Oral Pathogen Streptococcus Mutans Research Articles

Cloning and characterization of an α-enolase of the oral pathogen Streptococcus mutans that binds human plasminogen

Streptococcus mutans is the etiologic agent of dental caries and is a causative agent of infective endocarditis. While the mechanisms by which S. mutans cells colonize heart tissue is not clear, it is thought that bacterial binding to extracellular matrix and blood conponents is crucial in the development of endocarditis. Previously, we have demonstrated that S. mutans cells have the capacity to bind and activate plasminogen to plasmin. Here we report the first cloning and characterization of an α-enolase of S. mutans that binds plasminogen. The functional identity of the purified recombinant α-enolase protein was confirmed by its ability to catalyze the conversion of 2-phosphoglycerate to phosphoenolpyruvate. The protein exhibited a K m of 9.5 mM and a V max of 31.0 mM/min/mg. The α-enolase protein was localized in the cytoplasmic, cell wall and extracellular fractions of S. mutans. Binding studies using an immunoblot analysis revealed that human plasminogen binds to the enolase enzyme of S. mutans. These findings identify S. mutans α-enolase as a binding molecule used by this oral pathogen to interact with the blood component, plasminogen. Further studies of this interaction may be critical to understand the pathogenesis of endocarditis caused by S. mutans.

Age-dependent deficiency in saliva and salivary antibodies secretion in Down's syndrome

Objective Down's syndrome (DS) individuals suffer from an increased susceptibility to infections. Here, we assessed age-related changes in the salivary-specific humoral immunity of DS subjects. Design Parotid and whole saliva were collected from a young group of DS (YDS, n = 30, 23.3 ± 4 years), an older group of DS individuals (ODS, n = 10, 51.9 ± 8 years) and compared to two age-matched groups of healthy volunteers—a young group (YC, n = 29, 22.8 ± 5 years) and an older group (OC, n = 10, 48.4 ± 9 years). The levels of total IgA, and specific antibodies to three common oral pathogens ( Porphyromonas gingivalis, Actinobacillus ( Aggregatibacter) actinomycetemcomitans and Streptococcus mutans) were analysed. Results The limited increases in IgA concentrations could not compensate the dramatic reduction in the salivary flow rate observed in DS individuals. Therefore, the median secretion rates of the specific antibodies in whole and parotid saliva were 70–77% and 34–60% (respectively) lower in YDS individuals as compared to YC and farther 77–100% and 75–88% (respectively) lower in ODS compared to YDS. In contrast, the antibody secretion rates were similar for parotid saliva, or even increased for whole saliva of OC, compared with YC. Consequently, a dramatic cumulative extreme reduction (>92%) in the bacterial specific salivary antibodies differentiated the adult DS individuals from to their age-matched controls. Conclusions Our results indicate a severe immunodeficiency in the secretion rate of the specific salivary IgA response of in DS individuals which intensifies with age.

Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilms

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity.

Ent-Rosane and Labdane Diterpenoids from Sagittaria sagittifolia and Their Antibacterial Activity against Three Oral Pathogens

Seven new ent-rosane diterpenoids, sagittines A-G (1-7), together with one new labdane diterpene, 13-epi-manoyl oxide-19-O-alpha-l-2',5'-diacetoxyarabinofuranoside (8), were isolated from the whole plant of Sagittaria sagittifolia. The structures and relative configurations of 1-8 were characterized using spectroscopic means, chemical methods, and X-ray crystallography. Compounds 1-4 exhibited antibacterial activity against the oral pathogens Streptococcus mutans ATCC 25175 and Actinomyces naeslundiis ATCC 12104, with MIC values between 62.5 and 125 microg/mL. Compound 5 was active against only A. naeslundiis ATCC 12104, with an MIC value of 62.5 microg/mL.

LuxS controls bacteriocin production in Streptococcus mutans through a novel regulatory component

The oral pathogen Streptococcus mutans employs a variety of mechanisms to maintain a competitive advantage over many other oral bacteria which occupy the same ecological niche. Production of the bacteriocin, mutacin I, is one such mechanism. However, little is known about the regulatory mechanisms associated with mutacin I production. Previous work has demonstrated that the production of mutacin I greatly increased with cell density. In this study, we found that high cell density also triggered high level mutacin I gene transcription. However, this response was abolished upon deletion of luxS. Further analysis using real-time reverse transcription polymerase chain reaction (RT-PCR) demonstrated that in the luxS mutant transcription of both the mutacin I structural gene mutA and the mutacin I transcriptional activator mutR was impaired. Through microarray analysis, a putative transcription repressor annotated as Smu1274 in the Los Alamos National Laboratory Oral Pathogens Sequence Database was identified, which was strongly induced in the luxS mutant. Characterization of Smu1274, which we referred to as irvA, suggested that it may act as an inducible repressor to suppress mutacin I gene expression. A luxS and irvA double mutant regained the ability to produce mutacin I; whereas a constitutive irvA-producing strain was impaired in mutacin I production. These findings reveal a novel regulatory pathway for mutacin I gene expression, which may provide clues to the regulatory mechanisms of other cellular functions regulated by luxS in S. mutans.

Fluorescent Detection of Oral Pathogens by a Solid-Phase Immunoassay on PDMS.

We have developed an array of sensors for the oral pathogen Streptococcus mutans (S. mutans) using an enzymelinked linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) device. The model bacterial analyte, S. mutans, has been implicated in the initiation and progression of dental caries. The PDMS was modified with 3-aminopropyltriethoxysilance (APTS) and glutaraldehyde to covalently crosslink monoclonal anti-S. mutans immunoglobulin G (IgG) to the sensor surface. Successful IgG immobilization was verified by AFM and fluorescence imaging. Colloidal bacteria were captured on the sensor surface and labeled with immuno-active quantum dots (QDs), whose fluorescence was excited by an LED and detected by a CCD. The system was capable of detecting S. mutans concentrations as low as 6 106cells/ml in a 20 μl sample. This work represents a stable foundation for the development of a chair side diagnostic system capable of specific and sensitive detection of pathogens directly from oral fluid.

A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.

Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats. The S. mutans genome sequence was searched for ORFs containing 'A' repeats, and one novel gene, gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three 'A' repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K(D) of 2-3 nM. Construction of truncated derivatives of GbpD confirmed that the 'A' repeat region was essential for binding. Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the alpha/beta hydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site 'lipase box' and an 'oxyanion hole'. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S. mutans and another plaque bacterium, Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) of S. sanguinis, but had no effect on LTA from S. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm.

Antimicrobial constituents from goldenseal (the Rhizomes of Hydrastis canadensis) against selected oral pathogens.

Two new C-methyl flavonoids, 6,8-di- C-methylluteolin 7-methyl ether (1) and 6- C-methylluteolin 7-methyl ether (2), were isolated from a commercially available sample of the roots of Hydrastis canadensis, along with seven known compounds, berberine (3), beta-hydrastine (4), canadine (5), canadaline (6), isocorypalmine (7), canadinic acid (8), and beta-sitosterol 3- O-beta- D-glucoside (9). The structures of the new compounds 1 and 2 were elucidated on the basis of their spectral data including 1D and 2D NMR techniques. Of these isolates, berberine (3) and, to a lesser extent, 1 and 2, showed antimicrobial activity when evaluated against the oral pathogens Streptococcus mutans and Fusobacterium nucleatum. Berberine (3) exhibited an additive antimicrobial effect when tested against S. mutans in combination with 1.

Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes.

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn917-lac transposition occurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated. Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Tn 917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes

Previously, we demonstrated successful Tn 917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km r, repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn 917-lac (em r, β-gal +) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn 917-lac transposition occurred at a frequency of ca. 10 −6 with 20% of the resultant em r clones displaying varying levels of lacZ expression. Tn 917-lac mutants that expressed β-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42°C) temperature were isolated. Further characterization of one of the mutants with increased β-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The β-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3′-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn 917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Inhibitory Effect of Antibacterial Resin Composite against Streptococcus mutans

Dental resin composites with antibacterial activity may be useful for preventing the secondary caries frequently seen around restorations. Three types of silver–supported antibacterial materials (Novaron, Amenitop and AIS) inhibited the growth of the major oral pathogen Streptococcus mutans. Minimum inhibitory amounts in suspensions of Novaron, Amenitop and AIS against S. mutans were 40, 30 and 400 μg/ml, respectively. These antibacterial materials were incorporated into TEGDMA–UDMA–based light–activated resin composite, and the antibacterial activities of these composites were examined. Composites incorporating 5 wt% (N–5) or more of Novaron and 7 wt% (AM–7) or more of Amenitop inhibited the growth of S. mutans, whereas composites incorporating up to 10 wt% of AIS did not. No significant difference in either compressive or flexural strength was observed between the control and N–5 composites after 1 day and 6 months of storage in water. However, for AM–5 composite, there was a significant difference in either strength parameter between the two immersion periods. There was no or extremely little release of silver ions from the N–5 and AM–5 composites after 1 day or 6 months of immersion in water. These results indicated that a light–activated resin composite incorporating silver–supported antibacterial material such as Novaron may be clinically useful due to its inhibitory effect against S. mutans and favorable mechanical properties.

Construction and oral immunogenicity of a Salmonella typhimurium strain expressing a streptococcal adhesin linked to the [formula omitted] subunits of cholera toxin

A mafor adhesin from the oral pathogen Streptococcus mutans has been shown to be mucosally immunogenic upon genetic fusion with the cholera toxin A2 B subunits. To take advantage of the ability of Salmonella typhimurium to deliver cloned antigens to the mucosal inductive sites that would obviate the need for antigen purification, we expressed this chimeric construct in an attenuated S. typhimurium strain under the control of bacteriophage T7 transcription. Residual expression of the temperature-regulated T7 RNA polymerase at 30°C allowed production of the chimeric protein at 2–3% of the total soluble protein, but it was increased five to six times following induction at 37°C. Oral administration of a single dose of 10 9 recombinant Salmonella to mice resulted in serum IgG and salivary IgA antibody responses to Salmonella, cholera toxin, and the streptococcal adhesin, which were generally enhanced after a booster immunization.

Evidence that a low-affinity sucrose phosphotransferase activity in Streptococcus mutans GS-5 is a high-affinity trehalose uptake system

High-affinity sucrose uptake in the oral pathogen Streptococcus mutans is mediated by the phosphoenolpyruvate-dependent phosphotransferase system. In this report, we provide evidence that a lower-affinity sucrose phosphotransferase system in S. mutans GS-5, previously described by others, is in fact a high-affinity trehalose uptake system that also recognizes sucrose as a substrate.

Inhibition of Streptococcus mutans by the antibiotic streptozotocin: mechanisms of uptake and the selection of carbohydrate-negative mutants

The antibiotic streptozotocin [2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranoside], an analog of N-acetylglucosamine (NAG), has been shown to be useful for the selection of carbohydrate-negative and auxotrophic bacterial mutants. We have adapted this method for use with the oral pathogen Streptococcus mutans, a gram-positive, aerotolerant anaerobe that uses predominantly carbohydrates as carbon sources for growth. Streptozotocin selectively kills growing cells of S. mutans GS-5, and under appropriate conditions it can reduce the number of viable cells in actively growing cultures by a factor of 10(3) to 10(4). However, unlike in enteric bacteria, which take up this antibiotic by a single NAG-specific transport system, streptozotocin appears to be taken up in S. mutans by both a NAG-specific system and a relatively nonspecific system that is also involved in glucose, fructose, and mannose uptake. Combining streptozotocin selection and a screening procedure involving indicator plates containing triphenyl-tetrazolium chloride, we developed a general method for the isolation of carbohydrate-negative and auxotrophic mutants of S. mutans. A preliminary characterization of both pleiotropic and specific carbohydrate-negative mutants isolated by using this procedure is presented.

Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been indentified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.

ATP-dependent protein kinase activities in the oral pathogen Streptococcus mutans.

ATP-dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid-stable phosphoproteins were found. One was identified as HPr of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6-bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61-KD protein. In contrast, fructose 1-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61-KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho-(seryl)-HPr formed, but not that of the 61-KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP-dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase, is presented.

31P-NMR studies of the oral pathogen Streptococcus mutans: Observation of lipoteichoic acid

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg 2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.