Cryptococcus neoformans is a major pathogen in immunocompetant as well as immunocompromised patients including those with AIDS in both the developed as well as the developing world. Our long-term objective is to test the hypothesis that molecular regulators of the virulence factor laccase affects the virulence of Cryptococcus neoformans. The specific hypothesis behind the present proposal is that a virulence associated DEAD-box protein, Vad1, identified by insertional mutagenesis, is an important regulator of laccase and virulence in C. neoformans. This is based on the following observations. First, deletion of VAD1results in loss of virulence and accelerated clearance of C. neoformans from lung in mouse models. Second, differential display has shown that deletion of VAD1results in altered transcription of a number of genes in addition to laccase. Finally, deletion of one of the genes showing VAD1-dependent transcription, PCK1, exhibited attenuated virulence in a mouse model in spite of retained laccase activity.
In Specific Aim 1, we will assess for functional conservation of VAD1within the RCK/p54 protein subfamily of DEXD/H box proteins to which Vad1 exhibits significant homology. We plan to test this by overexpression of VAD1in an RCK/p54 mutant of yeast, testing the role of VAD1in haploid fruiting and mating, comparing the cellular localization of the Vad1 protein to members of the RCK/p54 subfamily and performing gel filtration of cellular extracts of cells expressing affinity-tagged Vad1 constructs to identify and characterize possible Vad1 multi-protein complexes.
In Specific Aim 2, we will further define the role of VAD1in laccase transcription. This will test our hypothesis that VAD1is required for optimal expression of laccase. We will analyze VAD1-mediated degradation of the transcriptional represser, NOT1, and the role of NOT1 in suppression of laccase activity.
In Specific Aim 3, we will analyze the host responses between wild-type and Avadl infections using an intratracheal model. Specifically, we will measure fungal cfu, leukocyte recruitment, cytokine production, antibody production and DTH response after infection with each strains.
In Specific Aim 4, we will assess the role of 5 M4D7-dependent genes in cryptococcal virulence using an intratracheal and an intravenous mouse model. Completion of these specific aims will provide an integrated approach to understanding the role of VAD1in the pathobiology of C. neoformans and may provide additional drug development targetsfor the treatment and prevention of cryptococcosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
3R01AI045995-08S1
Application #
7374111
Study Section
Special Emphasis Panel (ZRG1-IDM-E (02))
Program Officer
Duncan, Rory A
Project Start
1999-07-01
Project End
2010-02-28
Budget Start
2007-04-15
Budget End
2008-02-29
Support Year
8
Fiscal Year
2007
Total Cost
$102,235
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:
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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
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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
Chayakulkeeree, Methee; Johnston, Simon Andrew; Oei, Johanes Bijosono et al. (2011) SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans. Mol Microbiol 80:1088-101
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
Panepinto, John C; Misener, Amanda L; Oliver, Brian G et al. (2010) Overexpression of TUF1 restores respiratory growth and fluconazole sensitivity to a Cryptococcus neoformans vad1Delta mutant. Microbiology 156:2558-65

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