Insights into the Role of Copper and Zinc on the Uptake and Antifungal Activity of the Salivary Peptide Histatin-5

Abstract

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. The goal of this work is to gain insight into the role of metals in the biological activity Hist-5. Toward this goal, we developed and characterized a novel fluorescently labeled Hist-5 peptide (Hist-5*) to visualize changes in internalization and localization of the peptide in fungal cells as a function of metal availability in the growth medium. Here, we provide evidence for Zn-modulated antifungal activity of Hist-5 in which the availability of Zn2+ in the surrounding environment inhibits Hist-5 cellular uptake and cidality. Cellular growth assays revealed a concentration-dependent inhibitory effect of Zn2+ on Hist-5 antifungal activity. Imaging by confocal microscopy showed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. We found that modulation of extracellular Zn2+ concentration by metal chelating molecules or proteins reversed Zn-induced surface adhesion of Hist-5, leading us to propose a dynamic role for Zn2+ as an inhibitory switch to regulate Hist-5 fungicidal activity. We next present data to support the hypothesis that Hist-5 interacts with intracellular Cu to increase the fungicidal activity Hist-5. Combined fluorescence spectroscopy and microscopy experiments showed reversible Cu-dependent quenching of Hist-5* fluorescence, indicating a direct interaction between Hist-5 and intracellular Cu. X-ray fluorescence microscopy images revealed peptide-induced changes to cellular Cu distribution and cell-associated Cu content. Finally, we present progress towards expanding the scope in which we understand and assess Hist-5 biological activity by investigating the activity of the peptide under biologically relevant conditions and testing its fungicidal activity against other fungal species.