Downregulation of GbpB, a Component of the VicRK Regulon, Affects Biofilm Formation and Cell Surface Characteristics of Streptococcus mutans

Abstract

The virulence of the dental caries pathogen Streptococcus mutans relies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced by S. mutans participate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressed gbpB antisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation of gbpB by the two-component system VicRK was investigated, and phenotypic analysis of a vicK mutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrosedependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties.

Two-component signal transduction systems in oral bacteria

“…These findings suggest that VicRK Sm is activated not only by intrinsic peptidoglycan growth and cell division, but also by processes associated with sucrose-dependent biofilm initiation. Consistent with these data, VicR Sm directly induces expression of enzymes required for the synthesis of different sucrose-derived exopolysaccharides (GtfB, GtfC, Ftf) and glucan-binding (GbpB) [106,111,117]. …”

“…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].…”

“…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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“…PcsB/GbpB was implicated in the essentiality of VicRK in S. pneumoniae , where vicR deletion is possible only when pcsB is induced [119]. In S. mutans , GbpB/PcsB is required for surface binding to glucan exopolysaccharides synthesized from sucrose during biofilm formation [111]. …”

“…In S. mutans , GbpB/PcsB is required for surface binding to glucan exopolysaccharides synthesized from sucrose during biofilm formation [111]. …”