Identification of translocation mechanisms of Histatin 5 into Candida albicans
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Histatin 5 (Hst 5) is Salivary gland secreted cationic peptide which has a potent anti-fungal activity against Candida albicans at physiological concentrations. Its fungicidal mechanisms are initiated by cell surface docking to Ssa proteins, followed by intracellular transport to cytoplasmic effectors leading to the cell death. The translocation mechanism is speed limiting to the whole process and understanding the detail mechanism will benefit future drug design. Pull-down assays with purified recombinant Ssa1 or Ssa2 proteins suggested Ssa2p has much higher binding affinity than Ssa1p to Hst 5. To better understand the specific interaction between Ssa2p and Hst 5, Hst 5 binding sites on Ssa2p were identified. Hst 5 interactions with truncated Ssa2 proteins suggested ATPase is the essential domain for interaction. Limited digestion and peptide array further mapped the interaction epitopes are located in the Ssa2 126-156 and Ssa2 328-354 areas. Mutated Ssa2 proteins within these regions showed defect in Hst 5 functions, further proved the importance of these sites in Hst 5 interaction. Nucleotide can enhance Hst 5-Ssa2p interaction through FPLC assay. A polyamine transporter family member AGP2 has a potential to transport longer substrate (e.g. spermidine) which suggest a potential candidate for Hst 5 uptake on the cell membrane. agp2 null mutant was constructed through sequential deletion of both alleles of AGP2 genes. At lower Hst 5 concentration, agp2 null mutant was significantly more resistant to Hst 5 killing which suggests this transporter plays an important role in translocation of Hst 5 into the cytoplasm. Identification of Ssa2p interaction sites for Hst 5 as well as cell subsequent membrane transporter AGP2 provide valuable information for designing of therapeutic agents against oral candidasis.