Renata O. Mattos-Graner

Cloning of the Streptococcus mutans Gene Encoding Glucan Binding Protein B and Analysis of Genetic Diversity and Protein Production in Clinical Isolates

ABSTRACT Streptococcus mutans, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm. These include glucosyltransferases, their glucan products, and proteins that bind glucan. At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel. The gbpB gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus. Examination of gbpB genes from clinical isolates ofS. mutans revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein. The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay.

Characterization of Salivary Immunoglobulin A Responses in Children Heavily Exposed to the Oral Bacterium Streptococcus mutans: Influence of Specific Antigen Recognition in Infection

ABSTRACT The initial infection of children by Streptococcus mutans, the main pathogen of dental caries, depends on the ability of S. mutans to adhere and accumulate on tooth surfaces. These processes involve the adhesin antigen I/II (AgI/II), glucosyltransferases (GTF) and glucan-binding protein B (GbpB), each a target for anticaries vaccines. The salivary immunoglobulin A (IgA) antibody responses to S. mutans antigens (Ags) were characterized in 21 pairs of 5- to 13-month-old children. Pairs were constructed with one early S. mutans-infected and one noninfected child matched by age, racial background, number of teeth, and salivary levels of IgA. Specific salivary IgA antibody response and S. mutans infection levels were then measured during a 1-year follow-up. Robust responses to S. mutans were detected from 6 months of age. Salivary IgA antibody to AgI/II and GTF was commonly detected in salivas of all 42 children. However, GbpB-specific IgA antibody was seldom detected in the subset of infected children (38.1% at baseline). In contrast, most of the subset of noninfected children (76.2%) showed GbpB-reactive IgA antibody during the same period. Frequencies of GbpB responses increased with age, but differences in intensities of GbpB-IgA antibody reactions were sustained between the subsets. At baseline, GbpB-reactive IgA antibody accounted for at least half of the total salivary IgA S. mutans-reactive antibody in 33.3 and 9.5% of noninfected and infected children, respectively. This study provides evidence that a robust natural response to S. mutans Ags can be achieved by 1 year of age and that IgA antibody specificities may be critical in modulating initial S. mutans infection.

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

ABSTRACT 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.

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 g bpB 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 sucrose-dependent 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.

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.

Secretory immunity following mutans streptococcal infection or immunization.

Salivary IgA antibody responses to mutans streptococci can be observed in early childhood, sometimes even before permanent colonization of the oral biofilm occurs. Many of these early immune responses are directed to components thought to be essential for establishment and emergence of mutans streptococci in the oral biofilm. Initial responses are likely to be modulated by antigen dose, by immunological maturity, and by previous encounters with similar antigenic epitopes in the pioneer commensal flora. Our understanding of these modulating factors is modest and is an opportunity for continued investigation. Under controlled conditions of infection, experimental vaccine approaches have repeatedly shown that infection and disease can be modified in the presence of elevated levels of antibody in the oral cavity. Protection can be observed regardless of antibody isotype or method used to actively or passively provide the immune reagent. Limited clinical trials have supported the utility of both of these approaches in humans. Refinements in antigen formulation, delivery vehicles, enhancing agents and routes of application, coupled with approaches that are timed to intercept most vulnerable periods of infection of primary and permanent dentition may well provide the healthcare practitioner with an additional tool to maintain oral health.

Prospective study of potential sources of Streptococcus mutans transmission in nursery school children.

Transmission of Streptococcus mutans, a major dental caries pathogen, occurs mainly during the first 2.5 years of age. Children appear to acquire S. mutans mostly from their mothers, but few studies have investigated non-familial sources of S. mutans transmission. This study prospectively analysed initial S. mutans oral colonization in 119 children from nursery schools during a 1.5-year period and tracked the transmission from child to child, day-care caregiver to child and mother to child. Children were examined at baseline, when they were 5-13 months of age, and at 6-month intervals for determination of oral levels of S. mutans and development of caries lesions. Levels of S. mutans were also determined in caregivers and mothers. A total of 1392 S. mutans isolates (obtained from children, caregivers and mothers) were genotyped by arbitrarily primed PCR and chromosomal RFLP. Overall, 40.3 % of children were detectably colonized during the study, and levels of S. mutans were significantly associated with the development of caries lesions. Identical S. mutans genotypes were found in four nursery cohorts. No familial relationship existed in three of these cohorts, indicating horizontal transmission. Despite high oral levels of S. mutans identified in most of the caregivers, none of their genotypes matched those identified in the respective children. Only 50 % of children with high levels of S. mutans carried genotypes identified in their mothers. The results support previous evidence indicating that non-familial sources of S. mutans transmission exist, and indicate that this bacterium may be transmitted horizontally between children during the initial phases of S. mutans colonization in nursery environments.

CovR and VicRK Regulate Cell Surface Biogenesis Genes Required for Biofilm Formation in Streptococcus mutans

The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic) or covR (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA), or regulated only by VicR (SMU.2146c) or CovR (epsC) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.

Immunological and Microbiologic Changes during Caries Development in Young Children

We explored the association between caries development, colonization with caries-associated microflora, and immunity as children begin the transition to mixed dentition. Forty children received dental examinations at 3–4 years of age, repeated a year later. Children were grouped into caries-free (n = 23; CF) and caries-active (n = 17; CA ≧3 new lesions on follow-up). Salivary IgA and IgA antibody to Streptococcus mutans virulence epitopes were measured by Luminex assay. Mutans streptococci (MS), lactobacilli and total microorganisms were enumerated on selective media from plaque samples. There was no significant difference in baseline levels of MS or lactobacilli between CF and CA groups. However, both MS and lactobacilli levels were higher at follow-up in the CA group. Furthermore, children with detectable lactobacilli at baseline had significantly higher caries risk. Salivary IgA concentrations increased significantly in both groups during the study. Both CF and CA groups also displayed significant increases in salivary IgA antibody levels to glucosyltransferase, glucan-binding protein (Gbp) and antigen I/II salivary binding region. CF antibody levels to seven peptides associated with domains of biological importance increased at follow-up, in contrast to increases to only three peptides in CA saliva samples. Multivariate modeling showed that a lower baseline level of salivary IgA anti-GbpB was associated with higher caries risk. These data indicate that MS and lactobacilli are associated with caries in this population, that the secretory immune system is undergoing significant maturation during this period, and that the breadth of mucosal IgA response to epitopes of S. mutans virulence components may influence the degree to which these cariogenic microorganisms can cause disease.

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.