Activity of Streptococcus mutans VicR Is Modulated by Antisense RNA.

The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III-encoding gene ( rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (AS vicR) RNA was detected by Northern blotting and qRT-PCR assays. AS vicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An AS vicR RNA transcript was detected upstream of the rnc gene. Expression of the AS vicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of AS vicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of AS vicR RNA transcript and growth conditions. The biofilm biomass decreased in the AS vicR overexpression mutant. These data suggest a role for the AS vicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans.

Can insoluble polysaccharide concentration in dental plaque, sugar exposure and cariogenic microorganisms predict early childhood caries? A follow-up study.

BACKGROUND: Insoluble polysaccharide (IP) has been associated with caries prevalence in young children. However, the power of IP to predict ECC needs to be demonstrated. AIMS: To assess the relationships between early childhood caries (ECC) and extracellular insoluble polysaccharides (IP) in dental plaque, sugar exposure and cariogenic microorganisms. DESIGN: Visible plaque on maxillary incisors was recorded, followed by caries diagnosis in 65 preschoolers (3-4 years) at baseline and after 1 year. Plaque was collected for mutans streptococci (MS), total microorganism (TM) and lactobacilli (LB) enumerations in selective media, as well as for IP analysis, which was later assessed by colorimetry. Sugar/sucrose exposure was assessed by a diet chart. RESULTS: Positive correlations were found among the prevalence of caries and MS, TM, LB, solid sucrose and visible dental plaque. Additionally, children with IP concentrations in dental plaque higher than 2.36 µg/mg (odds ratio-OR=6.8), with visible plaque on maxillary incisors (OR=4.3), harbouring LB (OR=13) and exposed to solid sugar more than twice/day (OR=5) showed higher risk of developing caries (p<0.05). CONCLUSION: Extracellular insoluble polysaccharides, solid sugar/sucrose, visible dental plaque and cariogenic microorganisms could predict caries development, partially explaining the ECC pattern.

Influence of VicRK and CovR on the interactions of Streptococcus mutans with phagocytes.

OBJECTIVE: Streptococcus mutans are members of the oral microbiota that are implicated in dental caries and infective endocarditis. To adapt to environmental stresses encountered during host colonization, these bacteria employ two-component regulatory systems, which modulate global changes in gene expression. These include the systems VicRK and CovR. In this study, we investigate the influence of VicRK and CovR in S. mutans interactions with mononuclear and polymorphonuclear (PMN) phagocytes. METHODS: Patterns of S. mutans uptake by murine macrophages were determined in strains, which differ in the production of proteins regulated by VicRK and CovR. Bacterial uptake by murine macrophages and by PMN in human blood was analyzed in vicK and covR knockout mutants obtained in strains UA159 and LT11. RESULTS: Inactivation of covR did not affect uptake by macrophages, while vicK inactivation transiently reduced uptake only in LT11 (P < 0.05). In the two strains, inactivation of vicK and covR impaired uptake by PMN for a period of 1 h or more (P < 0.01-0.05). Mutant complementation with vicK or covR restored the PMN uptake phenotypes. CONCLUSION: This study indicates that VicRK and CovR regulate functions that influence bacterial susceptibility to phagocytosis, suggesting a novel role for these systems in the virulence of S. mutans.

Impact of Porphyromonas gingivalis inoculation on ligature-induced alveolar bone loss. A pilot study in rats.

BACKGROUND AND OBJECTIVE: Periodontitis is a polymicrobial infection characterized by the loss of connective tissue attachment, periodontal ligament and alveolar bone. The aim of this study was to evaluate the impact of Porphyromonas gingivalis inoculation on the ligature-induced alveolar bone loss (ABL) model in rats. MATERIAL AND METHODS: Forty male Wistar rats were randomly assigned to the following groups: G1, control (n = 10); G2, ligature-induced ABL (n = 15); and G3, ligature-induced ABL + P. gingivalis inoculation (n = 15). Rats in G2 and G3 were killed 15, 21 and 30 d after ligature placement, and the following parameters were assessed: microbiological load; ABL; and interleukin (IL)-1ß (Il1beta)/Il1ra, Il6/Il10 and Rankl/osteoprotegerin (Opg) mRNA ratios in the gingival tissues, as determined by quantitative PCR. RESULTS: Microbiological analyses demonstrated that rats in G1, G2 and G3 were positive for the presence of bacteria (determined using PCR amplification of the 16S gene), but that only the treatment sites of rats in G3 were positive for P. gingivalis at all time-points investigated. Histometrically, significant bone loss (p<0.001) was observed for both ligated groups (G2 and G3) compared with the nonligated group (G1), with higher ABL observed for G2 at all the experimental time-points. Furthermore, gene-expression analysis demonstrated that the presence of P. gingivalis in the dentogingival area significantly decreased the Il1ß/Il1ra, Il6/Il10 and Rankl/Opg mRNA ratios compared with ligature alone. CONCLUSION: Within the limits of this pilot study, it was concluded that inoculation of P. gingivalis affected the ligature-induced ABL model by the induction of an anti-inflammatory and antiresorptive host response.

Transcriptional analysis of gtfB, gtfC, and gbpB and their putative response regulators in several isolates of Streptococcus mutans.

BACKGROUND: Streptococcus mutans, a major dental caries pathogen, expresses several virulence genes that mediate its growth, accumulation on tooth surfaces, and acid-mediated tooth demineralization. GtfB and GtfC catalyze the extracellular synthesis of water-insoluble glucan matrix from sucrose, and are essential for accumulation of bacteria in the dental biofilm. GbpB, an essential protein of S. mutans, might also mediate cell-surface interaction with glucan. AIM/METHODS: In this study, we determined the transcription levels of gtfB, gtfC, and gbpB, and several putative transcriptional response regulators (rr) at different phases of planktonic growth in 11 S. mutans strains. RESULTS: Activities of gtfB and gtfC were growth-phase dependent and assumed divergent patterns in several strains during specific phases of growth, while gbpB activities appeared to be under modest influence of the growth phase. Transcription patterns of the rr vicR, covR, comE, ciaR, and rr1 were growth-phase dependent and some of these genes were expressed in a highly coordinated way. Each rr, except comE, was expressed by all the strains. Patterns of virulence and regulatory genes were, however, strain-specific. CONCLUSIONS: The findings suggest that mechanisms controlling virulence gene expression are variable among genotypes, providing the notion that the genetic diversity of S. mutans may have important implications for understanding mechanisms that regulate the expression of virulence genes in this species.