Defining the Role of the Streptococcus agalactiae Sht-Family Proteins in Zinc Acquisition and Complement Evasion

Abstract

Streptococcus agalactiae is not only part of the human intestinal and urogenital microbiota but is also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter and the three zinc-binding proteins, Lmb, AdcA, and AdcAII, S. agalactiae zinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess fivehistidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding the in vivo role of the Sht proteins in resisting complement᎑mediated clearance. IMPORTANCE This study examined the role of the two streptococcal histidine triad (Sht) proteins of Streptococcus agalactiae in zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor H in vitro this did not influence survival in human blood.

Identification of zinc-dependent mechanisms used by Group BStreptococcusto overcome calprotectin-mediated stress

“…Fig 1A). To confirm what has been previously described for GBS in zinc-limited chemically defined media (38,39), we observed that mutants lacking individual zinc-binding proteins exhibited similar calprotectin sensitivity as the WT GBS strain, while growth of a triple ΔadcAΔadcAIIΔlmb mutant was reduced (Supplemental Fig 1B). This is consistent with previous results that suggest functional redundancy exists between zinc-binding proteins.…”

“…The GBS zinc uptake machinery is encoded by four distinct operons and is under the regulation of the zinc-dependent AdcR repressor (38). AdcR is involved in maintaining intracellular zinc homeostasis, and has been shown to be important for growth in zinc-limiting conditions (38,39). This system is also significantly upregulated during GBS murine vaginal colonization and following incubation with human blood (71,72).…”

“…While there are a myriad of strategies employed to obtain zinc, the AdcABC/ZnuABC family of ATP-binding cassette transporters are present in most bacteria. Streptococcal pathogens S. pneumoniae and S. pyogenes encode two zinc-binding proteins AdcA and AdcAII, whereas GBS is particularly distinct as it possesses a third zinc-binding protein, Lmb (38,39). AdcA and AdcAII/Lmb have been shown to utilize distinct mechanisms to bind zinc ions and shuttle them through the AdcBC transporter and are important for growth in zinc-restricted environments and infection (40)(41)(42)(43).…”

Changes in transcription start sites of Zap1‐regulated genes during zinc deficiency: Implications for HNT1 gene regulation

“…The abundance of Hnt1 decreases markedly during deficiency (Wang et al, ). Moreover, members of the broader histidine triad superfamily have been implicated in zinc homeostasis suggesting a possible role for Hnt1 in these processes (Moulin et al, ). Future experiments will test these models to examine how HNT1 regulation impacts gene expression and cell physiology during zinc deficiency.…”

“…The fraction of intensity of each band relative to the total intensity of the three bands in each grouping is plotted. p values were less than .001 for when comparing the zinc-deficient samples to their control; no statistically significant differences were observed among the zincreplete samples Moreover, members of the broader histidine triad superfamily have been implicated in zinc homeostasis suggesting a possible role for Hnt1 in these processes (Moulin et al, 2019). Future experiments will test these models to examine how HNT1 regulation impacts gene expression and cell physiology during zinc deficiency.…”