Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis.

Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD(+) The oxidation of NADH to NAD(+) was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence.

Characterization of the arginolytic microflora provides insights into pH homeostasis in human oral biofilms.

A selected group of oral bacteria commonly associated with dental health is capable of producing alkali via the arginine deiminase system (ADS), which has a profound impact on the pH of human oral biofilms. An increased risk for dental caries has been associated with reduced ADS activity of the bacteria in oral biofilms. Arginolytic bacterial strains from dental plaque samples of caries-free and caries-active adults were isolated and characterized to investigate the basis for differences in plaque ADS activity between individuals. Fifty-six ADS-positive bacterial strains were identified by 16S rRNA gene sequencing, and their ADS activity levels were compared under standard growth conditions. The spectrum of bacterial ADS activity ranged from 45.2 to 688.0 units (mg protein)(-1). Although Streptococcus sanguinis was the most prevalent species, other Streptococcus sp. were also represented. Biochemical assays carried out using 27 ADS-positive strains under conditions known to induce or repress ADS gene expression showed substantial variation in arginolytic activity in response to pH, oxygen and the availability of carbohydrate or arginine. This study reveals that the basis for the wide spectrum of arginolytic expression observed among clinical strains is, at least in part, attributable to differences in the regulation of the ADS within and between species. The results provide insights into the microbiological basis for intersubject differences in ADS activity in oral biofilms and enhance our understanding of dental caries as an ecologically driven disease in which arginine metabolism moderates plaque pH and promotes dental health.

A tissue-dependent hypothesis of dental caries.

Current understanding of dental caries considers this disease a demineralization of the tooth tissues due to the acid produced by sugar-fermenting microorganisms. Thus, caries is considered a diet- and pH-dependent process. We present here the first metagenomic analysis of the bacterial communities present at different stages of caries development, with the aim of determining whether the bacterial composition and biochemical profile are specific to the tissue affected. The data show that microbial composition at the initial, enamel-affecting stage of caries is significantly different from that found at subsequent stages, as well as from dental plaque of sound tooth surfaces. Although the relative proportion of Streptococcus mutans increased from 0.12% in dental plaque to 0.72% in enamel caries, Streptococcus mitis and Streptococcus sanguinis were the dominant streptococci in these lesions. The functional profile of caries-associated bacterial communities indicates that genes involved in acid stress tolerance and dietary sugar fermentation are overrepresented only at the initial stage (enamel caries), whereas other genes coding for osmotic stress tolerance as well as collagenases and other proteases enabling dentin degradation are significantly overrepresented in dentin cavities. The results support a scenario in which pH and diet are determinants of the disease during the degradation of enamel, but in dentin caries lesions not only acidogenic but also proteolytic bacteria are involved. We propose that caries disease is a process of varying etiology, in which acid-producing bacteria are the vehicle to penetrate enamel and allow dentin degrading microorganisms to expand the cavity.

Potential Fatty Acid as Antibacterial Agent Against Oral Bacteria of Streptococcus mutans and Streptococcus sanguinis from Basil (Ocimum americanum): In vitro and In silico Studies.

BACKGROUND: Streptococcus mutans and Streptococcus sanguinis are Gram-positive bacteria that cause dental caries. MurA enzyme acts as a catalyst in the formation of peptidoglycan in bacterial cell walls, making it ideal as an antibacterial target. Basil (Ocimum americanum) is an edible plant that is diverse and has been used as a herbal medicine for a long time. It has been reported that basil has a pharmacological effect as well as antibacterial activity. The purpose of this study was to identify antibacterial compounds in O. americanum and analyze their inhibition activity on MurA enzyme. METHODS: Fresh leaves from O. americanum were extracted with n-hexane and purified by a combination of column chromatography on normal and reverse phases together with in vitro bioactivity assay against S. mutans ATCC 25175 and S. sanguinis ATCC 10556, respectively, while in silico molecular docking simulation of lauric acid (1) was conducted using PyRx 0.8. RESULTS: The structure determination of antibacterial compound by spectroscopic methods resulted in an active compound lauric acid (1). The in vitro evaluation of antibacterial activity in compound 1 showed Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of 78.13 and 156.3 ppm and 1250 and 2500 ppm against S. sanguinis and S. mutans, respectively. Further analysis and in silico evaluation determined lauric acid (1) as MurA Enzyme inhibitor. Lauric acid (1) showed a binding affinity of -5.2 Kcal/mol, which was higher than fosfomycin. CONCLUSION: Lauric acid showed the potential as a new natural antibacterial agent through MurA inhibition in bacterial cell wall biosynthesis.

Potential Allylpyrocatechol Derivatives as Antibacterial Agent Against Oral Pathogen of S. sanguinis ATCC 10,556 and as Inhibitor of MurA Enzymes: in vitro and in silico Study.

BACKGROUND: Streptococcus sanguinis is Gram-positive bacteria that contribute to caries. Many antibacterial agents are resistant against bacteria so that the discovery of new antibacterial agents is a crucial issue. Mechanism of antibacterial agents by disrupting cell wall bacteria is a promising target to be developed. One of the enzymes contributing to the cell wall is MurA enzyme. MurA is an enzyme catalyzing the first step of peptidoglycan biosynthesis in the cell wall formation. Inhibiting MurA is an effective and efficient way to kill the bacteria. Source of bioactive compounds including the antibacterial agent can be found in natural product such as herbal plant. Piper betle L. was reported to contain active antibacterial compounds. However, there is no more information on the antibacterial activity and molecular mechanism of P. betle's compound against S. sanguinis. PURPOSE: The study aims to identify antibacterial constituents of P. betle L. and evaluate their activities through two different methods including in vitro and in silico analysis. MATERIALS AND METHODS: The antibacterial agent was purified by bioactivity-guided isolation with combination chromatography methods and the chemical structure was determined by spectroscopic methods. The in vitro antibacterial activity was evaluated by disc diffusion and dilution methods while the in silico study of a compound binds on the MurA was determined using PyRx program. RESULTS: The antibacterial compound identified as allylpyrocatechol showed inhibitory activity against S. sanguinis with an inhibition zone of 11.85 mm at 1%, together with MIC and MBC values of 39.1 and 78.1 µg/mL, respectively. Prediction for molecular inhibition mechanism of allylpyrocatechols against the MurA presented two allylpyrocatechol derivatives showing binding activity of -5.4, stronger than fosfomycin as a reference with the binding activity of -4.6. CONCLUSION: Two allylpyrocatechol derivatives were predicted to have a good potency as a novel natural antibacterial agent against S. sanguinis through blocking MurA activity that causes disruption of bacterial cell wall.

Correlations of oral bacterial arginine and urea catabolism with caries experience.

BACKGROUND/AIM: Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects. METHODS: The subjects were divided into three groups according to caries status; 13 caries-free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries-active (CA) individuals (decayed teeth >or= 4); and 11 caries-experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real-time polymerase chain reaction was used to quantify the proportion of certain acid- or alkali-producing organisms in the samples. RESULTS: The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001). CONCLUSION: This study supports the theory that increased caries risk is associated with reduced alkali-generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali-generation in health and disease.

Role of Streptococcus sanguinis sortase A in bacterial colonization.

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis.

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

The origin and composition of multiple forms of dextransucrase from Streptococcus sanguis.

Multiple forms of purified dextransucrase have been observed in the presence of low detergent concentrations ( Luzio , G.A., Grahame , D. A. and Mayer, R.M. (1982) Arch. Biochem. Biophys. 216, 751-757). We now show these forms to arise partly as a result of proteolysis, and partly due to incomplete dissociation of the enzyme. Upon 25 degrees C incubation of the crude enzyme, several new bands appeared with little or no change in total activity. The electrophoretic pattern of aged, crude enzyme was similar to that of partially purified enzyme. Specific detection of dextransucrase on SDS gels revealed a single polypeptide of 174 kDa, which is converted to a 156 kDa protein during the aging process. The observation indicates the occurrence of proteolysis. The polypeptide composition of several of the enzyme forms was determined by two-dimensional electrophoresis. Forms Ia and IIa are composed exclusively of 174 kDa polypeptides. Forms III and IVa consist of 156 kDa units, as does the newly observed form Ic. It is likely that form Ib contains both 174 and 156 kDa polypeptides. The results indicate that incomplete dissociation of aggregates of the 174 kDa unit accounts for all of the bands observed on native gels run on fresh culture extracts. Additional enzyme forms result from aggregation of the 156 kDa proteolysis product alone, and from aggregation with unproteolyzed units to form hybrid aggregates.

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.

Inhibition of microbial IgA proteases by human secretory IgA and serum.

Microbial IgA proteases cleave human serum IgA1 immunoglobulin, but human secretory IgA is resistant to hydrolysis. We have found this resistance to be due to an inhibition of protease activity that is mediated by the Fab region of secretory IgA. The IgA proteases of the genus Neisseria are more sensitive to inhibition than is the protease of Streptococcus sanguis. There is also a serum inhibitor of Neisseria proteases that co-chromatographs with IgG. Monoclonal (myeloma) human IgG proteins and plasma protease inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin do not inhibit. Human sera do not contain inhibitor to S. sanguis protease activity. We conclude that microbial IgA proteases are subject to inhibition by IgA in secretions and IgG in serum, and this activity is most consistent with being an anti-enzyme antibody. The insensitivity of S. sanguis IgA protease to inhibition is unexplained but provides further evidence that the IgA proteases are structurally diverse.

Overproduction and characterization of the StsI restriction endonuclease.

The StsI restriction endonuclease (R-StsI), a class-IIS restriction endonuclease, found in Streptococcus sanguis 54, is a heteroschizomer of R-FokI, which recognizes 5'-GGATG-3'. To overproduce R-StsI in Escherichia coli, the coding region of R-StsI was joined to the tac promoter of an expression vector, pKK223-3. By introduction of the plasmid into E. coli UT481 cells expressing the fokIM gene, R-StsI activity was overproduced, from which R-StsI was purified homogeneously. We compared the properties of R-StsI with those of R-FokI. The optimum reaction conditions for R-StsI were quite different fron those for R-FokI. R-StsI is an acidic protein (pI 6.3). Anti-R-StsI serum did not cross-react with R-FokI, indicating three-dimensional structural dissimilarity. The domain structure of R-StsI was elucidated by digestion with trypsin. In the presence of substrate DNA, R-StsI was digested to yield 45-kDa N-terminal and 23-kDa C-terminal fragments. The amino-acid sequences around the trypsin cleavage sites of R-StsI and R-FokI were quite homologous.

Polymerase chain reaction amplification, cloning, sequence determination and homologies of streptococcal ATPase-encoding DNAs.

The highly conserved portion of the catalytic subunit (beta-subunit) of the membrane-bound, proton-translocating ATPase from three strains of oral streptococci has been amplified via the polymerase chain reaction. Hybridization studies demonstrated the existence of homology between Escherichia coli and Bacillus megaterium beta-subunit probes at the streptococcal DNA level. Highly degenerate primers, based on the E. coli and B. megaterium amino acid (aa) sequences, were used to amplify the homologues in Streptococcus mutans, S. sanguis and S. sobrinus. The 600 bp fragment from S. sobrinus has been cloned and its nucleotide (nt) sequence determined. Comparison of its nt and deduced aa sequence to that of E. coli and B. megaterium reveals a high degree of homology at the aa level.

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae.

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.

Purification and some properties of free and cell-associated dextransucrase from Streptococcus sanguis.

Dextransucrase of Streptococcus sanguis occurred in cell-free and cell-associated forms. Cell-free dextransucrase was purified by four successive chromatographies on Bio-Gel P 60, DEAE-cellulose, and Bio-Gel P 200 from the culture supernatant. The purification of cell-associated dextransucrase was made from the pellet of Streptococcus sanguis culture. Bacterial pellet was extracted with 1 M phosphate buffer (pH 6.0) and chromatographied by using an immunosorbent column. The two enzymes gave single bands in polyacrylamide gel electrophoresis. The molecular weight determined by sodium dodecyl sulfate polyacrylamide gel was about 100 000 daltons for the two forms of dextransucrases. The optimum pH of the cell-free and cell-associated enzymes was around 6 and the temperature optimum was broad for the two enzymes. The KM values for sucrose were respectively 2 mM and 3 mM for cell-free and cell-associated enzymes. When primer dextran was added, the reaction velocity increased but the KM for sucrose remained the same, and the KA for dextran was 200 muM for the two dextransucrases. Trehalose and maltose acted also as glucosyl residue acceptors. Purified enzymes had dextran synthesising activity and invertase-like activity. The same properties of the two forms of enzymes and the positive cross reaction against anti free and anti cell-associated globulins stongly suggest the identity of the two enzymes.

Arginine catabolism by strains of oral streptococci.

Arginine catabolism via the arginine deiminase pathway was found in Streptococcus sanguis 903. Citrulline and ornithine were released from resting cells incubated with arginine, arginine-containing peptides, or saliva. Maximum arginine catabolism by resting cells of S. sanguis 903 was found in the pH range 7-8 and at 45-48 degrees C. Arginine deiminase activity was found in the cytoplasm and in the cell-wall extract of this strain, while ornithine carbamoyltransferase activity was found in the cytoplasm and in extracts of cell walls and cytoplasmic membranes. Streptococcus mutans GS-5 and Streptococcus sobrinus strains OMZ 176 and 6715 could release arginine from salivary peptides but were incapable of significant arginine catabolism.

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin.

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.

Differentiation of Streptococcus sanguis and S. mitior by whole-cell rhamnose content and possession of arginine dihydrolase.

Whole-cell rhamnose concentrations were measured in 48 strains of streptococci resembling Streptococcus sanguis and S. mitior. Physiological characteristics were tested by the API-20/Strep system, and it was found that "typical" S. sanguis (arginine positive, aesculin positive) contained significant amounts of rhamnose, while "typical" S. mitior (arginine negative, aesculin negative) contained very low or undetectable amounts of rhamnose. Both groups contained dextran-positive and dextran-negative strains. Organisms that were more difficult to speciate (those giving positive results in the arginine or the aesculin test, but not in both) could also be divided into a rhamnose-positive and a rhamnose-negative group; with one exception, all of the rhamnose-positive strains gave a positive result with arginine in the API-20/Strep test. There were several discrepancies between the results of conventional tests for arginine and aesculin hydrolysis and those of the corresponding API test. The results of conventional tests for arginine hydrolysis did not correlate closely with rhamnose content, and conventional tests for aesculin hydrolysis were less sensitive than API tests. With the API-20/Strep system, S. sanguis can almost always be distinguished from S. mitior by its ability to hydrolyse arginine.

Changes of serological activity by alpha-L-fucosidase isolated from Streptococcus sanguis ATCC 10557.

alpha-L-Fucosidase isolated from the growth culture of Streptococcus sanguis ATCC 10557 acted on H- and Leb-blood group substances in porcine gastric lining, human gastric lining, human ovarian cyst fluid and human whole saliva, with consequent loss of H- and Leb -activities and a concomitant increase of Lea activity.

Purification and some properties of alpha-L-fucosidase isolated from Streptococcus sanguis.

alpha-L-Fucosidase acting on naturally occurring substrates was highly purified from the growth culture of Streptococcus sanguis ATCC 10557. The molecular weight of the enzyme was approximately 120,000 and the optimal pH was at 5.5. The purified enzyme showed specificity toward the linkage of alpha-(1 leads to 2) fucosides in oligosaccharides and glycoproteins. The enzyme released L-fucose from glycoprotein in human parotid saliva.