Two-component system vicrk regulates functions associated with establishment of streptococcus sanguinis in biofilms

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 (VicRKSs), 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 H2O2, 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 H2O2 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 VicRKSs 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.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 t821249414951FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIOR2009/54182-7; 2008/58333-7; 2009/50547-0sem informaçã

Salivary IgA antibody responses to Streptococcus mitis and Streptococcus mutans in preterm and fullterm newborn children

Objectives: The intensities and specificities of salivary IgA antibody responses to antigens of Streptococcus mutans, the main pathogen of dental caries, may influence colonization by these organisms during the first 1.5 year of life. Thus, the ontogeny of salivary IgA responses to oral colonizers continues to warrant investigation, especially with regard to the influence of birth conditions, e.g. prematurity, on the ability of children to efficiently respond to oral microorganisms. In this study, we characterised the salivary antibody responses to two bacterial species which are prototypes of pioneer and pathogenic microorganisms of the oral cavity (Streptococcus mitis and Streptococcus mutans, respectively) in fullterm (FT) and preterm (PT) newborn children. Methods: Salivas from 123 infants (70 FT and 53 PT) were collected during the first 10 h after birth and levels of IgA and IgM antibodies and the presence of S. mutans and S. mitis were analysed respectively by ELISA and by chequerboard DNA-DNA hybridization. Two subgroups of 24 FT and 24 PT children were compared with respect to patterns of antibody specificities against S. mutans and S. mitis antigens, using Western blot assays. Cross-adsorption of 10 infant's saliva was tested to S. mitis, S. mutans and Enterococcus faecalis antigens. Results: Salivary levels of IgA at birth were 2.5-fold higher in FT than in PT children (Mann-Whitney; P < 0.05). Salivary IgA antibodies reactive with several antigens of S. mitis and S. mutans were detected at birth in children with undetectable levels of those bacteria. Adsorption of infant saliva with cells of S. mutans produced a reduction of antibodies recognizing S. mitis antigens in half of the neonates. The diversity and intensity of IgA responses were lower in PT compared to FT children, although those differences were not significant. Conclusion: These data provide evidence that children have salivary IgA antibodies shortly after birth, which might influence the establishment of the oral microbiota, and that the levels of salivary antibody might be related to prematurity. (C) 2011 Elsevier Ltd. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [07/57346-5, 07/50807-7]Conselho Nacional de Pesquisa (CNPq) [472928/2007-4

The vaccination approach to control infections leading to dental caries

caries is a transmissible infectious disease, in which mutans streptococci (MS) play the role of main pathogens. This oral disease represents a public health problem worldwide, and despite the advances in dental health with the use of fluoride abroad, treatment of caries manifestations and their outcomes are still highly costly to public and private healthcare systems. Lack of treatment of dental caries ultimately may have serious systemic consequences. Mutans streptococci have a panel of virulence factors important for their establishment in the complex microbial community of the dental biofilm and in the induction of caries. In this review we discuss the advances in our understanding of the molecular mechanisms which underlie MS transmission, tooth colonization and virulence. Infection and disease take place in a milieu exposed to components of the mucosal and systemic immune systems of the host. Thus, inducing host responses which target aspects of mutans streptococcal colonization and disease may provide additional measures to modify dental caries. This review also describes current strategies for anti-caries vaccination efforts with regard to important bacterial targets, routes, adjuvants and delivery systems for active and passive immunization

Two-component signal transduction systems in oral bacteria

We present an overview of how members of the oral microbiota respond to their environment by regulating gene expression through two-component signal transduction systems (TCSs) to support conditions compatible with homeostasis in oral biofilms or drive the equilibrium toward dysbiosis in response to environmental changes. Using studies on the sub-gingival Gram-negative anaerobe Porphyromonas gingivalis and Gram-positive streptococci as examples, we focus on the molecular mechanisms involved in activation of TCS and species specificities of TCS regulons

The vaccination approach to controlinfections leading to dental caries

Dental caries is a transmissible infectious disease, in which mutans streptococci (MS) play the role of main pathogens. This oral disease represents a public health problem worldwide, and despite the advances in dental health with the use of fluoride abroad, treatment of caries manifestations and their outcomes are still highly costly to public and private healthcare systems. Lack of treatment of dental caries ultimately may have serious systemic consequences. Mutans streptococci have a panel of virulence factors important for their establishment in the complex microbial community of the dental biofilm and in the induction of caries. In this review we discuss the advances in our understanding of the molecular mechanisms which underlie MS transmission, tooth colonization and virulence. Infection and disease take place in a milieu exposed to components of the mucosal and systemic immune systems of the host. Thus, inducing host responses which target aspects of mutans streptococcal colonization and disease may provide additional measures to modify dental caries. This review also describes current strategies for anti-caries vaccination efforts with regard to important bacterial targets, routes, adjuvants and delivery systems for active and passive immunization

Functional Analysis of Glucan Binding Protein B from Streptococcus mutans

Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division

Comparative Analysis of Gtf Isozyme Production and Diversity in Isolates of Streptococcus mutans with Different Biofilm Growth Phenotypes

Streptococcus mutans is the main pathogenic agent of dental caries. Glucosyltransferases (Gtfs) produced by these bacteria are important virulence factors because they catalyze the extracellular synthesis of glucans that are necessary for bacterial accumulation in the dental biofilm. The diversity of GtfB and GtfC isozymes was analyzed in 44 genotypes of S. mutans that showed a range of abilities to form biofilms in vitro. Several approaches were used to characterize these isozymes, including restriction fragment length polymorphism analysis of the gtfB and gtfC genes, zymographic analysis of the identified GtfB and GtfC genotypes, and quantitation of isozyme production in immunoblot experiments with specific monoclonal antibodies. A high diversity of gtf genes, patterns of enzymatic activity, and isozyme production was identified among the isolates tested. GtfC and, to a lesser extent, GtfB were produced in significantly higher amounts by strains that had high biofilm-forming ability than by strains with low biofilm-forming ability. Biofilm formation was independent of the GtfB and GtfC genotype. Atypical strains that showed an apparent single Gtf isozyme of intermediate size between GtfB and GtfC were also identified. The results indicate that various expression levels of GtfB and GtfC isozymes are associated with the ability of distinct S. mutans genotypes to grow as biofilms, strengthening the results of previous genetic and biochemical studies performed with laboratory strains. These studies also emphasize the need to identify factors that control gtf gene expression