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.

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.

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 of S. 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.

Streptococcus mutans strains were isolated from cohorts of Brazilian nursery school children and genotyped by arbitrarily primed PCR and restriction fragment length polymorphism analysis. Of 24 children with two to five S. mutans isolates, 29% carried two or more genotypes. The presence of matching genotypes of S. mutans among children attending one nursery suggests horizontal transmission.Dental caries is a transmissible infectious disease in which mutans streptococci (MS) play the major role. Infective strains of Streptococcus mutans, the most prevalent species of the MS group, may persist for many years in the mouths of preschool children (1,6,22). Early colonization is related to high caries activity during childhood (3,7,11). The mechanisms by which MS colonize and accumulate on tooth surfaces are not completely clear. In addition to environmental and host factors, specific genotypes of S. mutans may be more aggressive colonizers. This is suggested by previous findings of positive relationships between the production of water-insoluble glucan from sucrose by glucosyltransferase (GTF) activities and the intensity of MS oral colonization and caries incidence (19).Essential to the development of strategies for the prevention of dental caries is the identification of sources of MS transmission. DNA fingerprinting studies indicate that vertical transmission of bacteria from mother to child is the major route for early acquisition (2,13,15,18,21). However, detection of genotypes in children that are not found in their mothers or other family members indicates that MS may also be acquired from other sources (13,21,22). Since the spread of infectious agents is likely to occur in a nursery environment, we investigated the genetic similarity of MS strains isolated from Brazilian children attending nursery schools. The production of GTF isozymes was also examined to validate the genotypic similarities that we identified.The study group included 35 MS-infected children between 12 and 30 months of age (mean Ϯ standard deviation ϭ 23 Ϯ 5 months). This group accounted for 49% of the MS-colonized children previously described in a larger population (20), and it was primarily selected for the study of MS virulence factors. These children attended nine nursery schools in the city of Piracicaba, São Paulo, Brazil, for 5 days per week, 10 h per day. A total of four sucrose-rich meals were provided daily in the nurseries. Clinical exams were performed to record the number of erupted teeth and manifest caries lesions as previously described (20). Written informed consent was obtained from the parents, and all consent and experimental procedures were approved by the institutional Ethical Committee of the University of São Paulo School of Dentistry.One to five isolates of MS were recovered from each of the 35 children. As previously described (19), oral samples were collected with tongue blades which were then pressed on the surface of mitis salivarius agar contact plates (Difco) (12) containing 2 IU of bacitracin (Sigma)/ml and 1...

Early mutans streptococci (MS) infection has been associated with higher caries activity in childhood. Since colonization with MS does not always lead to caries activity, additional factors may be involved in MS cariogenicity. For example, MS may differ in virulence traits such as the potential to synthesize glucan polymers from sucrose. In the present study, we tested the hypothesis that caries activity can be associated with variations in virulence factor expression of MS-infecting strains. At baseline, levels of MS obtained by the tongue-blade sampling method, and the presence of visible plaque on upper incisors, were measured in 101 12- to 30-month-old children. Dental caries lesions were diagnosed at baseline and after one year. Caries incidence data were then used to select ten caries-free and nine caries-active children from whom a total of 20 MS fresh isolates was studied. Water-insoluble glucan (WIG) synthesis, final pH, and sucrose-dependent adherence on glass surfaces were measured in these MS isolates. Concentrated culture supernatants were separated in duplicate SDS-PAGE gels, which were then either stained for protein or incubated with 5% sucrose. The intensities of the WIG bands developed in the 5% sucrose PAGE gels and the corresponding protein-stained GTF bands were measured by scanning densitometry. High MS levels (> or = 100 CFU) were associated with high caries incidence (p < 0.01). The presence of visible plaque did not correlate with caries incidence. The intensities of WIG bands were positively correlated with caries incidence (p < 0.05) and with the ability of MS to adhere to glass surfaces (p < 0.05). Analysis of our data suggests that the ability to synthesize WIG is an important virulence factor in initial caries development by increasing MS adherence and accumulation in the plaque of young children.