Gunnel Svensäter

Novel Two-Component Regulatory System Involved in Biofilm Formation and Acid Resistance in Streptococcus mutans

The abilities of Streptococcus mutans to form biofilms and to survive acidic pH are regarded as two important virulence determinants in the pathogenesis of dental caries. Environmental stimuli are thought to regulate the expression of several genes associated with virulence factors through the activity of two-component signal transduction systems. Yet, little is known of the involvement of these systems in the physiology and pathogenicity of S. mutans. In this study, we describe a two-component regulatory system and its involvement in biofilm formation and acid resistance in S. mutans. By searching the S. mutans genome database with tblastn with the HK03 and RR03 protein sequences from S. pneumoniae as queries, we identified two genes, designated hk11 and rr11, that encode a putative histidine kinase and its cognate response regulator. To gain insight into their function, a PCR-mediated allelic-exchange mutagenesis strategy was used to create the hk11 (Em(r)) and rr11 (Em(r)) deletion mutants from S. mutans wild-type NG8 named SMHK11 and SMRR11, respectively. The mutants were examined for their growth rates, genetic competence, ability to form biofilms, and resistance to low-pH challenge. The results showed that deletion of hk11 or rr11 resulted in defects in biofilm formation and resistance to acidic pH. Both mutants formed biofilms with reduced biomass (50 to 70% of the density of the parent strain). Scanning electron microscopy revealed that the biofilms formed by the mutants had sponge-like architecture with what appeared to be large gaps that resembled water channel-like structures. The mutant biofilms were composed of longer chains of cells than those of the parent biofilm. Deletion of hk11 also resulted in greatly diminished resistance to low pH, although we did not observe the same effect when rr11 was deleted. Genetic competence was not affected in either mutant. The results suggested that the gene product of hk11 in S. mutans might act as a pH sensor that could cross talk with one or more response regulators. We conclude that the two-component signal transduction system encoded by hk11 and rr11 represents a new regulatory system involved in biofilm formation and acid resistance in S. mutans.

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.

Multiple stress responses in Streptococcus mutans and the induction of general and stress-specific proteins.

The authors have previously demonstrated that Streptococcus mutans shows an exponential-phase acid-tolerance response following an acid shock from pH 7.5 to 5.5 that enhances survival at pH 3.0. In this study the response of S. mutans H7 to acid shock was compared with the responses generated by salt, heat, oxidation and starvation. Prior induction of the acid-tolerance response did not cross-protect the cells from a subsequent challenge by the other stresses; however, prior adaptation to the other stresses, except heat (42 degrees C), protected the cells during a subsequent acid challenge at pH 3.5. Starvation by fivefold dilution of the basal medium (BM) plus fivefold reduction of its glucose content increased the numbers of survivors 12-fold, whereas elimination of glucose from fivefold-diluted BM led to a sevenfold enhancement compared to the control cells; this indicated a relationship between the acid and starvation responses. The stress responses were further characterized by comparing the 2D electrophoretic protein profiles of exponential-phase cells subjected to the various stress conditions. Cells were grown to exponential phase at pH 7.5 (37 degrees C) and then incubated for 30 min under the various stress conditions in the presence of 14C-labelled amino acids followed by cell extraction, protein separation by 2D gel electrophoresis and image analysis of the resulting autoradiograms. Using consistent twofold or greater changes in IOD % as a measure, oxidative stress resulted in the upregulation of 69 proteins, 15 of which were oxidation-specific, and in the downregulation of 24 proteins, when compared to the control cells. An acid shock from pH 7.5 to 5.5 enhanced synthesis of 64 proteins, 25 of them acid-specific, while 49 proteins exhibited diminished synthesis. The dilution of BM resulted in the increased formation of 58 proteins, with 11 starvation-specific proteins and 20 showing decreased synthesis. Some 52 and 40 proteins were enhanced by salt and heat stress, with 10 and 6 of these proteins, respectively, specific to the stress. The synthesis of a significant number of proteins was increased by more than one, but not all stress conditions; six proteins were enhanced by all five stress conditions and could be classified as general stress proteins. Clearly, the response of S. mutans to adverse environmental conditions results in complex and diverse alterations in protein synthesis to further cell survival.

The effects of antimicrobials on endodontic biofilm bacteria

INTRODUCTION In the present study, confocal microscopy, a miniflow cell system, and image analysis were combined to test in situ the effect of antimicrobials and alkali on biofilms of Enterococcus faecalis, Lactobacillus paracasei, Streptococcus anginosus, and Streptococcus gordonii isolated from root canals with persistent infections. METHODS Biofilms formed for 24 hours were exposed for 5 minutes to alkali (pH = 12), chlorhexidine digluconate (2.5%), EDTA (50 mmol/L), and sodium hypochlorite (1%). The biofilms were then characterized by using fluorescent markers targeting cell membrane integrity (LIVE/DEAD) and metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride and fluorescein diacetate). In addition, the biofilm architecture and the extent to which coating of the substrate surface with collagen influenced the resistance pattern to the chemicals were also analyzed. RESULTS NaOCl (1%) affected the membrane integrity of all organisms and removed most biofilm cells. Exposure to EDTA (50 mmol/L) affected the membrane integrity in all organisms but failed to remove more than a few cells in biofilms of E. faecalis, L. paracasei, and S. anginosus. Chlorhexidine (2.5%) had a mild effect on the membrane integrity of E. faecalis and removed only 50% of its biofilm cells The effects were substratum-dependent, and most organisms displayed increased resistance to the antimicrobials on collagen-coated surfaces. CONCLUSIONS The biofilm system developed here was sensitive and differences in cell membrane integrity and removal of biofilm cells after exposure to antimicrobials commonly used in endodontics was discernible.

Acid Tolerance of Biofilm Cells of Streptococcus mutans

ABSTRACT Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.

The Acid-Tolerant Microbiota Associated with Plaque from Initial Caries and Healthy Tooth Surfaces

The intent of this study was to compare the inherent acid tolerance of bacteria in samples of dental plaque from tooth sites in subjects with and without initial caries. Plaque was collected from approximal surfaces showing early enamel caries and from healthy tooth surfaces in the same subjects, as well as from enamel surfaces of caries-free individuals. In addition to plating on blood agar, the plaque samples were plated directly on non-selective solid agar medium buffered to pH 7.0, 6.0, 5.5, 5.0, 4.5 and 4.0 to avoid any loss of adaptation to acid during primary isolation of plaque bacteria. The results showed that approximately 50% of the total cultivable plaque microbiota from caries, as well as healthy tooth sites, was able to grow at pH 5.5 and 1% at pH 5.0, pH values regarded as critical for the demineralization of tooth enamel. At pH 5.0, members of the genus Streptococcus were the dominant group, but mutans streptococci accounted for less than half of the streptococcal viable count. The other acid-tolerant streptococcal isolates included Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis,Streptococcus salivarius and Streptococcus sanguis. Analysis of the results indicated that the mutans streptococci in dental plaque were highly variable with respect to acid tolerance, and that both caries and healthy sites harboured significant numbers of mutans streptococci that were not acid-tolerant.

Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions.

Plasminogen binding by bacteria is a virulence factor important for the entry and dissemination of bacteria in the body. A wide variety of bacteria bind plasminogen, including both organisms causing disease and components of the normal oral flora. The purpose of this study was to examine the characteristics of plasminogen binding by six clinical isolates of oral streptococci from both dental plaque and inflammatory lesions. All the strains bound plasminogen with approximately the same affinity, and binding was specific and lysine-dependent as evidenced by its inhibition by epsilon-aminocaproic acid. All of the test strains were capable of activating bound plasminogen to plasmin without the addition of a plasminogen activator, and subsequent analysis revealed the presence of streptokinase in all strains. However, the streptococci exhibited fibrinolytic activity only in the presence of plasminogen and this could be inhibited by the addition of epsilon-aminocaproic acid. SDS-PAGE and 2D gel electrophoresis coupled with plasminogen ligand blotting showed that only a subset of the total proteins (2-15) were involved in the binding of plasminogen. Partial identification of the binding proteins revealed that four glycolytic enzymes, enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase, were predominant in binding plasminogen. The binding of plasminogen by bacteria from pus did not differ from that of the strains from supragingival plaque. The findings illustrate how apparently innocuous commensal bacteria are capable of utilizing a mechanism that is generally regarded as being of importance to pathogenicity and suggest an additional role of plasminogen binding.

Protein expression by Streptococcus mutans during initial stage of biofilm formation.

ABSTRACT Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a 14C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished ≥1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.

Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N(5)-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.