Genetic analysis of Cryptococcus neoformans virulence

Summary

Principal Investigator: JOSEPH B HEITMAN
Abstract: 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.
Funding Period: 2005-06-01 - 2010-02-28
more information: NIH RePORT

Top Publications

  1. pmc Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex
    Marianela Rodriguez-Carres
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America
    PLoS ONE 5:e9620. 2010
  2. pmc Cryptococcus gattii genotype VGI infection in New England
    Russell J McCulloh
    Division of Infectious Diseases, Rhode Island Hospital Brown University, Providence, RI 02903, USA
    Pediatr Infect Dis J 30:1111-4. 2011
  3. pmc Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex
    Keisha Findley
    Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA
    PLoS Genet 8:e1002528. 2012
  4. ncbi Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans
    Felicia J Walton
    Howard Hughes Medical Institute, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Mol Microbiol 57:1381-96. 2005
  5. pmc Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi
    Felicia J Walton
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Mol Biol Cell 17:3768-80. 2006
  6. pmc Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans
    Alexander Idnurm
    Room 322 CARL Building, Box 3546, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Eukaryot Cell 6:60-72. 2007
  7. pmc Eca1, a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, is involved in stress tolerance and virulence in Cryptococcus neoformans
    Weihua Fan
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Infect Immun 75:3394-405. 2007
  8. pmc Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the Tremellales
    Keisha Findley
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA
    Eukaryot Cell 8:353-61. 2009
  9. pmc Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis
    Alexander Idnurm
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA
    Eukaryot Cell 8:315-26. 2009

Scientific Experts

  • JOSEPH B HEITMAN
  • Alexander Idnurm
  • Russell J McCulloh
  • Keisha Findley
  • Felicia J Walton
  • Marianela Rodriguez-Carres
  • Sheng Sun
  • Fred S Dietrich
  • Weihua Fan
  • Yen Ping Hsueh
  • Anna Floyd Averette
  • Wenjun Li
  • James A Fraser
  • Banu Metin
  • Alvaro Fonseca
  • Johannes Kroiss
  • Rytas Vilgalys
  • Julia Breger
  • Eleftherios Mylonakis

Detail Information

Publications9

  1. pmc Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex
    Marianela Rodriguez-Carres
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America
    PLoS ONE 5:e9620. 2010
    ..depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species...
  2. pmc Cryptococcus gattii genotype VGI infection in New England
    Russell J McCulloh
    Division of Infectious Diseases, Rhode Island Hospital Brown University, Providence, RI 02903, USA
    Pediatr Infect Dis J 30:1111-4. 2011
    ..We report a case of an immunocompetent adolescent from New England who had severe pulmonary and central nervous system infection caused by the VGI genotype of C. gattii...
  3. pmc Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex
    Keisha Findley
    Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA
    PLoS Genet 8:e1002528. 2012
    ....
  4. ncbi Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans
    Felicia J Walton
    Howard Hughes Medical Institute, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Mol Microbiol 57:1381-96. 2005
    ..neoformans and provides further insight into the intimate relationship between metal ion homeostasis and melanin biosynthesis...
  5. pmc Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi
    Felicia J Walton
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Mol Biol Cell 17:3768-80. 2006
    ..neoformans but not in S. cerevisiae. These results indicate that conserved signaling pathways serve both similar and divergent cellular roles in morphogenesis in these divergent organisms...
  6. pmc Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans
    Alexander Idnurm
    Room 322 CARL Building, Box 3546, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Eukaryot Cell 6:60-72. 2007
    ..Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes...
  7. pmc Eca1, a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, is involved in stress tolerance and virulence in Cryptococcus neoformans
    Weihua Fan
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
    Infect Immun 75:3394-405. 2007
    ..Eca1 is likely involved in maintaining ER function, thus contributing to stress tolerance and virulence acting in parallel with Ca2+-calcineurin signaling...
  8. pmc Phylogeny and phenotypic characterization of pathogenic Cryptococcus species and closely related saprobic taxa in the Tremellales
    Keisha Findley
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA
    Eukaryot Cell 8:353-61. 2009
    ....
  9. pmc Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis
    Alexander Idnurm
    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA
    Eukaryot Cell 8:315-26. 2009
    ..Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals...