Yeast heat shock protein 70 Ssa family is required for docking and import of antifungal peptide histatin 5
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Salivary Histatins are a family of small histidine-rich peptides with potent antifungal activity against Candida species and other pathogenic yeasts. Their fungicidal mechanisms are initiated by cell surface docking to specific binding proteins, followed by intracellular transport to cytoplasmic effectors leading to cell death. In this project, we identified the yeast heat shock 70 proteins (Ssa1 and Ssa2) as Hst 5-binding proteins. They were co-localized on immunoblots of cell wall and cytoplasmic fractions. Yeast two-hybrid analysis confirmed the specific interactions between Ssa proteins and Hsts. To further assess the functional roles in vivo , the binding and fungicidal assays of Hst 5 were carried out in both S. cerevisiae and C. albicans ssa1 Δ/ssa2Δ mutants. Our results showed that binding and killing activities of Hst 5 were significantly decreased in S. cerevisiae ssa1ssa2Δ double mutant. In C. albicans strains, the Ssa2 protein plays a more critical function than Ssa1 protein in Hst 5 docking and import. The ssa2Δ mutant was highly resistant (23% sensitivity) to the candidacidal activity of Hst 5, which was correlated with a significant reduction in binding of 125 I-Hst 5. By contrast, the ssa1Δ mutant was indistinguishable from the wild-type strain. In addition, transferring of cells from room temperature to heat shock at 37°C increased the sensitivity to Hst 5 in both wild type and mutants. Our findings of Hsp70 Ssa proteins as the yeast cell wall binding and translocation components demonstrate a new function for this family. Moreover, identification of the SSA family as the specific cell surface target by Hst 5 provides valuable information for designing better therapeutic agents against oral candidiasis.