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.
