The Streptococcus mutans vicX Gene Product Modulates gtfB/C Expression, Biofilm Formation, Genetic Competence, and Oxidative Stress Tolerance

Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity

A VicRK Signal Transduction System in Streptococcus mutans Affects gtfBCD, gbpB, and ftf Expression, Biofilm Formation, and Genetic Competence Development

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic(+)). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic(+). Based on real-time PCR, Smuvic(+) showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans

<i>In vitro</i> Manganese-Dependent Cross-Talk between <i>Streptococcus mutans</i> VicK and GcrR: Implications for Overlapping Stress Response Pathways

<div><p><i>Streptococcus mutans</i>, a major acidogenic component of the dental plaque biofilm, has a key role in caries etiology. Previously, we demonstrated that the VicRK two-component signal transduction system modulates biofilm formation, oxidative stress and acid tolerance responses in <i>S. mutans</i>. Using i<i>n vitro</i> phosphorylation assays, here we demonstrate for the first time, that in addition to activating its cognate response regulator protein, the sensor kinase, VicK can transphosphorylate a non-cognate stress regulatory response regulator, GcrR, in the presence of manganese. Manganese is an important micronutrient that has been previously correlated with caries incidence, and which serves as an effector of SloR-mediated metalloregulation in <i>S. mutans</i>. Our findings supporting regulatory effects of manganese on the VicRK, GcrR and SloR, and the cross-regulatory networks formed by these components are more complex than previously appreciated. Using DNaseI footprinting we observed overlapping DNA binding specificities for VicR and GcrR in native promoters, consistent with these proteins being part of the same transcriptional regulon. Our results also support a role for SloR as a positive regulator of the <i>vicRK</i> two component signaling system, since its transcription was drastically reduced in a SloR-deficient mutant. These findings demonstrate the regulatory complexities observed with the <i>S. mutans</i> manganese-dependent response, which involves cross-talk between non-cognate signal transduction systems (VicRK and GcrR) to modulate stress response pathways.</p></div

<i>in vitro</i> phosphorylation of VicK in the presence of various metal cations.

<p>VicK (1 µM) was incubated in 100 mM Tris-HCl, pH 7.5 containing 1 mM of the designated cations and 0.10 µM [γ-³²P] ATP at room temperature for 15 minutes. The relative autophosphorylation of VicK was quantified using Image Quant 5.0 software (Molecular Dynamics) and is represented by the histogram above the scanned gel. The gels shown are representative of at least three independent experiments. Error bars represent ± std. errors of the average phosphorylation values derived from at least 3 independent experiments.</p

<i>In vitro</i> model of manganese-independent (A) and –dependent cross-regulation involving <i>S. mutans</i> SloR, VicRK and GcrR.

<p>A) In the absence of Mn²⁺ (approximating conditions of free-floating planktonic cells) <i>S. mutans gcrR</i> expression is de-repressed (VicR expression is not induced in the absence of Mn²⁺). Even though GcrR is the more abundant substrate, VicR is the favored species for VicK phosphorylation under these conditions. B) During this so-called “transition stage” (approximating conditions of an early biofilm) a “spike” in Mn²⁺ renders GcrR (still the predominant species) the favored substrate for phosphorylation by VicK, but only transiently. C) As the biofilm matures and Mn²⁺ concentrations increase, SloR is activated to repress <i>gcrR</i> expression, thereby reducing the availability of GcrR as a substrate. The activated SloR-Mn²⁺ complex encourages <i>vicR</i> expression, and hence VicR becomes the favored substrate for VicK phosphorylation once again.</p

VicK has a significant impact on transcription of known ATR-related genes in <i>S. mutans</i>.

<p>qRT-PCR was performed to reveal fold-change in gene expression at pH 5.5 versus 7.5 with cDNAs derived from <i>S. mutans</i> UA159 (solid black bars) and a <i>vicK</i> insertion-deletion mutant (SmuvicK) (striped bars). Error bars represent ± std. errors of the average expression values derived from at least 3 independent experiments. Student t-tests confirm that all genes are significantly down-regulated in the VicK mutant relative to the UA159 wild-type progenitor strain (p<0.001).</p

Construction of the <i>S. mutans</i> fusion strains GMS905, GMS906, and GMS907.

<p>The integration of the P<i>gcrR:cat</i> fusion that is resident on plasmid pLM1 occurred via a double cross-over event into the chromosome of <i>S. mutans</i> UA159, GMS584 and SmuvicK at the <i>phnA</i> and <i>mtlA</i> loci. Sequencing across the <i>S. mutans</i> chromosome-pLM1 junction confirmed appropriate insertion of the P<i>gcrR:cat</i> fusion.</p