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

Abstract

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.

Capping motifs in antimicrobial peptides and their relevance for improved biological activities

“…Nevertheless, at higher ratios (1:1 or higher), Zn 2+ ions seem to impede the antifungal activity of Hst-5 against C. albicans ( Campbell et al, 2022 ). According to Stewart et al (2023) , both higher and lower proportions of Zn 2+ in the Zn 2+ -Hst-5 complex do not exert an effect on improving or suppressing the effect on multiple species of Streptococcus , which normally colonize the oral cavity. Additionally, Hst-5 does not cause Zn 2+ starvation in this genus, as it does not compete for Zn 2+ binding with the Zn 2+ uptake protein AdcAI ( Stewart et al, 2023 ).…”

“…According to Stewart et al (2023) , both higher and lower proportions of Zn 2+ in the Zn 2+ -Hst-5 complex do not exert an effect on improving or suppressing the effect on multiple species of Streptococcus , which normally colonize the oral cavity. Additionally, Hst-5 does not cause Zn 2+ starvation in this genus, as it does not compete for Zn 2+ binding with the Zn 2+ uptake protein AdcAI ( Stewart et al, 2023 ). The above study, along with others, suggests that Hst-5 may play a role in the development of certain species of oral cavity microorganisms, exerting selective antimicrobial activity and maintaining microbial communities regulated through Zn 2+ ion concentrations ( Norris et al, 2021 ; Campbell et al, 2022 ; Stewart et al, 2023 ).…”

“…Additionally, Hst-5 does not cause Zn 2+ starvation in this genus, as it does not compete for Zn 2+ binding with the Zn 2+ uptake protein AdcAI ( Stewart et al, 2023 ). The above study, along with others, suggests that Hst-5 may play a role in the development of certain species of oral cavity microorganisms, exerting selective antimicrobial activity and maintaining microbial communities regulated through Zn 2+ ion concentrations ( Norris et al, 2021 ; Campbell et al, 2022 ; Stewart et al, 2023 ). With all this evidence, Hst-5 cannot be classified as belonging to class II; instead, it appears to be able to act in class I or III.…”