Identification and characterization of an autolysin gene, atlg, from Streptococcus sobrinus.

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans. In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA-inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA-deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus.

Detection of cariogenic bacterial genes by microchip electrophoresis.

Allele-specific PCR primers were designed, based on the dextranase (dex) gene, to identify Streptococcus mutans and Streptococcus sobrinus in dental plaque; subsequently, PCR products were detected via microchip electrophoresis (ME). In order to amplify the dex gene fragment of S. mutans and S. sobrinus, the following two PCR methods were established. Duplex allele-specific PCR primers were designed on a region of low DNA homology; furthermore, 211 and 126-bp fragments were amplified for S. mutans and S. sobrinus, respectively. Common PCR primer for single allele-specific PCR was designed so as to sandwich a region exhibiting high homology and amplify PCR product of different DNA size due to deletion of small DNA fragment in two dex genes. S. mutans and S. sobrinus were amplified, leading to the generation of 202 and 226-bp products, respectively. Analysis of DNA base size by ME in order to achieve efficient separation employed a polymer mixture consisting of hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO). In the presence of a polymer mixture of 0.125% PEO/0.6% HPMC, two PCR products were obtained, displaying degree of separation of 226 bp/202 bp of 2.67 (Rs). Reproducibility (CV%, n = 7) was 0.3%; additionally, separation time was approximately 85 s. This method was applied to the detection of S. mutans and S. sobrinus in dental plaque. Detection of the dex genes of S. mutans and S. sobrinus characterized by quickness, precision and high sensitivity was possible.

Membranotropic effects of the antibacterial agent Triclosan.

Triclosan is a broad-spectrum hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. To gain further insight into the mode of action of Triclosan we examined its effects on membranes by performing leakage titrations of different oral bacteria and studying its interaction with model membranes through the use of different biophysical techniques. There was negligible efflux of intracellular material from Streptococcus sobrinus at the minimal inhibitory concentration of Triclosan; whatever leakage did occur commenced only at much higher concentrations. In contrast, no leakage was observed at even the minimal bactericidal concentration for Porphyromonas gingivalis. Triclosan decreased the onset temperature of the gel to liquid-crystalline phase transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-3-[phospho-rac-glycerol] membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Steady-state fluorescence anisotropy measurements of different phospholipid/Triclosan samples using 3-(p-6-phenyl-1,3,5-hexatrienyl)-phenylpropionic acid were consistent with the calorimetric data. Incorporation of increasing amounts of Triclosan into 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) vesicles induced the nonlamellar H(II) hexagonal phase at low temperatures and new immiscible phases at temperatures below the main transition of DEPE. Taking these results together suggests that the antibacterial effects of Triclosan are mediated at least in part through its membranotropic effects, resulting in destabilized structures which compromise the functional integrity of cell membranes without inducing cell lysis.

A new mechanism of action of fluoride on streptococci.

Addition of fluoride to the growth medium of Streptococcus sobrinus resulted in a loss of glucan-binding lectin activity. Upon removal of fluoride, the bacteria regained their ability to bind glucan in about one generation. Chloramphenicol prevented recovery of ability to produce the lectin, showing the requirement for protein synthesis. Fluoride also caused a significant reduction in the tendency of the streptococci to form chains of cells, although the spent medium from fluoride-containing growth media did not dechain control cells. The fluoride thus does not activate autolytic enzymes. Importantly, 2-D electrophoresis and SDS-PAGE revealed several proteins were synthesized in the presence of fluoride that were not synthesized in its absence. It seems possible that fluoride places a stress on the bacteria, causing the synthesis of proteins that may play a role in protecting the cells against the stress. Numerous stress proteins are known for bacteria, including those resulting from heat, enzymes and osmotic shocks. The ability of fluoride to cause loss of glucan-binding may be related to its reported beneficial effects on oral health.

Immunolabeling of the major cell surface protein antigen of Streptococcus sobrinus with monoclonal antibody.

OBJECTIVE: The purpose of this study was to determine the accessibility of monoclonal antibody (McAb), specific for the major cell surface protein antigen (PAg) of Streptococcus sobrinus, to the surface of its native epitopes. MATERIALS AND METHODS: An indirect immunogold labeling technique was used to detect the reaction of McAb with S. sobrinus 6715. The reactions of polyclonal antibodies (PcAbs) against S. sobrinus 6715 or PAg with S. sobrinus 6715, S. mutans Ingbritt C and S. rattus BHT were studied as controls. RESULTS: The results indicated that PAg was localized on the outer cell surface of S. sobrinus, and McAb was reactive with only a few epitopes of the cell surface, whereas PcAbs were found to be reactive with more epitopes. CONCLUSIONS: McAb was specific for the PAg, but there was cross-reaction with S. mutans. Also there seemed to be an association between the fuzzy coat on the surface of S. sobrinus and PAg.

Concentrated bovine colostral whey proteins from Streptococcus mutans/Strep. sobrinus immunized cows inhibit the adherence of Strep. mutans and promote the aggregation of mutans streptococci.

The aim of this study was to examine the effect of bovine colostral whey proteins from cows immunized with Streptococcus mutans/Strep. sobrinus on the adherence and aggregation of caries-inducing bacteria, i.e., mutants streptococci. Both adherence and aggregation are important phenomena in the bacterial colonization of the human oral cavity. In all adherence experiments there was a significant difference between treatments by immune product (IP; from immunized cows) and a control product (CP; a similar product from non-immunized cows). The adherence of 35S-labelled Strep. mutans cells (serotype c) to parotid saliva-coated hydroxyapatite (SHA) was dose-dependently inhibited by both IP and CP if SHA was coated with either product before exposure to bacteria, but markedly lower concentrations of IP than CP were effective. When instead of SHA the bacterial cells were pretreated with IP or CP, only IP strongly and dose-dependently inhibited streptococcal adherence. When bacteria, IP or CP, and SHA were incubated simultaneously, a significant difference between IP and CP treatments was again found. Further, IP effectively aggregated both Strep. mutans and Strep. sobrinus cells, whereas hardly any effect was seen with CP. Both IP and CP aggregated the control bacterium Strep. sanguis, which affected the adherence of the pretreated bacteria.

Structural and antigenic characteristics of Streptococcus sobrinus glucan binding proteins.

Three purified glucan binding proteins (GBP-2, GBP-3, and GBP-5) from Streptococcus sobrinus 6715 were compared structurally by mass spectroscopy of tryptic fragments and antigenically by Western blot analysis with rat antisera to each GBP or to peptides containing putative glucan binding epitopes of mutans streptococcal glucosyltransferases. Structural and antigenic analyses indicated that GBP-3 and GBP-5 are very similar but that both are essentially unrelated to GBP-2. None of these S. sobrinus GBPs appeared to have a strong antigenic relationship with GBPs from Streptococcus mutans. Thus, S. sobrinus GBP-2 and GBP-3 appear to be distinct proteins with potentially different functions. S. sobrinus GBP-5 may be a proteolytic fragment of GBP-3, or, alternatively, the genes coding for these proteins may be closely related.

Whole-cell protein electrophoretic analysis of viridans streptococci: evidence for heterogeneity among Streptococcus mitis biovars.

One hundred reference strains representing all species belonging to the different phylogenetic lineages of the viridans streptococci were examined by means of one-dimensional whole-organism protein electrophoresis. For most of the species examined, multiple strains characterized by DNA-DNA hybridization were included and, wherever described, representatives of different biochemical variants were analysed. Most species were clearly differentiated. The data support the viewpoint that members of the Streptococcus anginosus group constitute a single species and indicate that Streptococcus mitis biovar 2 is a heterogeneous taxon comprising strains from several streptococcal species.

Purification, and biochemical and biological characterization of an immunosuppressive and lymphocyte mitogenic protein secreted by Streptococcus sobrinus.

An immunosuppressive/mitogenic (ISM) protein was purified from the supernatants of cultures of Streptococcus sobrinus with an isoelectric point of 4.75 and a relative molecular mass of 38 kDa (p38). Treatment of C57BL/6 mice with p38 induced an increase in the numbers of non-specific splenic Ig-secreting plaque-forming cells (PFC) with peak responses on day 3 for IgM-secreting PFC and on day 5 for IgG-secreting PFC, with an isotype pattern consisting predominantly of IgG2a and IgG2b. This increase was accompanied by a lymphocyte blastogenic response of both T and B lymphocytes. The in vitro effects of p38 on pure B, T and total splenic lymphocytes indicated that this ISM protein was primarily a B cell mitogen, being T cells activated subsequently by the generation of B blasts. Suppression of the murine primary immune response against sheep red blood cells was observed in C57BL/6 mice treated 4 days before with p38. The amino acid sequence of the N-terminus of p38 has a significant similarity with several enolases, particularly with rabbit enolase. However, the biological effects ascribed to p38 have not been detected after in vivo treatment with that enolase. The immunosuppressive effect of p38 was abrogated by depletion of IL-10 but not of IL-4. In agreement with this observation IL-10 was the only cytokine detected in serum of C57BL/6 mice after p38 treatment and the peak of serum levels was observed as soon as 2 h after treatment.

Multiple glucan-binding proteins of Streptococcus sobrinus.

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.

Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.

In vitro modulation of oral bacterial adhesion to saliva-coated hydroxyapatite beads by milk casein derivatives.

Bovine caseinate, derivatives of its glycosylated moiety [caseinoglycomacropeptide (CGP)], and caseinophosphopeptides were evaluated as inhibitors of adhesion of oral bacteria to saliva-coated hydroxyapatite beads (S-HA). All milk casein-derived components behaved as potent inhibitors of Streptococcus sanguis OMZ 9 and Streptococcus sobrinus OMZ 176 adhesion to S-HA, whereas neither bovine serum albumin nor polyethyleneglycol were able to interfere with the adhesion of these strains. By contrast, none of the molecular species tested was able to inhibit the attachment of Actinomyces viscosus Ny 1 to S-HA. On the other hand, casein derivatives were shown to displace human serum albumin from S-HA beads. They were also able to bind to the bacterial cell surface of all strains examined. Collectively, these findings suggest that interactions between acidic casein-derived milk components and the biological surfaces involved in bacterial adhesion to S-HA result in an inhibitory effect that is selective for the oral streptococci examined.

Grouping of oral streptococcal species using Fourier-transform infrared spectroscopy in comparison with classical microbiological identification.

The grouping and identification made by Fourier-transform infrared spectroscopy (FT-IR) of 40 oral streptococcal strains was compared with their known taxonomic positions. Grouping was obtained by cluster analysis on the spectral distances between the first derivative spectra of the strains. Spectra were divided into five spectral 'windows' and cluster analysis was either on either a combination of all five windows or on a selected window. The oral streptococci were divided into two main clusters, mutans streptococci and other streptococci. This division was based mainly on the spectral information contained in the window between 1300-900 cm-1, indicative of polysaccharide and phosphate-carrying compounds. In the grouping based on the entire spectrum, species clusters could be observed for Streptococcus crista, Strep. salivarius and Strep. rattus. A few strains were placed in wrong clusters because of intra-strain variance. It is concluded that the grouping of these oral streptococcal species was good and similar to the classical taxonomy and that FT-IR might develop into a new identification method for oral streptococci.

An 87-kilodalton glucan-binding protein of Streptococcus sobrinus B13.

An 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serotype d) was isolated and purified from extracellular culture supernatant by using affinity chromatography on Sephadex G-50 and elution with a guanidine HCl gradient. Western blot (immunoblot) analysis showed it to be antigenically related, but not completely identical, to the 74-kDa GBP of Streptococcus mutans Ingbritt. The 87-kDa GBP has no glucosyltransferase activity. A possible role for this GBP in the cariogenicity of S. sobrinus B13 is suggested.

Purification and properties of extracellular mutacin, a bacteriocin from Streptococcus sobrinus.

Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9.7%. The molecular mass of the purified mutacin preparation was estimated to be 6.5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 degrees C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by alpha-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.

Comparison of the physicochemical surface properties of Streptococcus rattus with those of other mutans streptococcal species.

Mutans streptococci comprise a group of seven closely related, yet distinct species. The distinction between the four species used in this study, namely Streptococcus sobrinus, Streptococcus cricetus, Streptococcus rattus, and Streptococcus mutans, has been made only recently on the basis of DNA homologies. In order to determine if there is a difference in the physicochemical surface properties of these species, strains were characterized by contact angles, zeta potentials and isoelectric points (IEP), elemental surface compositions by X-ray photoelectron spectroscopy, and molecular moieties by infrared spectroscopy. Contact angles, particularly when measured with water, can be considered a measure of cell surface hydrophobicity; zeta potentials reflect the charge of the outermost cell surface; X-ray photoelectron spectroscopy yields the relative abundance of carbon, oxygen, nitrogen, and phosphorus over the outer 5 nm of the bacterial cell surface; infrared spectroscopy enables a molecular characterization in terms of proteins, phosphates, and polysaccharides. All four species were homogeneous with regard to their physicochemical surface properties. However, the S. rattus species were clearly different from the others on the basis of the low water contact angle (21 +/- 2 vs. 26-31 degrees), highly negative zeta potential and lack of IEP, and high oxygen/carbon (0.50 +/- 0.02 vs. 0.41-0.43) and phosphorus/carbon (0.016 +/- 0.001 vs. 0.006-0.008) surface concentration ratios. Amongst the other differences observed, each species had a characteristic pH dependence of their zeta potential measured in phosphate buffer, yielding an IEP of 1.7, 2.1, and 2.5 for S. cricetus, S. sobrinus, and S. mutans, respectively. However, a cluster analysis on the zeta potential data showed only an isolated cluster for the S. rattus species. Thus it is likely that the higher cariogenicity of S. sobrinus with respect to S. cricetus and S. mutans is, in addition to a higher acidogenicity, due to the smaller negative surface charge, giving an increased adhesion to negatively charged tooth surfaces. S. rattus is expected to be the least adherent strain in the oral cavity as it carries a relatively high negative surface charge.