Salivary histatin 5 (Hst 5), a potent toxin for the human fungal pathogen Candida albicans, induces noncytolytic efflux of cellular ATP, potassium, and magnesium in the absence of cytolysis, implicating these ion movements in the toxin's fungicidal activity. Hst 5 action on Candida resembles, in many respects, the action of the K1 killer toxin on Saccharomyces cerevisiae, and in that system the yeast plasma membrane potassium channel, Tok1p, has recently been reported to be a primary target of toxin action. The question of whether the Candida homologue of Saccharomyces Tok1p might be a primary target of Hst 5 action has now been investigated by disruption of the C. albicans TOK1 gene. The resultant strains (TOK1/tok1) and (tok1/tok1) were compared with wild-type Candida (TOK1/TOK1) for relative ATP leakage and killing in response to Hst 5. Patch-clamp measurements on Candida protoplasts were used to verify the functional deletion of Tok1p and to provide its first description in Candida. Tok1p is an outwardly rectifying, noisily gated, 40-pS channel, very similar to that described in Saccharomyces. Knockout of CaTOK1 (tok1/tok1) completely abolishes the currents and gating events characteristic of Tok1p. Also, knockout (tok1/tok1) increases residual viability of Candida after Hst 5 treatment to 27%, from 7% in the wild type, while the single allele deletion (TOK1/tok1) increases viability to 18%. Comparable results were obtained for Hst-induced ATP efflux, but quantitative features of ATP loss suggest that wild-type TOK1 genes function cooperatively. Overall, very substantial killing and ATP efflux are produced by Hst 5 treatment after complete knockout of wild-type TOK1, making clear that Tok1p channels are not the primary site of Hst 5 action, even though they do play a modulating role.Antimicrobial defense mechanisms in the oral cavity are provided by innate nonimmune proteins and peptides of salivary gland origin, which include lactoperoxidase, lysozyme, lactoferrin, cathelicidins, and histatins (Hst's). These agents have overlapping antimicrobial activities and are key initial host defense molecules in limiting oral infections (15,26). The Hst's form a family of small, histidine-rich, cationic peptides secreted by human parotid and submandibular glands (27). Of this family, Hst 5 has the highest antifungal activity for Candida albicans and related yeasts (38). Oral candidiasis (thrush) is a common mucosal infection caused by C. albicans in patients undergoing chemotherapy or organ transplantation and in persons with immunodeficiency virus infections, underlying systemic disease states, or medications which reduce salivary flow.The spectrum of innate eukaryotic antifungal molecules includes a large array of peptides and proteins, varying widely in both structure and molecular mechanism of action. Many of these agents, but not all, directly target the plasma membrane and/or organellar membranes for disruption; others kill fungi via multitarget, multistep pathways. Chromofungin is an antifungal peptide derived ...
