Streptococcus mutans, a Gram-positive organism, is the primary causative agent in the formation of dental caries in humans. To persist in the oral cavity, S. mutans must be able to tolerate rapid environmental fluctuations and exposure to various toxic chemicals. However, the mechanisms underlying the ability of this cariogenic pathogen to survive and proliferate under harsh environmental conditions remain largely unknown. Here, we wanted to understand the mechanisms by which S. mutans withstands exposure to methyl viologen (MV), a quaternary ammonium compound (QAC) that generates superoxide radicals in the cell. To elucidate the essential genes for MV tolerance, screening of ∼3,500 mutants generated by ISS1 mutagenesis, revealed 15 MV-sensitive mutants. Among them, five and four independent insertions had occurred in SMU.905 and SMU.906 genes, respectively. These two genes are appeared to be organized in an operon and encode a putative ABC transporter complex; we designated the genes as vltA and vltB, for viologen transporter. To verify our results, vltA was deleted by using an antibiotic resistance marker; the mutant was just as sensitive to MV as the ISS1 insertion mutants. Furthermore, vltA and vltB mutants were also sensitive to other viologen compounds such as benzyl and ethyl viologens. Complementation assays were also carried out to confirm the role of VltA and VltB in viologen tolerance. Sensitivity to various drugs, including a wide range of QACs, was evaluated. It appears that a functional VltA is also required for full resistance toward acriflavin, ethidium bromide, and safranin; all are well-known QACs. These results indicate that VltA/B constitute a heterodimeric multidrug efflux pump of the ABC family. BLAST-P analysis suggests that homologs of VltA/B are widely present in streptococci, enterococci, and other important Gram-positive pathogens.

Two-component signal transduction systems (TCSs) in prokaryotes often regulate gene clusters that induce pathogenicity, and thus they have frequently been proposed as potential drug targets for attenuating the virulence of pathogens. The pathogenic potential of Streptococcus mutans, the major etiological pathogen of dental caries, is also regulated by its TCSs. The object of this study was to evaluate the effect of a histidine kinase (HK) inhibitor against two major virulence factors of S. mutans: biofilm formation and acid tolerance. Walkmycin C (WKM C), an HK inhibitor isolated from the screening of inhibitors against WalK HK in Bacillus subtilis, inhibited the in vitro autophosphorylation activity of three purified S. mutans HKs, i.e., VicK, CiaH, and LiaS. Although S. mutans does not have any essential HK but only an essential response regulator, VicR, WKM C showed an MIC of 6.25 μg/ml. This inhibitory effect of WKM C suggests that blocking the autophosphorylation of multiple HKs may inhibit phosphotransfer to VicR from VicK and other HKs. When WKM C was added at sub-MIC levels, the cells formed abnormal biofilms and also showed a defect in competence. When the cells were pretreated with WKM C, an increase in acid sensitivity was observed. Our results show that WKM C represses two pathogenic phenotypes of S. mutans, indicating the possibility of developing histidine kinase inhibitors into antivirulence drugs.

Commensal oral streptococci play critical roles in oral biofilm formation and promote dental health by competing with, and antagonizing the growth of, pathogenic organisms, such as Streptococcus mutans. Efficient utilization of the spectrum of carbohydrates in the oral cavity by commensal streptococci is essential for their persistence, and yet very little is known about the regulation of carbohydrate catabolism by these organisms. Carbohydrate catabolite repression (CCR) in the abundant oral commensal Streptococcus gordonii strain DL-1 was investigated using the exo-β-D-fructosidase gene (fruA) and a fructose/mannose sugar:phosphotransferase (PTS) enzyme II operon (levDEFG) as model systems. Functional studies confirmed the predicted roles of FruA and LevD in S. gordonii. ManL, the AB domain of a fructose/mannose-type enzyme II PTS permease, contributed to utilization of glucose, mannose, galactose, and fructose and exerted primary control over CCR of the fruA and levD operons. Unlike in S. mutans, ManL-dependent CCR was not sugar specific, and galactose was very effective at eliciting CCR in S. gordonii. Inactivation of the apparent ccpA homologue of S. gordonii actually enhanced CCR of fruA and levD, an effect likely due to its demonstrated role in repression of manL expression. Thus, there are some similarities and fundamental differences in CCR control mechanisms between the oral pathogen S. mutans and the oral commensal S. gordonii that may eventually be exploited to enhance the competitiveness of health-associated commensals in oral biofilms.

The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the -10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.

ABSTRACT: BACKGROUND: Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis. RESULTS: As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes alpha1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis. CONCLUSIONS: These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.

Recent analyses with ribosomal RNA-based technologies have revealed the diversity of bacterial populations within dental biofilms, and have highlighted their important contributions to oral health and disease. Dental biofilms are exceedingly complex and multispecies ecosystems, where oral bacteria interact cooperatively or competitively with other members. Bacterial interactions that influence dental biofilm communities include various different mechanisms. During the early stage of biofilm formation, it is known that planktonic bacterial cells directly attach to surfaces of the oral cavity or indirectly bind to other bacterial cells that have already colonized. Adherence through co-aggregation may be critical for the temporary retention of bacteria on dental surfaces, and may facilitate eventual bacterial colonization. It is likely that metabolic communication, genetic exchange, production of inhibitory factors (e.g., bacteriocins, hydrogen peroxide, etc.), and quorum-sensing are pivotal regulatory factors that determine the bacterial composition and/or metabolism. Since each bacterium can easily access a neighboring bacterial cell and its metabolites, genetic exchanges and metabolic communication may occur frequently in dental biofilms. Quorum-sensing is defined as gene regulation in response to cell density, which influences various functions, e.g., virulence and bacteriocin production. In this review, we discuss these important interactions among oral bacteria within the dental biofilm communities.

Induction of the agmatine deiminase system (AgDS) of Streptococcus mutans requires agmatine and is optimal at low pH. Here we show that the VicRK, ComDE and CiaRH two-component systems influence AgDS gene expression in response to acidic and thermal stresses.

Summary: Streptococci readily colonize mucosal tissues in the nasopharynx; the respiratory, gastrointestinal, and genitourinary tracts; and the skin. Each ecological niche presents a series of challenges to successful colonization with which streptococci have to contend. Some species exist in equilibrium with their host, neither stimulating nor submitting to immune defenses mounted against them. Most are either opportunistic or true pathogens responsible for diseases such as pharyngitis, tooth decay, necrotizing fasciitis, infective endocarditis, and meningitis. Part of the success of streptococci as colonizers is attributable to the spectrum of proteins expressed on their surfaces. Adhesins enable interactions with salivary, serum, and extracellular matrix components; host cells; and other microbes. This is the essential first step to colonization, the development of complex communities, and possible invasion of host tissues. The majority of streptococcal adhesins are anchored to the cell wall via a C-terminal LPxTz motif. Other proteins may be surface anchored through N-terminal lipid modifications, while the mechanism of cell wall associations for others remains unclear. Collectively, these surface-bound proteins provide Streptococcus species with a "coat of many colors," enabling multiple intimate contacts and interplays between the bacterial cell and the host. In vitro and in vivo studies have demonstrated direct roles for many streptococcal adhesins as colonization or virulence factors, making them attractive targets for therapeutic and preventive strategies against streptococcal infections. There is, therefore, much focus on applying increasingly advanced molecular techniques to determine the precise structures and functions of these proteins, and their regulatory pathways, so that more targeted approaches can be developed.

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the 'death effector'. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.

Introduction: In bacteria, two-component systems (TCS) involving the products of a histidine kinase gene (hk) and a response regulator gene (rr) play important roles in adaptation to environmental changes. Fourteen hk-rr homologs and one orphan rr homolog were identified in the Streptococcus mutans UA159 genome database. There have been no comprehensive evaluations of the roles of rr homologs in the acid tolerance of S. mutans. Methods: The TCS genes (tcs) of S. mutans were designated smtcs01-15. Mutants of S. mutans UA159 with deletions of rr and hk-rr were constructed. Acid tolerance was evaluated by comparing the doubling times at pH 7.2 and pH 5.5 between the wild-type and mutant strains. Results: Excluding smtcs10 and 12, for which viable mutants could not be obtained, a total of 13 rr deletion mutants were constructed. The rr deletions in smtcs03, 05, 08, and 13 resulted in diminished acid tolerance in comparison with UA159. The hk-rr double-mutants exhibited acid sensitivity levels similar to those of the corresponding rr mutants. The results of the present study reveal the involvement of the rr genes of smtcs03 and 05 in acid tolerance. Deletion of hk and/or rr in smtcs03 generated an acid-sensitive phenotype. In contrast, for smtcs05, while deletion of rr resulted in reduced acid tolerance, a single-deletion of hk had no effect on acid tolerance. Conclusions: We implicated two rr genes in the acid tolerance of S. mutans. In particular, smtcs05 is a novel tcs, the sole rr of which is involved in the acid tolerance of S. mutans.

Background/aim: Recombinase A (RecA) is essential for the transformation of both plasmid and chromosomal DNA in Streptococcus pneumoniae and is considered to be related to the SOS-response in Streptococcus mutans. Methods: In the present study, a RecA-deficient mutant strain (RAD) was constructed by insertional inactivation of the recA gene encoding the RecA protein in strain MT8148 of S. mutans, after which the biological functions of acid tolerance and biofilm formation were investigated. Results: RAD showed reduced acid tolerance and produced lower density biofilm compared with the wild-type strain. In addition, confocal microscopic observation indicated that the biofilm produced by RAD was composed of cells with significantly lower viability compared with that produced by strain MT8148. Conclusion: These results suggest that RecA has a relationship with biofilm formation.

Group B streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants have been identified which contribute to invasion of BMEC, little is know about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/H present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme and reactive oxygen species. Furthermore, competition experiments in mice showed that wild type GBS had a significant survival advantage compared to the GBSDeltaciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between GBS wild type and GBSDeltaciaR mutant bacteria revealed several CiaR-regulated genes that may contribute to GBS stress tolerance and subversion of host defenses. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.

The complete genome sequence of Streptococcus mutans, a bacterial pathogen commonly associated with human dental caries, was published in 2002. The streamlined genome (2.03 Mb) revealed an organism that is well adapted to its obligately host-associated existence in multispecies biofilms on tooth surfaces: a dynamic environment that undergoes rapid and substantial fluctuations. However, S. mutans lacks many of the sensing systems and alternative sigma factors that bacteria often use to coordinate gene expression in response to stress and changes in their environment. Over the past 7 years, functional genomics and proteomics have enhanced our understanding of how S. mutans has integrated the stress regulon and global transcriptional regulators to coordinate responses to environmental fluctuations with modulation of virulence in a way that ensures persistence in the oral cavity and capitalizes on conditions that are favourable for the development of dental caries. Here, we highlight advances in dissection of the stress regulon of S. mutans and its intimate interrelationship with pathogenesis.

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.

Streptococcus mutans is the primary causative agent involved in dental caries in humans. Among important virulence factors of this pathogen, its ability to form and sustain a polysaccharide-encased biofilm (commonly called dental plaque) is vital not only to its survival and persistence in the oral cavity, but also for its pathogenicity as well. This chapter focuses on the S. mutans' biofilm phenotype and how this mode of growth is regulated by its density-dependent quorum sensing (QS) system primarily comprised of the Competence Stimulating Peptide (CSP) and the ComD/ComE two-component signal transduction system. In addition to biofilm formation, the CSP-mediated QS system in S. mutans also affects its acidogenicity, aciduricity, genetic transformation and bacteriocin production. Interestingly, it has also been discovered that these properties are optimally expressed in cells derived from a biofilm as opposed to a free-floating planktonic mode of growth. Hence, strategies targeting S. mutans' QS system to attenuate biofilm formation and/or virulence are currently being used to develop therapeutic or preventative measures against dental caries. Recently, it was discovered that the addition of CSP in large concentrations (relative to amounts used for normal competence development) resulted in growth arrest and eventual cell death, thus paving way for CSP-mediated targeted killing of S. mutans. In addition to the QS system, effects of other two-component signal transduction systems on the biofilm phenotype of S. mutans are also discussed.

Previous studies identified irvA as a normally repressed, but highly inducible transcription regulator capable of repressing mutacin I gene expression in Streptococcus mutans. In this study, we aimed to identify and characterize the regulator(s) responsible for repressing the expression of irvA. An uncharacterized ORF (SMU.1398) located immediately adjacent to irvA and annotated as a putative transcription repressor was identified as a likely candidate. Mutation studies confirmed that the expression of irvA was greatly increased in the SMU.1398 background. Mutation of SMU.1398 ("irvR") abolished genetic competence and reduced the expression of the late competence genes/operons comEA, comY, and dprA without affecting the expression of the known competence regulators comC, comE/D, or comX. In addition, irvR was also found to be a potent negative regulator of dextran-dependent aggregation (DDAG) and gbpC expression. Each of these irvR mutant phenotypes could be rescued with a double mutation of irvA or complemented by introducing a wild type copy of irvR on a shuttle vector. These data indicate that the repression of irvA is critically dependent upon irvR and that irvA repression is essential for the development of genetic competence and the proper control of dextran-dependent aggregation in S. mutans.

Streptococcus mutans is a primary pathogen for dental caries in humans. CiaR and CiaH of S. mutans comprise a two-component signal transduction system (TCS) involved in regulating various virulent factors. However, the signal that triggers the CiaRH response remains unknown. In this study, we show that calcium is a signal for regulation of the ciaRH operon, and that a double-glycine-containing small peptide encoded within the ciaRH operon (renamed ciaX) mediates this regulation. CiaX contains a serine-aspartate (SD) domain that is shared by calcium-binding proteins. A markerless in-frame deletion of ciaX reduced ciaRH operon expression and diminished the calcium repression of operon transcription. Point mutations of the SD-domain resulted in the same phenotype as the in-frame deletion, indicating that the SD-domain is required for CiaX function. Further characterization of ciaX demonstrated that it is involved in calcium mediated biofilm formation. Furthermore, inactivation of ciaR or ciaH led to the same phenotype as the in-frame deletion of ciaX, suggesting that all three genes are involved in the same regulatory pathway. Sequence analysis and real-time RT-PCR identified a putative CiaR binding site upstream of ciaX. We conclude that the ciaXRH operon is a three-component, self-regulatory system modulating cellular functions in response to calcium.

Streptococcus mutans, a major oral pathogen responsible for dental caries formation, possess a variety of mechanisms for survival in the human oral cavity, where the conditions of the external environment are diverse and in a constant state of flux. Formation of biofilms, survival under acidic pH, and the production of mutacins are considered to be important virulence determinants displayed by this organism. Biofilm formation is facilitated by the production of GbpC, an important cell surface-associated protein that binds to glucan, a adhesive polysaccharide produced by the organism itself. To better understand the nature of the environmental cues that induce GbpC production, we examined the role of 14 sensor kinases on the expression of gbpC in S. mutans strain UA159. We found that only the LiaS sensor kinase regulates gbpC expression, while the other sensor kinases had little or no effect on gbpC expression. We also found that while LiaS negatively regulates gbpC expression, inactivation of its cognate response regulator, LiaR, does not appear to affect expression of gbpC. Since both gbpC expression and mutacin IV production are regulated by a common regulatory network, we also tested the effect of liaS mutation on mutacin production, and found that LiaS positively regulates mutacin IV production. Furthermore, RT-PCR analysis suggests that LiaS does so by regulating the expression of nlmA, which encodes a peptide component of mutacin IV, and nlmT, which encodes an ABC transporter. As with expression of gbpC, LiaR did not have any apparent effect on mutacin IV production. Based on the results of our study, we speculate that LiaS is engaged in cross-talk with one or more response regulators belonging to the same family as LiaR, enabling LiaS to regulate the expression of several genes coding for virulence factors.

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.