CANDIDACIDAL MECHANISMS OF SALIVARY HISTATINS

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Salivary histatins are a family of at least 12 polypeptide (7-38 amino acids) which have been shown to be effective candidacidal molecules as well as being active inhibitors of yeast cell growth and germination. Sequence and conformational data is available for the members of the histatin family and the minimal peptide portion of the molecule required for complete candidacidal activity has been identified. However, unlike most other antimycotic drugs where the mechanism of action is well characterized, very little is known about the molecular basis of histatins' antifungal activity. A more complete understanding of the molecular mechanism of action of these salivary molecules is required in order to design methods of delivery of histatins to selected oral sites (linkage to solid surfaces or associated with the oral mucosa), as well as designing synthetic peptides with enhanced function. Like the imidazole antimycotic, histatins appear to affect both candidal cell membrane function as well as ergosterol (a key fungal sterol) biosynthesis. Histidine groups in the highly conserved candidacidal domains may provide docking or intracellular translocation functions. The validity of assignment of these functional domains and the specific mechanisms of action of histatins will examined using the following approaches. Four model molecules consisting if the candidacidal domain (A16) (GYKRKFHEKHHSHRGY) alone, A16 and N-terminal domains (histatin 5), A16 and C-terminal domains (histatin 4), and A16 and both C- and N- terminal domains (histatin 3) will be compared in their abilities to affect cell membrane integrity, ergosterol biosynthesis and inhibition of adhesion. In addition, a model peptide having amino acid substitutions at the active site and a constrained helical peptide will be tested for candidacidal activity. Specifically, membrane effects will be examined by comparing the ability of these model peptides to release intracellular dye from Candida albicans whole cells and spheroplast (cell wall free); and in vitro by analysis of liposome-peptide interactions. In vitro model membrane interactions will be tested with three complementary assays: a) histatin induced liposome disruption as measured by release of encapsulated dye; b) measurement of change in fluidity of liposomes using differential scanning calorimetry (DSC); and c) liposome induced conformational change of histatin peptides measured by circular dichroism (CD). Inhibition of ergosterol of radiolabeled precursors into ergosterol following treatment with the four model histatin and amino acid substituted peptides. And finally, inhibition of adhesion of C. albicans to saliva coated poly (methyl methacrylate) (PMMA) by pretreating cells with sublethal doses of histatins will be assessed.