Chitin synthesis is an attractive target for antifungal drugs because chitin is necessary for fungal growth and viability and because the polymer is not present in host cells. Fungal chitin synthesis is complex and requires multiple chitin synthase isozymes each of which is necessary for chitin synthesis at a specific cellular location or in a particular cell type during the life cycle. Specific chitin synthase isozymes hat are necessary for vegetative (hyphal) growth are potential targets for novel drugs. Thus the precise developmental function of each chitin synthase is the key to determining which isozymes are useful drug targets. Five chitin synthase genes from Aspergillus nidulans have been identified and disrupted. At least two isozymes, chsB and chsD, are promising drug targets because inactivation of the enzymes by gene disruption leads to extremely slow hyphal growth or to cell lysis. The chsB isozyme synthesizes a chitin subfraction that is necessary for normal hyphal organization and morphology but not for osmotic integrity. The chsD isozyme synthesizes a chitin subfraction that is necessary for osmotic integrity but not for normal hyphal morphology. The N-terminus of the chsD isozyme is homologous to myosin. The data obtained from the proposed studies will contribute to the understanding of the growth morphogenesis and development of filamentous fungi and to the assessment of specific chitin synthases as drug targets.
Three specific aims are outlined.
Specific Aim 1 is to complete the sequencing and disruption of the five known chitin synthase genes. We will attempt to identify additional chitin synthases genes. Considerable progress completion toward this goal has already been made and is presented.
Specific Aim 2 involves the characterization of the phenotypes of the chitin synthase disruptant strains in order to assess the developmental role for each enzyme.
Specific Aim 3 is to assess the functions of the N-terminus of the chsD isozyme with special emphasis on the ability of N-terminal sequences to bind to cytoskeletal proteins.
In Specific Aim 4, the enzymology of the chsB and chsD enzymes will be examined and aspects of the temporal and spatial regulation of these two enzymes will be examined. The transcriptional levels of chitin synthase genes will be determined throughout the sexual and asexual life cycles. The chsB and chsD genes will be modified to encode epitope tagged enzymes that retain the ability to complement the corresponding chitin synthase null strain. The enzymes will be purified and their cellular localization will be determined using monoclonal antibody to the epitope. The processing of chsB and chsD enzymes will examined both in vivo and in vitro.
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