Cryptococcus neoformans is a fungal pathogen that infects the human brain. The organism has a defined sexual cycle involving haploid and a strains, and the mating-type is linked to virulence and differentiation. Most environmental and clinical isolates are mating-type, and strains can be more virulent than congenic a strains and preferentially penetrate the CNS during co-infection with a strains by a process involving pheromone signaling similar to quorum sensing in bacteria and other fungi. strains also undergo monokaryotic fruiting and produce basidiospores via a modified sexual cycle. Thus, the structure, function, and evolution of the mating-type locus (MAT) and its links to sexual development, virulence, and ecology are of considerable interest. In studies supported by this award, in aim 1 the MAT locus was cloned, sequenced, and annotated from both mating-types and four divergent Cryptococcus species/varieties, leading to a detailed evolutionary model.
In aim 2 on MAT functions, the MAT allele specific genes SXI1 and SXI2a were shown to encode homeodomain proteins that establish cell identity and enable sexual reproduction. MAT encoded genes were shown to play roles in sexual reproduction, virulence, and essential functions.
In aim 3 PCR fingerprinting and a MAT locus microarray were developed and provide comparative genomic analysis approaches to elucidate MAT structure and evolution. This progress, combined with recent advances involving genome sequences, microarrays, and discovery of fertile strains and development of congenic strains, allows us to propose new hypotheses and aims. Four interrelated specific aims are proposed.
Aim 1 focuses on the sequence of MAT, and extends the analysis to all molecular and mating-types. We propose to sequence MAT from species related to the Cryptococcus pathogenic species cluster;preliminary results indicate this will reveal key evolutionary events that fashioned this large gene complex. Studies to recreate and analyze the hypothesized tripolar intermediate and tetrapolar ancestor by reconfiguring the genomic location of the SXI1/2 genes are also proposed.
Aim 2 focuses on the functions of MAT, including roles of pheromones and Ste3 receptor homologs in virulence via processes similar to quorum sensing.
Aim 3 focuses on the structure and function of MAT-linked recombinational activators, proposed to have played key roles in MAT origins and evolution.
Aim 4 will focus on the structure and evolution of the MAT locus by CGH analysis. These studies will expand understanding of this unique genomic region and its roles in virulence, and serve as a paradigm for gene cluster and sex chromosome evolution and function.

Public Health Relevance

This project focuses on the structure, function, and evolution of the mating type locus, a specialized region of the fungal genome linked to mating, differentiation, and virulence. In particular, the role of genes encoding cell-cell communication molecules involved in the infectious process will be a focus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI050113-09
Application #
8196847
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Duncan, Rory A
Project Start
2001-07-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
9
Fiscal Year
2012
Total Cost
$382,239
Indirect Cost
$137,214
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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Goodenough, Ursula; Heitman, Joseph (2014) Origins of eukaryotic sexual reproduction. Cold Spring Harb Perspect Biol 6:
Lee, Soo Chan; Sun, Sheng; Heitman, Joseph (2014) Unseen sex in ancient virgin fungi. New Phytol 201:3-5
Janbon, Guilhem; Ormerod, Kate L; Paulet, Damien et al. (2014) Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet 10:e1004261
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Phadke, Sujal S; Feretzaki, Marianna; Clancey, Shelly Applen et al. (2014) Unisexual reproduction of Cryptococcus gattii. PLoS One 9:e111089
Gioti, Anastasia; Nystedt, Bjorn; Li, Wenjun et al. (2013) Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis. MBio 4:e00572-12
Ni, Min; Feretzaki, Marianna; Li, Wenjun et al. (2013) Unisexual and heterosexual meiotic reproduction generate aneuploidy and phenotypic diversity de novo in the yeast Cryptococcus neoformans. PLoS Biol 11:e1001653
Kozubowski, Lukasz; Yadav, Vikas; Chatterjee, Gautam et al. (2013) Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans. MBio 4:e00614-13

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