Natural genomic variants that influence cryptococcal pathogenicity Cryptococcus neoformans is a global pathogen responsible for hundreds of thousands of deaths yearly in HIV+ individuals and increasing morbidity in non-AIDS patient populations. C. neoformans strains collected from pa- tients with cryptococcal meningitis have been used to elucidate C. neoformans evolution and in efforts to corre- late disease outcome with in vitro measures such as virulence factor production or fungal growth. We know that distinct genome sequences are associated with varied clinical outcomes. However, we do not know which natural genomic variants are responsible for the differential virulence of clinical isolates. Our long-term goal is to understand the molecular mechanisms by which natural variation in the cryptococcal genome determines pathogenicity. A first step toward this goal is to identify naturally occurring variants that can be experimentally proven to influence virulence, at single-gene resolution. The next step is to characterize the impact of variants in these genes on cryptococcal biology and disease progression. We hypothesize that we can combine genetic and genomic methods with experimental follow-up to identify, validate, and characterize natu- rally occurring variants that significantly influence cryptococcal virulence. This hypothesis is supported by our compelling preliminary data, in which two distinct genetic methods implicate variants in the same genomic region as influencing infectivity. We propose to achieve our goals by pursuing three Aims:
Aim 1 is to use two distinct, complementary statistical genetics methods to discover genomic regions containing naturally occurring variants that influence fungal viru- lence.
Aim 2 is to further apply genetics, supported by expression analysis and mutant studies, to refine these regions and identify specific high-priority genes within them.
Aim 3 is to use genome engineering to validate the influence of selected variant genes on virulence and to assess their effects on fungal gene expression, physiol- ogy, and pathogenicity in a mouse model. Applying an unbiased strategy to identify genes harboring natural variants that influence virulence will highlight new potential targets for antifungal therapy. Characterizing these variants will also pave the way for future inves- tigations of the mechanistic basis of their effects, which will elucidate key features of cryptococcal biology and pathogenesis. Our methods are applicable to other fundamental questions in cryptococcal biology, and poten- tially to other microbial pathogens. Finally, our studies will produce valuable reagents and data sets that will enhance investigations by other C. neoformans researchers.
The pathogenic fungus Cryptococcus neoformans causes a meningoencephalitis that kills almost 200,000 peo- ple per year. Striking differences in virulence are observed among naturally occurring strains of this microbe. The goal of this research is to identify the regions of the genome, and specific changes in DNA sequence, that are responsible for these differences. Successful completion of these studies will establish powerful new meth- ods and significant community resources for research, increase fundamental understanding of this organism and the disease it causes, and potentially impact antifungal drug development.
