The bacterial cell wall is a traditional target for anti-infective drugs, but only recently have analogous antifungal drugs (echinocandins) been developed. While promising, echinocandins have limited applicability for fungi that do not rely heavily on beta-(1,3)-glucan for cell integrity. A similar but completely unexplored target is alpha-(1,3)-glucan, a structural feature of the cell wall of all fungal pathogens other than Candida spp. Since mutants lacking this polysaccharide are avirulent but viable, forward genetics becomes a means to identify candidate drug targets, and it is also far easier to study the role and regulation of the discovered genes. In this research plan, we will begin to test the hypothesis that genes and gene products involved in the synthesis, processing, and regulation of alpha-(1,3)-glucan can serve as selective targets for antifungal agents. As our model system, we will use H. capsulatum, in which the role of alpha-(1,3)-glucan in virulence and biology has been most thoroughly studied.
Our specific aims are to: I. Identify genes involved in the production of alpha-(1,3)-glucan. H. capsulatum mutants, generated by Agrobacterium-mediated mutagenesis, will be screened for deficiency of alpha-(1,3)-glucan or for loss of regulation of AGS1 (which encodes alpha-(1,3)-glucan synthase). DNA flanking the insertions in all of these mutants will be recovered and sequenced, and the interrupted genes will be identified from the sequenced Histoplasma genome. Genes selected for further study will be those that are conserved among a wide variety of other fungi. II. Validate and characterize genes implicated in production of alpha-(1,3)-glucan. For each of the mutants of interest, we will validate that the mutation is responsible for the phenotype by either (i) complementing the mutant with the wild-type gene the mutation; or (ii) using RNAi to silence that gene in a wild-type strain. The mutants will be evaluated for relative virulence in cultured macrophages and in a mouse model of pulmonary infection. Genes with the highest potential as drug targets will be selected for more detailed analysis of gene function and expression, using defined mutants and reporter gene constructs.
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