Streptococcus mutans Proteases Degrade Dentinal Collagen.

Here, we explored the role of S. mutans's whole cell and discrete fractions in the degradation of type I collagen and dentinal collagen. Type I collagen gels and human demineralized dentin slabs (DS) were incubated in media alone or with one of the following: overnight (O/N) or newly inoculated (NEW) cultures of S. mutans UA159; intracellular proteins, supernatant or bacterial membranes of O/N cultures. Media from all groups were analyzed for protease-mediated release of the collagen-specific imino acid hydroxyproline. Images of type I collagen and DS were analyzed, respectively. Type I collagen degradation was highest for the supernatant (p < 0.05) fractions, followed by intracellular components and O/N cultures. Collagen degradation for DS samples was highest for O/N samples, followed by supernatant, and intracellular components (p < 0.05). There was lower detectable degradation for both type I collagen and DS from NEW culture samples (p < 0.05), and there was no type I collagen or DS degradation detected for bacterial membrane samples. Structural changes to type I collagen gel and dentinal collagen were observed, respectively, following incubation with S. mutans cultures (O/N and NEW), intracellular components, and supernatant. This study demonstrates that intracellular and extracellular proteolytic activities from S. mutans enable this cariogenic bacterium to degrade type I and dentinal collagen in a growth-phase dependent manner, potentially contributing to the progression of dental caries.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

Role of Dilution Rate and Nutrient Availability in the Formation of Microbial Biofilms.

We revisited the mathematical model of the chemostat and examined consequences of considerably decreasing the concentration of limiting nutrient in the inflow for the growth of both the planktonic and biofilm cells in the chemostat tank (fermenter). The model predicts a substantially lower steady-state biomass of planktonic cells in response to decreasing inflowing nutrient concentration. Contrarily, the steady-state concentration of nutrient inside the fermenter is expected to remain the same, as long as the inflowing concentration does not fall below its value. This allows the biofilm cells to grow at a rate regulated only by the exchange rate of the medium (dilution rate). We maintained a strain of Enterococcus faecalis in a chemostat of our own design with limiting nutrient in the inflow set near saturation constant at three dilution rates (0.09, 0.28, and 0.81 h-1). The highest dilution rate was near the critical rate calculated by the model. The one-day total biofilm buildup was 21× larger and its estimated growth rate 2.4× higher at highest dilution rate than at the lowest one. This increased biofilm formation with increased dilution rates is in agreement with previously published data on pure and mixed continuous flow cultures.

Gene expression and protein synthesis of esterase from Streptococcus mutans are affected by biodegradation by-product from methacrylate resin composites and adhesives.

An esterase from S. mutans UA159, SMU_118c, was shown to hydrolyze methacrylate resin-based dental monomers. OBJECTIVE: To investigate the association of SMU_118c to the whole cellular hydrolytic activity of S. mutans toward polymerized resin composites, and to examine how the bacterium adapts its hydrolytic activity in response to environmental stresses triggered by the presence of a resin composites and adhesives biodegradation by-product (BBP). MATERIALS AND METHODS: Biofilms of S. mutans UA159 parent wild strain, SMU_118c knockout strain (ΔSMU_118c), and SMU_118c complemented strain (ΔSMU_118cC) were incubated with photo-polymerized resin composite. High performance liquid chromatography was used to quantify the amount of a universal 2,2-Bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bisGMA)-derived BBP, bishydroxy-propoxy-phenyl-propane (bisHPPP) in the media. Fluorescence in situ hybridization (FISH) and quantitative proteomic analysis were used to measure SMU_118c gene expression and production of SMU_118c protein, respectively, from biofilms of S. mutans UA159 wild strain that were cultured with bisHPPP. RESULTS: The levels of bisHPPP released from composite were similar for ΔSMU_118c and media control, and these were significantly lower compared to the parent wild-strain UA159 and complemented strain (ΔSMU_118cC) (p < 0.05). Gene expression of SMU_118c and productions of SMU_118c protein were higher for bisHPPP incubated biofilms (p < 0.05). SIGNIFICANCE: This study suggests that SMU_118c is a dominant esterase in S. mutans and capable of catalyzing the hydrolysis of the resinous matrix of polymerized composites and adhesives. In turn, the bacterial response to BBP was to increase the expression of the esterase gene and enhance esterase production, potentially accelerating the biodegradation of the restoration, adhesive and restoration-tooth interface, ultimately contributing to premature restoration failure. STATEMENT OF SIGNIFICANCE: We recently reported (Huang et al., 2018) on the isolation and initial characterization of a specific esterase (SMU_118c) from S. mutans that show degradative activity toward the hydrolysis of dental monomers. The current study further characterize this enzyme and shows that SMU_118c is a dominant degradative esterase activity in the cariogenic bacterium S. mutans and is capable of catalyzing the hydrolysis of the resinous matrix of polymerized composites and adhesives. In turn, the bacterial response to biodegradation by-products from composites and adhesives was to increase the expression of the esterase gene and enhance esterase production, accelerating the biodegradation of the restoration, adhesive and the restoration-tooth interface, potentially contributing to the pathogenesis of recurrent caries around resin composite restorations.

Biodegradation of resin-dentin interfaces is dependent on the restorative material, mode of adhesion, esterase or MMP inhibition.

OBJECTIVE: To measure the effect of simulated human salivary esterases (SHSE) and metalloproteinases (MMP) inhibition on the integrity of restoration-tooth interfaces made from traditional or polyacid-modified resin composites bonded to human dentin by either total-etch or self-etch adhesives. METHODS: Resin-dentin specimens, made from traditional (Z250) or polyacid-modified (Dyract-eXtra) composites were bonded to human dentin using total-etch (TE-Scotchbond) or self-etch (SE-EasyBond) adhesives. TE was applied with or without the MMP inhibitor galardin. Specimens were incubated in phosphate-buffer or SHSE (37°C/pH=7.0) for up to 180 days, then suspended in a continuous flow biofilm fermenter cultivating biofilms of Streptococcus mutans UA159. Interfacial bacterial penetration, biofilm biomass and viability were measured by confocal laser scanning microscopy and biomarker dyes and used as interfacial biodegradation markers. RESULTS: All specimens showed increased biofilm penetration and biomass with time regardless of incubation condition. SHSE increased bacterial penetration in all experimental samples after 180days (p<0.05). Galardin reduced interfacial bacterial ingress and bacterial biomass vs. non-MMP-inhibited TE-bonded specimens (p<0.05). TE interfaces showed lower interfacial bacterial biomass vs. SE after 90-day and 180-day (p<0.05). Dyract-eXtra specimens showed lower bacterial cell viability within the interface vs. Z250 (p<0.05). SIGNIFICANCE: The biodegradation of resin-tooth interfaces is accelerated by esterases, modulated by MMP inhibition and is dependent on the material's chemistry and mode of adhesion. The in vitro bacterial growth model used in this study facilitates the elucidation of differences in interfacial integrity and biostability between different materials and techniques and is suitable for assessment of their performance prior to clinical evaluation.

Esterase from a cariogenic bacterium hydrolyzes dental resins.

OBJECTIVES: To identify and characterize specific esterases from S. mutans with degradative activity toward methacrylate-based resin monomers. METHODS: Out of several putative esterases, an esterase encoded in an Open Reading Frame as SMU_118c (The National Center for Biotechnology Information, NCBI), was found to have true hydrolase activities. SMU_118c was cloned, expressed, purified and further characterized for its respective hydrolytic activity towards ester-containing nitrophenyl substrates and the universal resin monomers bis-phenyl-glycidyl-dimethacrylate (bisGMA) and triethyleneglycol dimethacrylate (TEGDMA) at neutral (7.0) or cariogenic (5.5) pH. Mass spectrometry (MS) was used to verify the expression of SMU_118c protein in S. mutans UA159. RESULTS: Similar to the whole cell activity of S. mutans, SMU_118c showed the highest affinity toward para-nitrophenyl acetate (pNPA) and para-nitrophenyl butyrate (pNPB) vs. ortho-nitrophenyl butyrate (oNPB) and butyrylthiocholine iodide (BTC) (p < 0.05). The esterase retained 60% of its activity after 21 days and hydrolyzed bisGMA at a higher rate than TEGDMA at both neutral and cariogenic pH (p < 0.001), similarly to the predominant human salivary esterase degradative activity. MS confirmed that SMU_118c is an intracellular protein in S. mutans UA159 and expressed under pathogenic (pH 5.5) growth conditions. SIGNIFICANCE: The similarity in the activity profile to the whole S. mutans bacterial cell, the stability over time at cariogenic pH, the preference to hydrolyze bisGMA and confirmed expression profile suggest that SMU_118c could be a significant contributor to the whole bacterial degradative activity of S. mutans toward the degradation of resin composites, adhesives and the restoration-tooth interface, potentially accelerating restoration's failure. STATEMENT OF SIGNIFICANCE: The current study builds upon our highly-cited previous study by Bourbia et al., (JDR, 2013) that reported on that the cariogenic bacterium, S. mutans has esterase-like activities that enable the bacterium to degrade dental composites and adhesives. The current submission is the first to report on the isolation and characterization of the specific esterase activity (SMU_118c) from S. mutans that is a significant contributor to the whole bacterial degradative activity toward the hydrolysis of dental resins. This activity compromises the restoration-tooth interface, increases interfacial bacterial microleakage (Kermanshahi et al., JDR 2010), potentially contributing to the pathogenesis of recurrent caries around resin composite restorations. This represent a significant contribution to the field of biomaterials and their clinical performance.

K+ modulates genetic competence and the stress regulon of Streptococcus mutans.

Potassium (K+) is the most abundant cation in dental plaque fluid. Previously, we reported the link between K+ transport via Trk2 in Streptococcus mutans and its two critical virulence attributes: acid tolerance and surface adhesion. Herein, we build further on the intimate link between K+ levels and S. mutans biology. High (>25 mM) versus low (≤5 mM) K+ concentrations in the growth medium affected conformational epitopes of cell surface-localized adhesin P1. At low K+, the expression of stress response elements gcrR and codY, cell-adhesion-associated genes such as spaP and metabolism-associated genes such as bglP was induced at stationary phase (P<0.05), suggesting that K+-mediated regulation is growth phase-dependent and stress-sensitive. Production of the newly discovered secretory protein encoded by SMU_63c was strongly dependent on the availability of K+ and growth phase. This protein is a newly discovered regulator of genetic competence and biofilm cell density. Thus, the influence of K+ on DNA transformation efficiency was also examined. Compared with 25 mM K+ concentration, the presence of low K+ reduced the transformation frequency by 100-fold. Genetic transformation was abolished in a strain lacking a Trk2 system under all K+ concentrations tested. Consistent with these findings, repression of competence-associated genes, comS and comX, was observed under low environmental K+ conditions and in the strain lacking Trk2. Taken together, these results highlight a pivotal role for environmental K+ as a regulatory cation that modulates stress responses and genetic transformation in S. mutans.

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c.

Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1- and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

Transcriptional Profiling of the Oral Pathogen Streptococcus mutans in Response to Competence Signaling Peptide XIP.

In the cariogenic Streptococcus mutans, competence development is regulated by the ComRS signaling system comprised of the ComR regulator and the ComS prepeptide to the competence signaling peptide XIP (ComX-inducing peptide). Aside from competence development, XIP signaling has been demonstrated to regulate cell lysis, and recently, the expression of bacteriocins, small antimicrobial peptides used by bacteria to inhibit closely related species. Our study further explores the effect of XIP signaling on the S. mutans transcriptome. RNA sequencing revealed that XIP induction resulted in a global change in gene expression that was consistent with a stress response. An increase in several membrane-bound regulators, including HdrRM and BrsRM, involved in bacteriocin production, and the VicRKX system, involved in acid tolerance and biofilm formation, was observed. Furthermore, global changes in gene expression corresponded to changes observed during the stringent response to amino acid starvation. Effects were also observed on genes involved in sugar transport and carbon catabolite repression and included the levQRST and levDEFG operons. Finally, our work identified a novel heat shock-responsive intergenic region, encoding a small RNA, with a potential role in competence shutoff. IMPORTANCE Genetic competence provides bacteria with an opportunity to increase genetic diversity or acquire novel traits conferring a survival advantage. In the cariogenic pathogen Streptococcus mutans, DNA transformation is regulated by the competence stimulating peptide XIP (ComX-inducing peptide). The present study utilizes high-throughput RNA sequencing (RNAseq) to provide a greater understanding of how global gene expression patterns change in response to XIP. Overall, our work demonstrates that in S. mutans, XIP signaling induces a response that resembles the stringent response to amino acid starvation. We further identify a novel heat shock-responsive intergenic region with a potential role in competence shutoff. Together, our results provide further evidence that multiple stress response mechanisms are linked through the genetic competence signaling pathway in S. mutans.

Mechanistic, genomic and proteomic study on the effects of BisGMA-derived biodegradation product on cariogenic bacteria.

OBJECTIVES: Investigate the effects of a Bis-phenyl-glycidyl-dimethacrylate (BisGMA) biodegradation product, bishydroxypropoxyphenyl-propane (BisHPPP), on gene expression and protein synthesis of cariogenic bacteria. METHODS: Quantitative real-time polymerase chain reaction was used to investigate the effects of BisHPPP on the expression of specific virulence-associated genes, i.e. gtfB, gtfC, gbpB, comC, comD, comE and atpH in Streptococcus mutans UA159. Possible mechanisms for bacterial response to BisHPPP were explored using gene knock-out and associated complemented strains of the signal peptide encoding gene, comC. The effects of BisHPPP on global gene and protein expression was analyzed using microarray and quantitative proteomics. The role of BisHPPP in glucosyltransferase (GTF) enzyme activity of S. mutans biofilms was also measured. RESULTS: BisHPPP (0.01, 0.1mM) up-regulated gtfB/C, gbpB, comCDE, and atpH most pronounced in biofilms at cariogenic pH (5.5). The effects of BisHPPP on the constructed knock-out and complemented strains of comC from quorum-sensing system, implicated this signaling pathway in up-regulation of the virulence-associated genes. Microarray and proteomics identified BisHPPP-regulated genes and proteins involved in biofilm formation, carbohydrate transport, acid tolerance and stress-response. GTF activity was higher in BisHPPP-exposed biofilms when compared to no-BisHPPP conditions. SIGNIFICANCE: These findings provide insight into the genetic and physiological pathways and mechanisms that help explain S. mutans adaptation to restorative conditions that are conducive to increased secondary caries around resin composite restorations and may provide guidance to clinicians' decision on the selection of dental materials when considering the long term oral health of patients and the interactions of composite resins with oral bacteria.

Triethylene Glycol Up-Regulates Virulence-Associated Genes and Proteins in Streptococcus mutans.

Triethylene glycol dimethacrylate (TEGDMA) is a diluent monomer used pervasively in dental composite resins. Through hydrolytic degradation of the composites in the oral cavity it yields a hydrophilic biodegradation product, triethylene glycol (TEG), which has been shown to promote the growth of Streptococcus mutans, a dominant cariogenic bacterium. Previously it was shown that TEG up-regulated gtfB, an important gene contributing to polysaccharide synthesis function in biofilms. However, molecular mechanisms related to TEG's effect on bacterial function remained poorly understood. In the present study, S. mutans UA159 was incubated with clinically relevant concentrations of TEG at pH 5.5 and 7.0. Quantitative real-time PCR, proteomics analysis, and glucosyltransferase enzyme (GTF) activity measurements were employed to identify the bacterial phenotypic response to TEG. A S. mutans vicK isogenic mutant (SMΔvicK1) and its associated complemented strain (SMΔvicK1C), an important regulatory gene for biofilm-associated genes, were used to determine if this signaling pathway was involved in modulation of the S. mutans virulence-associated genes. Extracted proteins from S. mutans biofilms grown in the presence and absence of TEG were subjected to mass spectrometry for protein identification, characterization and quantification. TEG up-regulated gtfB/C, gbpB, comC, comD and comE more significantly in biofilms at cariogenic pH (5.5) and defined concentrations. Differential response of the vicK knock-out (SMΔvicK1) and complemented strains (SMΔvicK1C) implicated this signalling pathway in TEG-modulated cellular responses. TEG resulted in increased GTF enzyme activity, responsible for synthesizing insoluble glucans involved in the formation of cariogenic biofilms. As well, TEG increased protein abundance related to biofilm formation, carbohydrate transport, acid tolerance, and stress-response. Proteomics data was consistent with gene expression findings for the selected genes. These findings demonstrate a mechanistic pathway by which TEG derived from commercial resin materials in the oral cavity promote S. mutans pathogenicity, which is typically associated with secondary caries.

Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance.

UNLABELLED: Potassium (K(+)) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K(+) and a variety of K(+) transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K(+) acquisition in Streptococcus mutans and the importance of K(+) homeostasis for its virulence attributes. The S. mutans genome harbors four putative K(+) transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K(+) cotransporter (GlnQHMP), and a channel-like K(+) transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K(+)] less than 5 mM eliminated biofilm formation in S. mutans. The functionality of the Trk2 system was confirmed by complementing an Escherichia coli TK2420 mutant strain, which resulted in significant K(+) accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K(+)-dependent cellular response of S. mutans to environment stresses. IMPORTANCE: Biofilm formation and stress tolerance are important virulence properties of caries-causing Streptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment of S. mutans. K(+) is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K(+) transporters in S. mutans. We identified the most important system for K(+) homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K(+) for the activity of biofilm-forming enzymes, which explains why such high levels of K(+) would favor biofilm formation.

The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans.

UNLABELLED: In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ΔcopYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation. IMPORTANCE: S. mutans is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide. S. mutans is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of S. mutans include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of copYAZ (the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in S. mutans. Manipulating the pathways affected by copper and the copYAZ system may help to develop potential therapeutics to prevent S. mutans infection in and beyond the oral cavity.

In vitro manganese-dependent cross-talk between Streptococcus mutans VicK and GcrR: implications for overlapping stress response pathways.

Streptococcus mutans, a major acidogenic component of the dental plaque biofilm, has a key role in caries etiology. Previously, we demonstrated that the VicRK two-component signal transduction system modulates biofilm formation, oxidative stress and acid tolerance responses in S. mutans. Using in vitro phosphorylation assays, here we demonstrate for the first time, that in addition to activating its cognate response regulator protein, the sensor kinase, VicK can transphosphorylate a non-cognate stress regulatory response regulator, GcrR, in the presence of manganese. Manganese is an important micronutrient that has been previously correlated with caries incidence, and which serves as an effector of SloR-mediated metalloregulation in S. mutans. Our findings supporting regulatory effects of manganese on the VicRK, GcrR and SloR, and the cross-regulatory networks formed by these components are more complex than previously appreciated. Using DNaseI footprinting we observed overlapping DNA binding specificities for VicR and GcrR in native promoters, consistent with these proteins being part of the same transcriptional regulon. Our results also support a role for SloR as a positive regulator of the vicRK two component signaling system, since its transcription was drastically reduced in a SloR-deficient mutant. These findings demonstrate the regulatory complexities observed with the S. mutans manganese-dependent response, which involves cross-talk between non-cognate signal transduction systems (VicRK and GcrR) to modulate stress response pathways.

An intimate link: two-component signal transduction systems and metal transport systems in bacteria.

Bacteria have evolved various strategies to contend with high concentrations of environmental heavy metal ions for rapid, adaptive responses to maintain cell viability. Evidence gathered in the past two decades suggests that bacterial two-component signal transduction systems (TCSTSs) are intimately involved in monitoring cation accumulation, and can regulate the expression of related metabolic and virulence genes to elicit adaptive responses to changes in the concentration of these ions. Using examples garnered from recent studies, we summarize the cross-regulatory relationships between metal ions and TCSTSs. We present evidence of how bacterial TCSTSs modulate metal ion homeostasis and also how metal ions, in turn, function to control the activities of these signaling systems linked with bacterial survival and virulence.

A biochemical characterization of the DNA binding activity of the response regulator VicR from Streptococcus mutans.

Two-component systems (TCSs) are ubiquitous among bacteria and are among the most elegant and effective sensing systems in nature. They allow for efficient adaptive responses to rapidly changing environmental conditions. In this study, we investigated the biochemical characteristics of the Streptococcus mutans protein VicR, an essential response regulator that is part of the VicRK TCS. We dissected the DNA binding requirements of the recognition sequences for VicR in its phosphorylated and unphosphorylated forms. In doing so, we were able to make predictions for the expansion of the VicR regulon within S. mutans. With the ever increasing number of bacteria that are rapidly becoming resistant to even the antibiotics of last resort, TCSs such as the VicRK provide promising targets for a new class of antimicrobials.

Bioactive chitosan nanoparticles and photodynamic therapy inhibit collagen degradation in vitro.

INTRODUCTION: Collagen is the major structural protein of human dentin. Degradation of collagen by bacterial enzymes can facilitate microbial penetration, compromise structural/interfacial integrity, and lower resistance to fracture of dentin. We evaluated the ability of photodynamic therapy (PDT), bioactive chitosan nanoparticles (CSnp), or PDT in combination with CSnp to inhibit bacterial collagenase-mediated degradation of collagen. METHODS: Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT (rose bengal activated with 540 nm light at 40 J/cm(2)), or 1% CSnp followed by PDT. Samples, except those used as untreated controls, were exposed to Clostridium histolyticum collagenase (125 CDU/mL) for 24 hours. The soluble digestion products were assessed by hydroxyproline assay, and the remaining adherent collagen was quantified by picrosirius red staining. Fourier transform infrared spectroscopy, immunoblotting, and scanning electron microscopy were used to study the interaction between CSnp/PDT with type 1 collagen. The data were analyzed by 1-way analysis of variance and post hoc Tukey test. RESULTS: As assessed by hydroxyproline release into the medium, collagen treated with CSnp, PDT, or a combination of CSnp and PDT exhibited less degradation than untreated controls (3.6-fold, 1.7-fold, and 7.9-fold reduction, respectively; P < .05). Compared with all other treatments, GD-treated collagen was the most resistant to collagenolytic degradation (239.6-fold reduction, P < .05). The abundance of post-treatment residual collagen, as measured by picrosirius red staining, was inversely related to the extent of collagen degradation. Analysis of collagen cross-links with Fourier transform infrared spectroscopy showed that PDT or GD treatments enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls (P < .05). CONCLUSIONS: Combined photochemical cross-linking of rat tail collagen by PDT and binding to CSnp inhibit collagenolytic activity.

Structural and functional analysis of the N-terminal domain of the Streptococcus gordonii adhesin Sgo0707.

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

Molecular and structural basis of glutathione import in Gram-positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans.

Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-I-leg of GSSG is accommodated tightly via extensive interactions contributed by the N- and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine.

Mechanistic insights revealed by the crystal structure of a histidine kinase with signal transducer and sensor domains.

Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.