The cell wall of the fungal pathogen C. neoformans is an essential organelle that incorporates chitin as a rigid, insoluble, polysaccharide polymer. De-acetylation converts chitin to chitosan, a more flexible and soluble polymer. While chitin is an essential component of fungal cell walls, the importance of chitosan to cell wall integrity is unclear. The overall goal of this project is to identify key steps in Cryptococcus cell wall biosynthesis that will make attractive targets for antifungal drugs. Our preliminary data indicate that chitosan is an abundant component of Cryptococcus cell walls, both during vegetative growth and during infection of mouse lungs. We have explored genes that contribute to chitosan production, and shown that only one of eight putative chitin synthases, and one of three putative chitin synthase regulators substantially impact chitosan levels. In addition, we have demonstrated that three of four potential polysaccharide deacetylases produce chitosan. The shared phenotypes among strains deleted of genes in the chitosan biosynthetic pathway, include defects in cell integrity and leaky melanin, and suggest that these phenotypes are the result of substantial reduction of chitosan. Data obtained from this project will establish the role of chitosan in C. neoformans cell wall integrity and virulence, how chitosan is attached to the cell wall, and identify critical steps in this aspect of cell wall biosynthesis that could serve as antifungal targets. This project has three specific aims. In the first, we will determine the composition of chitosan and how it is linked to the cell wall during growth in the mammalian host. In the second, we will test the roles of the three chitin deacetylases in chitosan biosynthesis, cell wall integrity and virulence. In the third aim, we will test our current model that one chitin synthase and its regulator together synthesize the chitin that is deacetylated by the three chitin deacetylases to generate chitosan and determine the interactions among the these proteins that are critical for chitosan production. The results of these studies should provide a better understanding of chitosan biosynthesis and function in C. neoformans, and determine if chitosan production would be a viable antifungal target for novel anti-cryptococcal therapy. Fungal infections have become more prevalent in recent years due to the increase in the immunocompromised patient population from AIDS, organ transplants and chemotherapies. Systemic fungal infections are serious health threats, and safe, highly effective antifungal therapies are not available. Biosynthesis of the fungal cell wall is an attractive target for antifungal therapies because the cell wall is an essential organelle that is not present in the human host, and this project will delineate the biosynthesis of the cell wall of a fungal pathogen, Cryptococcus neoformans, and determine if one of its components, chitosan, is a potential antifungal target.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI072195-04
Application #
7739458
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Duncan, Rory A
Project Start
2007-12-15
Project End
2011-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
4
Fiscal Year
2010
Total Cost
$343,778
Indirect Cost
Name
Washington University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Upadhya, Rajendra; Lam, Woei C; Maybruck, Brian T et al. (2017) A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome. Fungal Genet Biol 108:13-25
Upadhya, Rajendra; Lam, Woei C; Maybruck, Brian et al. (2016) Induction of Protective Immunity to Cryptococcal Infection in Mice by a Heat-Killed, Chitosan-Deficient Strain of Cryptococcus neoformans. MBio 7:
Loures, Flávio V; Röhm, Marc; Lee, Chrono K et al. (2015) Recognition of Aspergillus fumigatus hyphae by human plasmacytoid dendritic cells is mediated by dectin-2 and results in formation of extracellular traps. PLoS Pathog 11:e1004643
Loures, Flávio V; Levitz, Stuart M (2015) XTT Assay of Antifungal Activity. Bio Protoc 5:
Wiesner, Darin L; Specht, Charles A; Lee, Chrono K et al. (2015) Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection. PLoS Pathog 11:e1004701
Specht, Charles A; Lee, Chrono K; Huang, Haibin et al. (2015) Protection against Experimental Cryptococcosis following Vaccination with Glucan Particles Containing Cryptococcus Alkaline Extracts. MBio 6:e01905-15
Bueter, Chelsea L; Lee, Chrono K; Wang, Jennifer P et al. (2014) Spectrum and mechanisms of inflammasome activation by chitosan. J Immunol 192:5943-51
Bueter, Chelsea L; Specht, Charles A; Levitz, Stuart M (2013) Innate sensing of chitin and chitosan. PLoS Pathog 9:e1003080
Lam, Woei C; Gerik, Kimberly J; Lodge, Jennifer K (2013) Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 signaling pathway in response to thermal stress. Eukaryot Cell 12:118-31
Baker, Lorina G; Lodge, Jennifer K (2012) Galactose-Inducible promoters in Cryptococcus neoformans var. grubii. Methods Mol Biol 845:211-26

Showing the most recent 10 out of 17 publications