Zinc Binding Inhibits Cellular Uptake and Antifungal Activity of Histatin-5 in

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro and metals have been shown to alter the fungicidal activity of the peptide. Previous reports on the effect of Zn(2+) on Hist-5 activity have been varied and seemingly contradictory. Here we present data elucidating the dynamic role Zn(2+) plays as an inhibitory switch to regulate Hist-5 fungicidal activity. A novel fluorescently labeled Hist-5 peptide (Hist-5*) was developed to visualize changes in internalization and localization of the peptide as a function of metal availability in the growth medium. Hist-5* was verified for use as a model peptide and retained antifungal activity and mode of action similar to native Hist-5. Cellular growth assays showed that Zn(2+) had a concentration-dependent inhibitory effect on Hist-5 antifungal activity. Imaging by confocal microscopy revealed that equimolar concentrations of Zn(2+) kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. However, the Zn-induced decrease in Hist-5 activity and uptake was rescued by decreasing Zn(2+) availability upon addition of a metal chelator EDTA or S100A12, a Zn-binding protein involved in the innate immune response. These results lead us to suggest a model wherein commensal C. albicans may exist in harmony with Hist-5 at concentrations of Zn(2+) that inhibit peptide internalization and antifungal activity. Activation of host immune processes that initiate Zn-sequestering mechanisms of nutritional immunity could trigger Hist-5 internalization and cell killing.