Salivary Antimicrobial Peptide Histatin-5 Does Not Display Zn(II)-Dependent or -Independent Activity against Streptococci

[Image: see text] Histatin-5 (Hst5) is a member of the histatin superfamily of cationic, His-rich, Zn(II)-binding peptides in human saliva. Hst5 displays antimicrobial activity against fungal and bacterial pathogens, often in a Zn(II)-dependent manner. In contrast, here we showed that under in vitro conditions that are characteristic of human saliva, Hst5 does not kill seven streptococcal species that normally colonize the human oral cavity and oropharynx. We further showed that Zn(II) does not influence this outcome. We then hypothesized that Hst5 exerts more subtle effects on streptococci by modulating Zn(II) availability. We initially proposed that Hst5 contributes to nutritional immunity by limiting nutrient Zn(II) availability and promoting bacterial Zn(II) starvation. By examining the interactions between Hst5 and Streptococcus pyogenes as a model Streptococcus species, we showed that Hst5 does not influence the expression of Zn(II) uptake genes. In addition, Hst5 did not suppress growth of a ΔadcAI mutant strain that is impaired in Zn(II) uptake. These observations establish that Hst5 does not promote Zn(II) starvation. Biochemical examination of purified peptides further confirmed that Hst5 binds Zn(II) with high micromolar affinities and does not compete with the AdcAI high-affinity Zn(II) uptake protein for binding nutrient Zn(II). Instead, we showed that Hst5 weakly limits the availability of excess Zn(II) and suppresses Zn(II) toxicity to a ΔczcD mutant strain that is impaired in Zn(II) efflux. Altogether, our findings led us to reconsider the function of Hst5 as a salivary antimicrobial agent and the role of Zn(II) in Hst5 function.