Cryptococcus neoformans is a major pathogen in AIDS patients causing life-threatening meningoencephalitis in a large number of HIV+ individuals. Furthermore, groups classically at risk for cryptococcosis, the elderly, transplant recipients and those immunosuppressed with steroids or chemotherapy, are continually rising. Previous studies by us and others have shown a strong association between laccase-dependent melanin production by C. neoformans in vitro and virulence of the organism in experimental animals. We have shown that laccase is a cell wall-associated virulence factor by multiple lines of evidence: 1) localization by immuno-electron microscopy using an anti-laccase monoclonal antibody, 2) localization to the periphery of a green-fluorescent protein-tagged recombinant laccase by confocal microscopy, 3) localization of enzyme activity and laccase immunoreactive protein to cell wall preparations of C. neoformans. We have also developed a method of insertional mutagenesis and have isolated numerous laccase-deficient mutants. The overall objective of this proposal is to test the hypothesis that accurate cell wall targeting of laccase is required for the virulence of Cryptococcus neoformans. Detailed understanding of virulence-related trafficking may allow the identification of other novel virulence factors which share these same pathways and may allow the identification of unique trafficking inhibitors that disrupt the function of multiple virulence factors with a single agent.
In Specific Aim 1, we will identify targeting-specific amino acid sequences in laccase. We will use confocal microscopy to compare the cellular localization of expressed green fluorescent protein-tagged laccase in C. neoformans with an identical fusion protein containing deletions of the putative N-terminal vacuolar targeting sequence E25SPT or C-terminal clathrin-coated pit sequence Y426GFNNI. Additional deletions will be performed within the laccase N-terminus and C-terminus of the fusion protein to determine if other targeting sequences may be present.
In Specific Aim 2, we will identify genes and inhibitors involved in laccase trafficking by studying laccase trafficking after mutation of sentinel genes involved in actin-dependent, clathrin-dependent and vacuolar-dependent clathrin-independent trafficking pathways.
In Specific Aim 3 we will identify genes involved in laccase trafficking by studying laccase-deficient insertional mutants which show secretory defects in laccase secretion. Each of the mutants identified in Specific Aims 2 and 3 will then be assessed for production of other virulence-related factors such as capsule, urease, phospholipase and virulence in a mouse model.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
3R01AI049371-03S1
Application #
6925206
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Duncan, Rory A
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2004-07-01
Budget End
2005-03-31
Support Year
3
Fiscal Year
2004
Total Cost
$45,059
Indirect Cost
Name
University of Illinois at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Williamson, Peter R (2015) Post-infectious inflammatory response syndrome (PIIRS): Dissociation of T-cell-macrophage signaling in previously healthy individuals with cryptococcal fungal meningoencephalitis. Macrophage (Houst) 2:
Park, Yoon-Dong; Shin, Soowan; Panepinto, John et al. (2014) A role for LHC1 in higher order structure and complement binding of the Cryptococcus neoformans capsule. PLoS Pathog 10:e1004037
Albuquerque, Patrícia; Nicola, André M; Nieves, Edward et al. (2013) Quorum sensing-mediated, cell density-dependent regulation of growth and virulence in Cryptococcus neoformans. MBio 5:e00986-13
Raja, Meera R; Waterman, Scott R; Qiu, Jin et al. (2013) A copper hyperaccumulation phenotype correlates with pathogenesis in Cryptococcus neoformans. Metallomics 5:363-71
Qiu, Jin; Olszewski, Michal A; Williamson, Peter R (2013) Cryptococcus neoformans growth and protection from innate immunity are dependent on expression of a virulence-associated DEAD-box protein, Vad1. Infect Immun 81:777-88
Waterman, Scott R; Park, Yoon-Dong; Raja, Meera et al. (2012) Role of CTR4 in the Virulence of Cryptococcus neoformans. MBio 3:
Adler, Amos; Park, Yoon-Dong; Larsen, Peter et al. (2011) A novel specificity protein 1 (SP1)-like gene regulating protein kinase C-1 (Pkc1)-dependent cell wall integrity and virulence factors in Cryptococcus neoformans. J Biol Chem 286:20977-90
Park, Yoon-Dong; Panepinto, John; Shin, Soowan et al. (2010) Mating pheromone in Cryptococcus neoformans is regulated by a transcriptional/degradative ""futile"" cycle. J Biol Chem 285:34746-56
Casadevall, Arturo; Nosanchuk, Joshua D; Williamson, Peter et al. (2009) Vesicular transport across the fungal cell wall. Trends Microbiol 17:158-62
Hu, Guowu; Hacham, Moshe; Waterman, Scott R et al. (2008) PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans. J Clin Invest 118:1186-97

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