Two-Component System VicRK Regulates Functions Associated with Establishment of Streptococcus sanguinis in Biofilms

Abstract

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRK Ss ), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H 2 O 2 , a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H 2 O 2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRK Ss regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.

Two-component signal transduction systems in oral bacteria

“…Consistently, atypical long-chain phenotypes are found in v icK isogenic mutants obtained in S. pnemoniae , S. mutans , and S. sanguinis [90,105,106,111]. In VicR regulons, the most conserved target gene encodes PcsB (protein required for cell separation of group B Streptococcus ) that was first identified in group B streptococci [114] and orthologue GbpB (glucan-binding protein B in S. mutans ) [115,116].…”

“…However, molecular studies indicate that even highly conserved TCS, e.g. TCS VicRK, have species-specific functions in bacterial colonization and virulence as addressed below [90,91]. …”

“…VicR is in the OmpR/PhoB family of regulators in which the output domain forms a DNA-binding winged-helix domain. In B. subtilis and Staphylococcus aureus , both VicR and VicK are essential for bacterial viability [100]; on the other hand, only vicR is essential in S. pneumoniae [105], S. mutans [106,107], and S. sanguinis [90], and both vicR and vicK can be deleted in S. gordonii [91]. In S. pyogenes , insertional inactivation of vicR was possible, but the mutant was not viable in a mice model of infection [108].…”

“…In S. pyogenes , insertional inactivation of vicR was possible, but the mutant was not viable in a mice model of infection [108]. The exact reasons for VicR and/or VicK essentially in different species are unknown, but a possibility is their role in the regulation of peptidoglycan (murein) biosynthesis, cell division and cell wall homeostasis [90,98,103,107,109–111]. Because vicK can be deleted in S. pneumoniae, S. mutans , and S. sanguinis , one possibility is that the VicR of these species could be phosphorylated by alternative pathways.

Figure 3.VicRK orthologues of oral streptococcal species.

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“…VicRK plays an important role in modulating cell division, cell-wall biosynthesis and homeostasis, and membrane integrity, as evidenced by the presence of several genes encoding autolysins and other murein hydrolases in the VicR regulons of B. subtilis [112], S. aureus [109], S. pneumoniae [105], S. mutans [107,113], and S. sanguinis [90]. Consistently, atypical long-chain phenotypes are found in v icK isogenic mutants obtained in S. pnemoniae , S. mutans , and S. sanguinis [90,105,106,111].…”