Cryptococcus neoformans is a pathogenic fungus that is found world-wide and causes meningioencephalitis, particularly in immunocompromised individuals. It is invariably fatal unless treated, and the current antifungals are inadequate to effectively cure this disease, due to inherent toxicities or the inability to kill the fungus and prevent relapse. Recent studies have indicated that there are over 1,000,000 new cases of cryptococcosis in the world each year, which results in over 600,000 deaths. New agents to treat Cryptococcus are needed, and the fungal cell wall is an attractive target, since it is unique to fungi and absent in humans. We have shown that chitosan, the deacetylated form of chitin, is a critical component of the Cryptococcal cell wall and is absolutely required for virulence using a mouse model of cryptococcosis. It is also necessary for persistence in the mouse - strains lacking chitosan were cleared within 48 hrs, much more rapidly than other major virulence factors. Our studies have further identified the key enzymes required for chitosan production in this fungus. Because Cryptococcus is a manipulable organism, with a robust genetic system and solid animal models, it is an excellent model system to study chitosan biosynthesis and the role of chitosan in disease progression. In this renewal application, we propose to extend our studies on chitosan biosynthesis and chitosan's role in pathogenesis, with the long-term goal of delineating the necessary components and their interactions for chitosan biosynthesis. In three specific aims, we will address the following questions:
Aim 1. How is specificity achieved to deacetylate the chitin produced by the single chitin synthase (out of eight) and the single chitin synthase regulator (out of three)? Aim 2. Which proteins are necessary for chitosan production, and how do they interact? We have already identified some of the proteins, and will test whether any of them are limiting, and whether they are co-localized and form complexes. We will also explore whether there are additional proteins that are necessary for chitosan production.
Aim 3. Why are chitosan deficient mutants cleared so quickly? Is the host response to the chitosan deficient mutants different than the host response to wild type Cryptococcus and/or are the chitosan deficient cells more susceptible to killing by the host?
Fungal infections have become more prevalent in recent years due to the increase in the immune-compromised patient populations from AIDS, organ transplants and chemotherapies. Cryptococcus neoformans is a pathogenic fungus which kills over 600,000 people per year, mostly in underdeveloped countries and current therapies are inadequate because they are not effective enough or have significant toxicity. 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. Therefore, understanding the components of the wall, the basic biosynthetic mechanisms responsible for making these components, and how the host responds to the wall components is important. Chitosan is a polysaccharide found in cryptococcus that is essential for growth of the fungus in the mammalian host. This project will help us understand 1) the mechanisms of chitin and chitosan biosynthesis, which will contribute to drug development efforts, and 2) the role of chitosan in cryptococcal disease development.
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