FUNGAL CELL WALL ENZYMOLOGY--(1-3)-B-GLUCAN SYNTHASE

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Fungi are medically and economically very important, contributing to waste, spoilage and disease. Treatment of human fungal disease, especially in immunodeficient patients, for example those suffering from Acquired Immunodeficiency Syndrome, is hampered by very few clinically useful drugs. Filamentous fungal morphogenesis and growth are the direct result of polysaccharide polymers and other materials being sequentially deposited at each hyphal apex leading to the formation of a mature cell wall. Cell-wall assembly results in a wall containing chitin, beta(1-3) glucan, and other beta- and alpha- linked glucans, proteins and lipids. A key step, perhaps the key step, in fungal cell wall assembly is the synthesis of beta(1-3) glucan and its subsequent incorporation into the cell wall. This proposal has as its aim, the detailed understanding of (1-3) - beta-D-glucan synthase, the enzyme responsible for glucan synthesis. 1.The development of in vitro conditions for the assay of (1-3)-beta-glucan synthase activity of a number of human pathogenic fungi, e.g., Histoplasma capsulatum, Cryptococcus neoformans, Aspergillus nidulans, A. fumigatus, Trichophyton mentagrophytes, Sporothrix schneckii, and Blastomyces dermatitidis. 2.Detailed comparison of (1-3)-beta-glucan synthase activities from the above pathogenic fungi and those of Neurospora crassa and Candida albicans. This will include the determination of various kinetic parameters (substrate, ions, pH optimum, various inhibitors and activators) and detailed product characterization. The GTP-binding regulatory protein will be purified and characterized. 3.Isolation, molecular cloning, and characterization of the gene(s) coding from (1-3)-beta-glucan synthase activity of Neurospora crassa.