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J. Biol. Chem., Vol. 278, Issue 31, 28553-28561, August 1, 2003

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Candida albicans Ssa1/2p Is the Cell Envelope Binding Protein for Human Salivary Histatin 5^*

Xuewei S. Li , Molakala S. Reddy , Didi Baev  and Mira Edgerton   ¶

From the Departments of Oral Biology and Restorative Dentistry, School of Dental Medicine, State University of New York, Buffalo, New York 14214

Salivary histatins are a family of small histidine-rich peptides with potent antifungal activity. We previously identified a 70-kDa cell envelope protein in Candida albicans and Saccharomyces cerevisiae that mediates binding of histatin (Hst) 5. Isolation of Hst 5-binding protein followed by matrix-assisted laser desorption ionization mass spectrometry analysis identified this protein as the heat shock protein Ssa1p. Ssa protein and Hst 5-binding protein were found to be co-localized on immunoblots of yeast -mercaptoethanol cell wall extracts and cytosolic fractions. Yeast two-hybrid analysis showed strong interactions between Ssa1p and both Hst 3 and Hst 5. To assess functional roles of Ssa proteins in the Hst 5 antifungal mechanism in vivo, both binding and fungicidal assays were carried out using S. cerevisiae isogenic SSA1/SSA2 mutants. ¹²⁵I-Hst 5 binding assays showed saturable binding (K_d = 2.57 x 10^–6 M) with the wild-type SSA1/SSA2 strain; however, Hst 5 binding with the ssa1ssa2 double mutant was reduced (K_d = 1.25 x 10^–6 M). Cell wall HSP70 proteins were also diminished, but still detectable, in S. cerevisiae ssa1ssa2 cells and are likely to be Ssa3p or Ssa4p. Hst 5 (31 µM) killed 80% of the wild-type cells in fungicidal assays at room temperature. However, only 50–60% killing of the single mutants (ssa1 and ssa2) was observed, and fungicidal activity was further reduced to 20–30% in the ssa1ssa2 double mutant. Incubation of cells under heat shock conditions increased the sensitivity of cells to Hst 5, which correlated with increased Hst 5-binding activity in ssa1ssa2 cells, but not in wild-type cells. This study provides evidence for a novel function for yeast Ssa1/2 proteins as cell envelope binding receptors for Hst 5 that mediate fungicidal activity.

Received for publication, January 21, 2003 , and in revised form, May 19, 2003.

^* This work was supported by United States Public Health Service Grants DE10641 and DE00406 from NIDCR, National Institutes of Health (to M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: SUNY at Buffalo Main Street Campus, 310 Foster Hall, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-3067; Fax: 716-829-3942; E-mail: edgerto{at}buffalo.edu.

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