BrpA is involved in regulation of cell envelope stress responses in Streptococcus mutans

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.     

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Functional Genomics Approach to Identifying Genes Required for Biofilm Development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS_Sm) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans

The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.     

LuxS-mediated signaling in Streptococcus mutans is involved in regulation of acid and oxidative stress tolerance and biofilm formation

LuxS-mediated quorum sensing has recently been shown to regulate important physiologic functions and virulence in a variety of bacteria. In this study, the role of luxS of Streptococcus mutans in the regulation of traits crucial to pathogenesis was investigated. Reporter gene fusions showed that inactivation of luxS resulted in a down-regulation of fructanase, a demonstrated virulence determinant, by more than 50%. The LuxS-deficient strain (TW26) showed increased sensitivity to acid killing but could still undergo acid adaptation. Northern hybridization revealed that the expression of RecA, SmnA (AP endonuclease), and Nth (endonuclease) were down-regulated in TW26, especially in early-exponential-phase cells. Other down-regulated genes included ffh (a signal recognition particle subunit) and brpA (biofilm regulatory protein A). Interestingly, the luxS mutant showed an increase in survival rate in the presence of hydrogen peroxide (58.8 mM). The luxS mutant formed less biofilm on hydroxylapatite disks, especially when grown in biofilm medium with sucrose, and the mutant biofilms appeared loose and hive-like, whereas the biofilms of the wild type were smooth and confluent. The mutant phenotypes were complemented by exposure to supernatants from wild-type cultures. Two loci, smu486 and smu487, were identified and predicted to encode a histidine kinase and a response regulator. The phenotypes of the smu486 smu487 mutant were, in almost all cases, similar to those of the luxS mutant, although our results suggest that this is not due to AI-2 signal transduction via Smu486 and Smu487. This study demonstrates that luxS-dependent signaling plays critical roles in modulating key virulence properties of S. mutans.     

Malolactic fermentation by Streptococcus mutans

Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation of L-malate to yield L-lactic acid and concomitant reduction in acidity. The activity was inducible by L-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add standardized HCl solution to maintain a set pH value in pH-stat experiments. The net conclusion is that malate has the potential to be effective for alkalinization of dental plaque, although the fermentation is sensitive to fluoride and triclosan, which are commonly added to oral care products.     

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.     

Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms

Fluoride and zinc, alone or in combination at concentrations of 0.2 mM, inhibited production-secretion of glucosyltranferases by Streptococcus mutans UA159 growing in suspension cultures. Inhibition did not involve growth inhibition or starvation. Fluoride and zinc also inhibited glucan production, especially insoluble glucan, in fed-batch biofilms. Inhibition of biofilms appeared to be associated with starvation as indicated by markedly decreased ATP pools and iodophilic polysaccharide levels in biofilm cells. As insoluble glucans are important for virulence of mutans streptococci, the inhibitory actions of fluoride and zinc could significantly affect cariogenicity.     

生物膜定量分析仪对不同细菌早期生物膜形成能力的比较研究

目的通过生物膜定量分析仪来观察铜绿假单胞菌(Pseudomonas aeruginosa PAO1),变形链球菌(Streptococcus mutans UA159)以及大肠埃希菌(Escherichia coli MG1655)生物膜形成能力的不同,并以各菌株的吸光度值A600为参考,对3种菌株早期生物膜形成能力进行比较。方法通过向生物膜培养悬液中加入与细菌直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较3种菌株在生物膜形成上的差别。结果实验发现铜绿假单胞菌PAO1和大肠埃希菌MG1655的细菌增长速度基本相同,但铜绿假单胞菌PAO1的生物膜形成明显快于大肠埃希菌MG1655。大肠埃希菌MG1655和变形链球菌UA159的生物膜形成速度基本相同,但大肠埃希菌MG1655的细菌增长速度明显高于变形链球菌UA159。结论不同细菌有各自的生物膜形成模式。生物膜定量分析仪作为一种高效简便的检测手段,可用于生物膜早期形成的动态分析。     

Acid tolerance response of biofilm cells of Streptococcus mutans

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.     

The anti-biofouling effect of polyphenols against Streptococcus mutans

Biofouling is a process of surface colonization by microorganisms through cell adhesion and production of extracellular polymers (polysaccharides and proteins). It often causes serious problems in the chemical, medical and pharmaceutical industries. Recently, it was demonstrated that some natural phenolic compounds found in plants and vegetables have an antibiofouling effect, reducing formation of biofilm by Gram-negative bacteria. In this study, Streptococcus mutans, a Gram-positive bacterium was investigated for the antibiofouling effect of polyphenols. It was hypothesized that the two enzymes, glucosyltransferase and fructosyltransferase, produced by S. mutans, would be inhibited by the natural phenolic compounds. When these two enzymes were inhibited, less (or no) biofilms were formed. Enzymes were separated from a S. mutans culture medium, and their activities were measured with five different polyphenols using microtiter-plates and high-performance liquid chromatography. The results of minimum inhibitory concentration (MIC) were used to determine the enzyme inhibition effect of polyphenols on biofilm formation without killing the cells. Most of the polyphenols used showed considerable reduction of biofilm formation. Gallic acid and tannic acid showed significant enzyme inhibition effects below their MICs.     

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.     

Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.