in the studies proposed for R. oryzae, we will establish a typing system to classify R. oryzae and R. delemar that are two sub-species in the R. oryzae complex. The two sub-species will be discriminated at high resolution based on MLST and lactate dehydrogenase gene sequences. Our preliminary studies indicate that R. oryzae is sexual and saprobic, whereas R. delemar is infertile under laboratory conditions and may be more virulent. We will also further test sexuality of the two sub-species as well as virulence in the wax moth and the mouse to investigate links between sexuality and virulence. Understanding the life cycles and detailed phylogeny of these pathogens will greatly enhance our knowledge to develop new drug targets and diagnostic typing approaches, and may provide the means to control human pathogenic zygomycetes. PUBLIC HEALTH RELEVANCE: Mucormycosis is an opportunistic fungal infection recently recognized as emerging cause of infectious disease with a high mortality rate. However, little is known about the relationship of sexual reproduction to pathogenesis in the zygomycetes compared to other fungal pathogens. This project will provide insight to develop novel drug targets, typing methodology to assign species causing infection, and information that can be applied to control zygomycete infections.

ABSTRACT The need to train scientists to conduct fungal research is greater than ever. Worldwide estimates of human fungal disease include over a billion people with invasive, allergic, or chronic fungal diseases. Plant diseases, caused predominantly by fungi, are estimated to reduce global food yields by 20-40%. These striking statistics showcase the immense human and financial tolls imparted by fungal diseases. Our objective is to develop molecular mycology scientists trained in the latest methods of laboratory, translational, or clinical research who are fully prepared to pursue independent research careers investigating the many aspects of fungal diseases. The Tri-Institutional Molecular Mycology and Pathogenesis Training Program (Tri-I MMPTP) has been funded since 2003 to recruit, support, and train promising postdoctoral scientists and physicians to develop productive research careers. It is the only mycology-focused postdoctoral training program in existence, and it has been highly successful in training the next generation of outstanding scientists. The tremendous productivity of this training program is predicated on its unique design, leveraging the proximity of three prominent research universities: Duke University, the University of North Carolina at Chapel Hill, and North Carolina State University. These institutions offer arguably the highest geographic concentration of researchers who study fungi in the country. The Tri-I MMPTP has trained or is currently training a total of 43 post-doctoral fellows, many of whom have gone on to lead independent NIH-funded laboratories, lead government research efforts, work in industry on cutting-edge projects, and assume positions of leadership in academia, government, and industry. At many institutions, faculty who investigate medical fungi have little contact with geneticists who work on model fungi, biochemists who study cellular mechanisms, or infectious diseases physicians who care for immunocompromised patients. Similarly, those who study phytopathogens are intellectually, and often physically, removed from biomedical researchers. A proven concept underlying our multidisciplinary interaction is that clinical and basic researchers, and plant and animal mycologists, together discover new approaches that are mutually beneficial. With this integrated design, our trainees become broadly knowledgeable, versatile, and more attractive to prospective employers. Several outstanding training themes are responsible for our continued success: careful selection from a large pool of competitive applicants, promotion of diversity (including 3 of 7 current trainees from under-represented backgrounds), and centralized courses on scientific and grant writing. These opportunities are all connected through trainee Individual Research Advisory Committees. At the core is our dedicated mentorship, including a pathway to independence mentality and a formalized continued mentorship program for three years after program completion. Our interconnected strategy has generated highly successful scientists for 15 years, and our constant programmatic updates via trainee and mentor feedback have developed exciting approaches to fill the need for molecular mycologists.

DESCRIPTION (provided by applicant): Cryptococcus neoformans and Cryptococcus gattii frequently infect humans to cause life-threatening pneumonia and meningitis resulting in considerable morbidity and mortality in both immunocompromised and immunocompetent hosts. More than one million cases of cryptococcal infection occur globally annually, resulting in >620,000 deaths and up to one-third of all AIDS-associated deaths. An outbreak of C. gattii in the Pacific Northwest involves non-AIDS patients, the majority of whom are immunocompetent, and our investigations document this outbreak has expanded into the US and novel hypervirulent genotypes have emerged. Both Cryptococcus species have a defined sexual cycle involving two opposite mating types (a,a). However, because the vast majority of clinical and environmental isolates are a mating type, it had been unclear if sex occurs in nature and how aerosolized spores thought to be infectious propagules arise. We discovered a novel sexual cycle involving only one mating type, termed a-a same-sex mating or unisexual reproduction. Our population genetic studies provide substantial evidence that unisexual reproduction is a predominant route generating genetic diversity in nature. Our studies further demonstrate that spores produced by sex are infectious following inhalation, and thus unisexual reproduction may be the source of human infections. Candida albicans was also recently discovered to undergo same-sex mating, and cryptic sexual cycles are being found for other fungal and parasite eukaryotic pathogens. Thus, our studies on the roles of sex in pathogen evolution and emergence are of general importance. Our studies supported by this award have defined conditions and genetic circuits governing a-a opposite and a-a unisexual reproduction of Cryptococcus. We have 1) shown that sex occurs in common environmental niches (pigeon guano, plants/trees) and in response to nutrient limitation, light/darkness, carbon dioxide, and inositol; 2) elucidated same-sex genetic cascades involving the novel cell surface receptor Cpr2 and the nuclear transcription factors Mat2 and Znf2; 3) adduced robust population genetic evidence that unisexual reproduction occurs in nature; and 4) shown that sexual reproduction has shaped the C. gattii population structure with implications for pathogen emergence. Our hypothesis is that a-a unisexual reproduction generates genetic diversity and infectious spores. Our recent studies reveal novel features of sexual reproduction allowing us to propose three new aims to test these hypotheses. Aim 1 is to elucidate how the RNAi-dependent sex-induced silencing pathway functions to guard genome integrity. Aim 2 focuses on the de novo generation of genotypic and phenotypic plasticity by sexual reproduction involving aneuploidy and diploidy. Aim 3 addresses sexual reproduction and the basis of hypervirulence in the C. gattii Pacific Northwest outbreak. These studies will advance understanding of how microbial pathogens evolve and emerge via sexual reproduction with implications for treatment and prevention. PUBLIC HEALTH RELEVANCE: These studies focus on the human fungal pathogens Cryptococcus neoformans and Cryptococcus gattii, fungi that infect the lungs and brain to cause life-threatening pneumonia and meningitis. C. neoformans causes >1,000,000 infections annually, and >620,000 deaths and up to one-third of all AIDS related deaths. It is now a greater cause of AIDS related morbidity in Africa than tuberculosis. C. gattii is causing an outbreak in the Pacific Northwest in largely immunocompetent individuals, and has expanded into the United States. Our studies focus on the role of sexual reproduction in Cryptococcus, leading to genetic diversity and the production of infectious spores.

DESCRIPTION (provided by applicant): Invasive fungal infections are a leading cause of death in immunocompromised patients. Current antifungals have limited clinical efficacy, are poorly fungicidal, are in some cases toxic, and are increasingly ineffective due to emerging resistance. We have established that the conserved phosphatase calcineurin is broadly required for invasive fungal disease. The FDA-approved calcineurin inhibitor FK506 is active in vitro against major invasive fungal pathogens, but also suppresses host immunity. Our approach seeks to overcome the fungal versus human specificity barrier to significantly advance antifungal treatment. The objective of this study is to utilize a structural biology-based strategy to define fungal-mammalian calcineurin structural differences, validate fungal-specific targets, and generate and optimize novel FK506 analogs to treat invasive fungal diseases. Our central hypothesis is that by employing a structural biological approach using both crystallography and NMR spectroscopy that we will define novel targetable fungal-specific areas in the calcineurin complex critical for fungal pathogenesis. For maximum clinical breadth, we will focus on the two major clinical pathogens: the yeast Candida albicans and the mold Aspergillus fumigatus. Our preliminary studies document proof of principle non-immunosuppressive FK506 analogs with robust antifungal activity. We hypothesize that structures of the calcineurin A and B complex, coupled with calmodulin and the immunophilin complex (FKBP12-FK506), will reveal novel fungal-specific targets for inhibition. We have recently solved the structure for C. albicans, and will now solve structures for the calcineurin heterodimer alone and complexed with FKBP12-FK506/analogs from A. fumigatus. The multiple molecular views will allow identification of sites that are distinct between human and fungal calcineurin complexes that can be exploited for targeted inhibitor development. Protein regions that are dynamic or resist crystallization will be structurally characterized by NMR. Putative inhibitory domains will be validated via genetic and biochemical assays, utilizing site-directed mutagenesis of key contact and surface residues to examine structural stability, fungal phenotype, and drug action/resistance. Non-immunosuppressive fungal-specific FK506 analogs will be generated by Amplyx Pharmaceuticals based on the SAR results of our first iteration. This will guide the production of second generation analogs optimized for retention of antifungal activity and abrogation of immunosuppression by capitalizing on the unique structural differences between the host and fungal enzymes. Medicinal chemistry and inhibitor docking experiments will be conducted to alter analogs based on screening results. Lead compounds will be tested using an iterative approach both in vitro and in murine models of invasive candidiasis and invasive aspergillosis. We will capitalize on structural biology as a new approach to targeting calcineurin by defining fungal-specific features with no mammalian counterpart to generate novel antifungal therapeutics.

ABSTRACT: Project 1 ? Crystallography and NMR-guided Generation of Fungal Calcineurin Inhibitors The conserved phosphatase calcineurin is broadly necessary for invasive fungal disease. FDA-approved calcineurin inhibitors (FK506, cyclosporine A (CsA)) are active against invasive fungal pathogens, but they also suppress host immunity. Our preliminary studies document proof of principle compounds that have antifungal activity in vitro and in vivo and are non-immunosuppressive. The goals of this proposal are to identify fungal- specific targets in the calcineurin pathway via fungal-mammalian structural differences and develop novel inhibitors. The central hypothesis is that a structural biological approach through crystallography and NMR spectroscopy will define novel and targetable fungal-specific areas in the calcineurin circuit critical for fungal pathogenesis. We hypothesize that structures of the calcineurin AB complex, coupled with calmodulin and the immunophilin complexes (cyclophilin A-CsA, FKBP12-FK506), will reveal novel fungal-specific targets for inhibition. Our successful preliminary studies have already solved structures of the calcineurin AB complex for Candida albicans and Aspergillus fumigatus. To extend maximal clinical breadth, this proposal will now focus on Cryptococcus neoformans/gattii, Candida glabrata, and Mucor circinelloides. Cryptococcal meningitis is the most common etiology of fungal central nervous system disease worldwide, Candida glabrata is emerging as both an azole- and echinocandin-resistant species, and mucormycosis is the 3rd most common invasive fungal infection in all transplant recipients. We will solve structures for the calcineurin heterodimer alone and complexed with FKBP12-FK506/analogs and cyclophilin A-CsA/analogs for these three pathogens. The multiple molecular views provided will allow identification of sites for chemical targeting that are distinct between human and fungal calcineurin complexes. Protein regions that are dynamic or resist crystallization will be structurally characterized by NMR. Putative inhibitory domains will be validated via genetic and biochemical assays, utilizing site-directed mutagenesis of key contact and surface residues to examine structural stability, fungal phenotype, and drug action/resistance. Non-immunosuppressive fungal-specific calcineurin inhibitors will be generated by: 1) screening libraries of CsA analogs (Scynexis, Novartis), FK506 analogs (Amplyx Pharmaceuticals), and FK506-tripeptide conjugates (Jun Liu, Johns Hopkins) to provide the structural basis to pursue novel chemical modifications based on known drug platforms; and 2) conducting medicinal chemistry in collaboration with investigators at Amplyx and Scynexis to alter analogs based on our screening/structural results. This will provide insight to produce second-generation inhibitors optimized for potentiation of antifungal activity and abrogation of immunosuppression by capitalizing on unique structural differences between the host and fungal enzymes. Non-immunosuppressive calcineurin inhibitors will advance to the Inhibitor Testing Core for antifungal activity screening, cell-based toxicity assays, and animal model efficacy testing. Results will feedback to this Project for any additional medicinal chemistry modifications.

Project Summary The fungal pathogen Cryptococcus neoformans is estimated to cause disease in more than 1,000,000 people worldwide every year, resulting in greater than 600,000 deaths. However, not all Cryptococcus isolates are killers; some are relatively benign while oth- ers are hypervirulent. Differences in pathogenicity are often correlated with variation in key morphological and physiological features such as the ability to grow at high tem- peratures, the size of the protective polysaccharide capsule surrounding the yeast cell, or resistance to antifungal drugs. In order to develop better ways to prevent and treat cryptococcal disease we need to understand the underlying genetic differences that lead to changes in virulence and virulence-related traits. We also need to understand how frequently and how wide spread these genetic differences are in different parts of the world. To tackle these problems, the proposed research will use a combination of experi- mental and statistical approaches to dissect the causal genetic basis of variation in vir- ulence and virulence-related traits. In Aim 1 the investigators will employ a statistical technique called Quantitative Trait Locus (QTL) mapping, that exploits genotypic and phenotypic differences among offspring derived from genetic crosses to identify regions of the genome (loci) and DNA changes (alleles) that contribute to differences in virulence traits. The investigators will validate the contributions of the loci identi?ed in this man- ner using gene deletions and related techniques. In Aim 2 the investigators will subject genetically diverse populations of Cryptococcus to selection in animal hosts. Following selection, pooled genome sequencing will be used to identify loci and alleles that are favoring during infection. This technique provides an unbiased approach for discover- ing loci that contribute to virulence, and will both complement and extend the results of Aim 1. In Aim 3 the investigators will put their ?ndings in the context of natural populations of Cryptococcus by studying the frequency and geographic distributions of virulence alleles identi?ed in Aims 1 and 2. This aim will employ a large, world-wide sample of Cryptococcus strains isolated both from clinical settings and from the natural environment. The investigators will use this information to identify regions of the world where there are high frequencies of virulent genotypes, or where there is potential for in- creased virulence through processes like recombination or hybridization. 1

Project Summary The fungal pathogen Cryptococcus neoformans is estimated to cause disease in more than 1,000,000 people worldwide every year, resulting in greater than 600,000 deaths. However, not all Cryptococcus isolates cause lethal infections; some are relatively benign while others are hypervir- ulent. Differences in pathogenicity are often correlated with variation in speci?c morphological and physiological features such as the ability to grow at high temperatures, the size of the pro- tective polysaccharide capsule surrounding the yeast cell, or resistance to antifungal drugs. In order to develop better ways to prevent and treat cryptococcal disease we need to understand the underlying genetic differences that lead to changes in virulence and virulence-related traits. We also need to understand the frequency and distribution of these genetic differences in different parts of the world. To tackle these problems, the proposed research will use a combination of experimental and statistical approaches to dissect the causal genetic basis of variation in virulence and virulence- related traits. In Aim 1 we will employ a statistical technique called Quantitative Trait Locus (QTL) mapping, that exploits genotypic and phenotypic differences among offspring derived from genetic crosses to identify regions of the genome (loci) and DNA changes (alleles) that con- tribute to differences in virulence traits. We will validate the contributions of the loci identi?ed in this manner using gene replacements and related techniques. In Aim 2 we will subject ge- netically diverse populations of Cryptococcus to selection in animal hosts. Following selection, pooled whole genome sequencing will be used to identify loci and alleles that are favored during infection. This technique provides an unbiased approach for discovering loci that contribute to virulence, and will both complement and extend the results of Aim 1. In Aim 3 we will frame our ?ndings in the context of natural populations of Cryptococcus by studying the frequency and geo- graphic distributions of virulence alleles identi?ed in Aims 1 and 2. This aim will employ a large, global sample of Cryptococcus strains isolated from both clinical settings and the natural environ- ment. This information will be used to carry out Genome-Wide Association (GWAS) mapping of virulence traits and to identify regions of the world where there are higher frequencies of virulent genotypes or where there is potential for increased virulence through recombination.

Abstract Cryptococcus neoformans and Cryptococcus gattii are globally distributed fungal pathogens that cause >220,000 life-threatening infections each year in immunocompromised and immunocompetent patients, leading to >180,000 deaths, >15% of all HIV/AIDS-related deaths, and >70% mortality in low-income countries. In studies supported by this award, the scope and functions of a-? and ?-? sexual reproduction on the evolution and pathogenesis of Cryptococcus were defined. Sexual reproduction governs population structure, enabling genetic exchange and promoting clonality, and results in the production of infectious spores. During this sustained funding period, significant advances were achieved characterizing the mechanisms and impact of sexual reproduction on these pathogens and their virulence: 1) discovery of roles of RNAi in controlling transposon movement and transgene silencing during mitosis and meiosis and characterization of the consequences of RNAi loss on genome structure and stability; 2) studies on generation and affect of aneuploidy on drug resistance, pathogenicity, and development, and engineering of a ploidy sensor to analyze genetic factors contributing to endoreplication during unisexual reproduction; 3) delineation of isolates causing the C. gattii outbreak in the Pacific Northwest and closely aligned hypermutator lineages, and 4) demonstration that unisexual reproduction generates genetic diversity de novo and enhances competitiveness for nutrients and mating partners. In the current proposal, we hypothesize 1) RNAi has been retained and lost in unique isolates and species, influencing genomic stability and driving evolution of drug resistance and pathogenesis, and 2) unisexual reproduction is a major force in evolution, global distribution, and impact of Cryptococcus pathogens. Our studies reveal unique facets of RNAi and sexual reproduction enabling us to propose new aims to test these hypotheses. Aim 1 focuses on RNAi in genome stability, elucidating components and mechanisms by which RNAi controls transposons and silences repetitive DNA sequences, and studying the short-term and long- term consequences of RNAi loss on drug resistance and pathogenesis in C. neoformans hypermutator clinical isolates and the RNAi-deficient C. deuterogattii outbreak species. Aim 2 will elucidate unisexual reproduction pathways and mechanisms involving 1) unisexual reproduction occurring in mixed mating-type populations and in strains with rearranged genomes as a model of speciation, 2) genetic and environmental factors that promote unisexual reproduction of global serotype A lineages and analysis of infectious spores in animal models, and 3) overcoming the Hill-Robertson effect to enable linkage of beneficial mutations and separation of detrimental and beneficial mutations to enhance fitness and pathogenesis. These studies will advance the understanding of how genome stability and unisexual reproduction drive pathogen evolution and population structure, development of drug resistance, and infectious spore production and virulence, with direct implications for infectious disease evolution, treatment, and prevention.