title: Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5.
abstract: 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 beta-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 (Kd = 2.57 x 10(-6) m) with the wild-type SSA1/SSA2 strain; however, Hst 5 binding with the Deltassa1ssa2 double mutant was reduced (Kd = 1.25 x 10(-6) m). Cell wall HSP70 proteins were also diminished, but still detectable, in S. cerevisiae Deltassa1ssa2 cells and are likely to be Ssa3p or Ssa4p. Hst 5 (31 microm) killed 80% of the wild-type cells in fungicidal assays at room temperature. However, only 50-60% killing of the single mutants (Deltassa1 and Deltassa2) was observed, and fungicidal activity was further reduced to 20-30% in the Deltassa1ssa2 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 Deltassa1ssa2 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.
authors.affiliation: Department of Oral Biology and Restorative Dentistry, School of Dental Medicine, State University of New York, Buffalo, New York 14214, USA.
authors.forename: Xuewei S
authors.initials: XS
authors.lastname: Li
authors.forename: Molakala S
authors.initials: MS
authors.lastname: Reddy
authors.forename: Didi
authors.initials: D
authors.lastname: Baev
authors.forename: Mira
authors.initials: M
authors.lastname: Edgerton
mesh_terms: D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D002176:Candida albicans / Q000737:chemistry*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054808:Histatins; D006358:Hot Temperature; D006801:Humans; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D000935:Antifungal Agents; D002352:Carrier Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C068075:HTN3 protein, human; D054808:Histatins; D010446:Peptide Fragments; C542288:SSA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012471:Salivary Proteins and Peptides; D008623:Mercaptoethanol; D014357:Trypsin; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae