AIDS Opportunistic Pathogens/Fungal Cell Wall Drug Targets

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DESCRIPTION (provided by applicant): Oral candidiasis is a hallmark of HIV infection. A third of HIV sero-positive and 90% of AIDS patients will present with oropharyngeal candidiasis. Few antifungal agents are available for treatment and to compound the problem, some species of Candida, notably Candida glabrata, are innately resistant to the important triazole class of antifungals. This grant proposes to identify potential targets in yeast cell wall biogenesis for development of novel antifungal agents. The Candida cell wall is a proven drug target: the echinocandin class of drugs (of which Merck's Cancidas is a member) target beta1,3 glucan synthase and can be successfully used against multiple species of Candida including Candida albicans and C. glabrata. The other major glucan in the cell wall is beta1-6 glucan. Less is understood at a biochemical level about beta1-6 biosynthesis, although it is normally essential for the viability of the cell. One of beta1-6 glucan's essential functions is to covalently crosslink proteins into the cell wall: the vast majority of cell wall proteins in S. cerevisiae and in C. albicans and C. glabrata are so called GPI-cell wall proteins (or GPI-CWPs) and are covalently attached through the remnant of the GPI anchor to beta1-6 glucan. The process by which GPI-proteins are attached to the cell wall is not understood in detail at all. We have established reagents for analyzing GPI-protein crosslinking to the cell wall and propose using these to identify and analyze mutants of S. cerevisiae in which GPI-CWP processing and crosslinking to beta1-6 glucan is compromised. Specifically, we are generating a series of temperature sensitive alleles of genes known or predicted to be involved in GPI-CWP biogenesis. We will use these temperature sensitive mutant strains to analyze intermediates in the GPI-CWP crosslinking pathway to more precisely define the role of these genes in the maturation and crosslinking of GPI-CWPs. The yeast cell wall is essential for yeast viability, and GPI-CWP function is essential to cell wall integrity. Identification of the enzymes responsible for GPI-CWP crosslinking will allow chemotherapeutic targeting of a fungal specific, essential and proven target for development of new antifungal therapy.