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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063443-04
Application #
7371944
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Duncan, Rory A
Project Start
2005-06-01
Project End
2010-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
4
Fiscal Year
2008
Total Cost
$413,973
Indirect Cost
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Findley, Keisha; Sun, Sheng; Fraser, James A et al. (2012) Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex. PLoS Genet 8:e1002528
McCulloh, Russell J; Phillips, Raina; Perfect, John R et al. (2011) Cryptococcus gattii genotype VGI infection in New England. Pediatr Infect Dis J 30:1111-4
Rodriguez-Carres, Marianela; Findley, Keisha; Sun, Sheng et al. (2010) Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex. PLoS One 5:e9620
Findley, Keisha; Rodriguez-Carres, Marianela; Metin, Banu et al. (2009) Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the Tremellales. Eukaryot Cell 8:353-61
Idnurm, Alexander; Walton, Felicia J; Floyd, Anna et al. (2009) Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis. Eukaryot Cell 8:315-26
Fan, Weihua; Idnurm, Alexander; Breger, Julia et al. (2007) Eca1, a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, is involved in stress tolerance and virulence in Cryptococcus neoformans. Infect Immun 75:3394-405
Idnurm, Alexander; Giles, Steven S; Perfect, John R et al. (2007) Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans. Eukaryot Cell 6:60-72
Walton, Felicia J; Heitman, Joseph; Idnurm, Alexander (2006) Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi. Mol Biol Cell 17:3768-80
Walton, Felicia J; Idnurm, Alexander; Heitman, Joseph (2005) Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 57:1381-96