Streptococcus Sanguis Cells Research Articles

Adherent bacteria cells in five dental materials: sonication effect.

Adherent bacterial cells on the surfaces of two dental porcelain ceramics, three composite resins and human enamel were examined using four types of bacteria strains. Their adherent cells were counted on saliva-coated and uncoated material surfaces after sonication, and contact angle and zeta potential were measured for each adherent cell tested. A correlation between contact angle and bacterial cells on an uncoated surface was found to be higher in two Streptococcus sanguis cells than in S. mutans Ingbritt and S. sobrinus OMZ 176, whereas there appeared to be a higher correlation between S. mutans Ingbritt or S. sobrinus OMZ 176 and zeta potential on the uncoated surface. On the saliva-coated surface, a significantly high correlation was found between the adherent cells, with the exception of S. sanguis ATCC 10 557, and the zeta potential. Contact angle and zeta potential values were small when the surfaces of the materials were coated with saliva, as compared with those on the uncoated surface. The sonication condition (120 s) of adherent cells on the surface of the material significantly depended on the types of bacteria cells, showing that S. mutans Ingbritt (>50-60%) had a greater removal percentage than the others (<50%).

Unexpectedly high levels of some presumably protective secretory immunoglobulin A antibodies to dental plaque bacteria in salivas of both caries-resistant and caries-susceptible subjects.

The role of salivary antibodies in protection against cariogenic bacteria is actually a matter of debate. Correlation between caries experience and naturally occurring antibodies was extensively investigated. Comparison of salivary antibodies from 21 caries-resistant and 22 caries-susceptible subjects was carried out by using a new quantitative method. Secretory immunoglobulin A (S-IgA) antibodies to Streptococcus sobrinus and Streptococcus sanguis cells were detected in all salivas and at similar levels in both groups. When assayed with two major antigens from S. sobrinus, i.e., protein antigen I/II and cell wall carbohydrates, only specific activities of antibodies to the protein component were increased (P < 0.01), but this occurred unexpectedly in the caries-susceptible group. Western blot (immunoblot) analysis with the culture supernatant and cell wall proteins from S. sobrinus showed the same antibody specificity in both groups. No selective increase of the protease-resistant S-IgA2 subclass was found, and avidities of antibodies to both antigen I/II and cell wall carbohydrates were similar. Our results demonstrate that naturally induced S-IgA antibodies against S. sanguis, S. sobrinus, and the major antigens of the latter are not sufficient to inhibit caries development.

Purification and partial characterization of a 65-kDa platelet aggregation-associated protein antigen from the surface of Streptococcus sanguis.

Cells of Streptococcus sanguis express a collagen-like immunodeterminant (class II antigen) on their cell walls that induces aggregation of platelets in plasma. These platelet aggregation-associated proteins (PAAPs) are recovered in cell-free preparations obtained from cells of S. sanguis after 5 min of sonic or limited trypsin treatment. Pretreatment of platelet-rich plasma with these soluble preparations selectively inhibits platelet aggregation in response to S. sanguis cells. A PAAP antigen was isolated and purified from minimal tryptic digests of S. sanguis cells using (i) immunoaffinity chromatography or (ii) gel filtration and ion-exchange chromatography. A monospecific rabbit antiserum was prepared against PAAP (from procedure ii) and used to verify identity with PAAP fragments in different preparations. Criteria of purity included single precipitins in immunoelectrophoresis and crossed immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblot, and COOH (Lys)- and NH2 (Pro)-terminal analyses. The 65-kDa (p65) antigen isolated by immunoaffinity chromatography had 50-fold greater specific inhibitory activity in S. sanguis-induced PRP aggregation than the original tryptic digest and about 1.4 times that recovered by sequential column chromatography. Amino acids of the p65 PAAP fragment constituted 89.5% of the total dry weight, with glycine, lysine, and glutamic acid predominant. Lesser amounts of proline were also noted. Monosaccharides, including glucose and galactose, comprised 4.0% of the total. A platelet interactive determinant of p65 was localized to a 23-kDa tryptic fragment after further trypsin treatment. Amino acids of this 23-kDa fragment constituted 99.8% of the total dry weight. In their native state on the cell wall of platelet-interactive strains of S. sanguis, platelet aggregation-associated proteins are probably assembled on fibrils as polyvalent agonists.

Properties of a phosphocarrier protein (HPr) extracted from intact cells of Streptococcus sanguis.

Cells of Streptococcus sanguis strain Challis were incubated with sodium lauroylsarcosinate to extract surface proteins. A polypeptide of apparent molecular mass 16 kDa comprising about 12% of the extract was purified using anion-exchange chromatography. The polypeptide was shown to be a phosphocarrier protein (HPr) that could also be found in the soluble (cytoplasmic) fraction from cells broken by homogenization with glass beads. In vivo labelling of S. sanguis cells with 32Pi showed that the polypeptide carried a heat- and acid-stable phosphorylation and that during sucrose starvation the HPr became dephosphorylated. Antiserum raised to the S. sanguis HPr reacted on Western blots with HPrs from all oral streptococci tested, together with strains of S. pyogenes and S. salivarius, but not with HPrs from S. faecalis or S. bovis, nor with proteins from Staphylococcus aureus, Bacillus subtilis, Actinomyces viscosus and various lactobacilli. The S. sanguis HPr had a high content of alanine (17.2%) and was similar in overall amino acid composition to the HPrs from S. mutans an S. salivarius. The N-terminal residues (to 37) of the S. sanguis HPr showed strong sequence identity (82%) with the N-terminal sequence of S. faecalis HPr. It is suggested that HPr in S. sanguis is associated closely with the cytoplasmic membrane. Non-disruptive methods of removing cell-surface proteins from streptococci effect release of HPr and possibly other cytoplasmic components.

Cell-surface Proteins of Streptococcus sanguis Associated with Cell Hydrophobicity and Coaggregation Properties

Incubating cells of Streptococcus sanguis with sodium lauroyl sarcosinate, under conditions that did not cause lysis, solubilized material comprising 5-8% of the cell dry weight. The treatment reduced cell hydrophobicity, and reduced the ability of the cells to coaggregate with Actinomyces spp. The extract contained about 20 polypeptides and these were identified as being cell-surface components on the basis of one or more of the following criteria: being degraded when cells were incubated with protease; being labelled when cells were iodinated using a lactoperoxidase-catalysed reaction; reacting with antibodies raised to fixed whole cells. Eight of the polypeptides accounted for more than 70% of the total protein extracted, and one component (molecular mass 16 kDa) was hydrophobic. The cell-surface proteins described are implicated in cell hydrophobicity and coaggregation.

Streptococcal R plasmid pIP501: endonuclease site map, resistance determinant location, and construction of novel derivatives.

The streptococcal resistance plasmid pIP501 (30 kilobase pairs [kb]) encodes resistance to chloramphenicol (Cmr) and erythromycin (Emr) and is capable of conjugative transfer among numerous streptococcal species. By using a streptococcal host-vector recombinant DNA system, the Cmr and Emr determinants of pIP501 were localized to 6.3-kb HindIII and 2.1-kb HindIII-AvaI fragments, respectively. pIP501 was lost at a frequency of 22% in Streptococcus sanguis cells grown at 42 degrees C but was stable in cells grown at 37 degrees C (less than 1% frequency of loss). Sequences from a cryptic multicopy plasmid, pVA380-1, were substituted for the pIP501 Emr determinant in vitro, and the resulting recombinant plasmid, designated pVA797, was recovered in transformed S. sanguis cells. The replication of pVA797 was governed by the pVA380-1 sequences based on temperature-stable replication and incompatibility with pVA380-1-derived replicons. The self-ligation of partially cleaved HindIII pIP501 DNA fragments allowed the localization of a pIP501 region involved in autonomous plasmid replication. A small pIP501 derivative (pVA798) obtained from this experiment had a greatly increased copy number but was unstably inherited. Our data indicate that the sequences encoding the resistance determinants and some of the plasmid replication machinery are relatively clustered on the pIP501 molecule. The properties of pVA797 and pVA798 indicate that these molecules will enhance current streptococcal genetic systems from the standpoint of conjugative mobilization (pVA797) and gene amplification (pVA798).

Degradation of the microbial and salivary components participating in human dental plaque formation by proteases elaborated by plaque bacteria

Twenty-eight strains of facultative, Gram-positive, sporulating bacilli which produce caseinolytic enzymes were isolated from human early dental plaque. A major component of the extracellular caseinolytic enzymes elaborated by strong producers seemed to be neutral zinc proteases. The extracellular proteases inactivated glucosyltransferase of Streptococcus mutans and inhibited the synthesis of adherent glucans from sucrose. The enzymes also degraded the Strep. mutans cell-surface receptor for dextran and glucan, the receptor for salivary agglutinins, located on Streptococcus sanguis cells, and the surface component of Actinomyces viscosus cells involved in co-agglutination with Strep. sanguis cells. The enzymes hydrolysed human whole saliva proteins, which seemed to result in loss of the ability to agglutinate Strep. sanguis cells.

Heterofermentative glucose metabolism by glucose transport-impaired mutants of oral streptococcal bacteria during growth in batch culture

Spontaneous mutants defective in a membrane component of the phosphoenolpyruvate-glucose phosphotransferase system were isolated by plating cells of Streptococcus sanguis 10556, Streptococcus mutans GS5-2 and NCTC 10449 on agar containing lactose and 2-deoxyglucose. Toluenized cells of these mutants were defective in their ability to catalyse the phosphoenolpyruvate-dependent phosphorylation of 2-deoxyglucose. The parental strains were mainly homofermentative when grown in batch culture in the presence of various sugars. Nevertheless, the mutants produced acetate, formate and ethanol when cultured in the presence of glucose but were homofermentative when grown in the presence of lactose or maltose. Analysis of one mutant isolated from Strep. sanguis (mutant GS26) revealed normal levels of glucokinase, glucose-6-phosphate dehydrogenase, puruvate kinase and lactate dehydrogenase. This last enzyme was dependent on fructose 1,6-diphosphate for catalytic activity. The determination of the intracellular level of fructose 1,6-diphosphate (FDP) during growth of the cells in batch culture showed that the mutant strains contained 2 to 15 times less FDP than the parental strains. Growth experiments performed at pH 6.0 and 7.0 with Strep. sanguis and its PTS-negative mutant GS26 suggested that the regulation of pyruvate metabolism in this bacterium include the intracellular level of FDP and the initial hydrogen concentration of the growth medium. The results also suggested that, in these bacteria, an active PTS is required to maintain the intracellular concentration of FDP high enough to keep the cell homofermentative during growth in batch culture.

Characterization of a galactose-specific lectin from Actinomyces viscosus by a model aggregation system.

A simple model system has been developed in which lectin-mediated aggregation of glycoprotein-coated beads can be monitored by following the decrease in light scattering at 650 nm. Aggregation has been characterized with the lectin of Actinomyces viscosus T14V. Its dependence on pH, temperature, and stirring rate was examined, and the number of bacterial cells in relation to the number of latex beads resulting in optimal aggregation was established. This system has the advantage of permitting the study of a single ligand of defined structure. The ligand density was determined with radiolabeled glycoproteins. Under the conditions of the assay, ligand leakage was less than 3%, and ligands were not displaced from the beads by various proteins, glycoproteins, or by other components present in the assay mixture. Latex beads coated with asialofetuin aggregate upon the addition of A. viscosus T14V cells. By contrast, when asialofetuin was first extensively treated with purified galactose oxidase, no aggregation occurred. Only after reduction with NaBH(4) was aggregation restored, demonstrating that galactose termini of asialofetuin are essential for the binding of A. viscosus lectin. An absolute requirement for calcium was also demonstrated. Various sugars inhibited aggregation in the following order, starting with the most effective: lactose, methyl-beta-D-galactopyranoside, galactose, N-acetylgalactosamine, methyl-alpha-D-galactopyranoside. Beads coated with fimbriae from A. viscosus coaggregated with neuraminidase-treated human erythrocytes and with Streptococcus sanguis cells. In each instance the aggregation was inhibited by lactose, indicating that the A. viscosus lectin is located in the fimbriae. Cells grown under different conditions differed in their effectiveness in aggregating glycoprotein-coated beads, suggesting differences in lectin density or accessibility. Two different experimental designs were used to establish the minimum ligand density for aggregation to occur. In one type of experiment, a threshold concentration was found for asialo alpha(1)-acid glycoprotein, but not for asialofetuin. With an alternate approach in which a different population of galactose residues was exposed, a threshold phenomenon was also demonstrated for asialofetuin. The importance of structural ligand features in the aggregation assay is discussed in view of these findings.

Blood-group-reactive glycoprotein from human saliva interacts with lipoteichoic acid on the surface of Streptococcus sanguis cells

[ 3H]-Glycerol-labelled lipoteichoic acid (LTA) was extracted from Streptococcus sanguis cells using aqueous phenol. Chemical analysis of the LTA yielded phosphate: glycerol: glucose: fatty acids in the mole ratio 1.0:0.97:0.76:0.03. The LTA inhibited the interaction between Strep, sanguis cells and a high mol. wt blood-group-reactive glycoprotein (BGR-glycoprotein) isolated from human saliva and reduced Strep. sanguis-mediated haemagglutination activity. Purified LTA from Strep. mutons strains OMZ61 and HS6, which have been shown not to interact with the BGR-glycoprotein, also inhibited the BGR-glycoprotein mediated aggregation of Strep. sanguis, as did an antiserum prepared against Lactobacillus casei LTA. It is proposed that the binding of the salivary glycoprotein to Strep. sanguis cells is achieved through LTA associated with bacterial surface fibrils.

Attachment of oral Cytophaga species to hydroxyapatite-containing surfaces.

Model systems simulating the cementum portion of teeth were used to characterize the attachment process by which certain species of oral Cytophaga initiate the colonization of the tooth root surface in vitro. The adsorption of these bacteria to spheroidal hydroxyapatite beads and mechanically powdered root material followed Langmuir isotherm kinetics. From such data, the number of binding sites per 20 mg of substrate and the affinity constants were evaluated for two strains of Cytophaga sp. Resting cells of the two strains tested adhered relatively tenaciously to hydroxyapatite beads in numbers similar to those observed with cells of Streptococcus sanguis. Attachment of bacteria to the substrates was partially inhibited by (i) coating the substrates with human serum or saliva, (ii) pretreating cell suspensions with proteinase K or phospholipase C or D, or (iii) exposing the cells to temperatures greater than 60 degrees C for 15 min. Treating resting cell suspensions with pronase, neuraminidase, phospholipase A2, or 0.1 M ethylenediaminetetraacetic acid had no effect on the attachment process.

Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates.

A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by compentent S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is competitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin.

Enamel microhardness and fluoride uptake underneath fermenting and non-fermenting artificial plaque.

Washed cells of Streptococcus sanguis were used to form artificial plaque on the surface of bovine enamel and incubated underneath buffer solutions, initial pH 6, for 36 h at 37 degrees C. The decrease in the microhardness of the enamel surface under fermenting "plaque" could be prevented with fluoride. Enamel under a fermenting "plaque" took up significantly more (P less than 0.0u) fluoride than enamel under a non-fermenting "plaque" (initial F- in buffer: 10 parts/10(6)). The artificial plaque did not accumulate fluoride. Within fermenting "plaques/, the pH decreased significantly more without flouride (P less than 0.01) than with fluoride. Fluoride combined with sucrose more than negated the softening of the enamel caused by sucrose fermentation, i.e. it increased the hardness above the original values. The diffusion of fluoride through the fermenting artificial plaque was more rapid than through a non-fermenting plaque. These findings suggest that caries-conducive circumstances may promote fluoride uptake by enamel compared with non-caries-conducive circumstances.

Tolerant response of Streptococcus sanguis to beta-lactams and other cell wall inhibitors.

In contrast to group A streptococci or Streptococcus pneumoniae, cells of Streptococcus sanguis (group H) do not exhibit the irreversible effects of penicillin treatment, such as loss of viability or lysis. On the other hand, the same bacteria show typical effects of penicillin, such as morphological alterations, reduction in the rate of cell wall synthesis, and secretion of murein and lipoteichoic acid polymers into the medium. A novel effect of cell wall inhibitors was also noted: treatment with beta-lactams or with fosfomycin, d-cycloserine, or beta-halogeno-d-alanine caused the release of substantial amounts of glycerol lipids into the growth medium. The antibiotic "tolerance" of S. sanguis is interpreted in terms of the hypothesis that the activity of bacterial murein hydrolases is essential for the irreversible effects of cell wall inhibitors.

Characterization of an antibody directed against a surface component of normal and pleomorphic cells of Streptococcus sanguis.

Whole cells of Streptococcus sanguis were utilized as an immunoadsorbent to purify large quantities of an antibody (S1) directed against a cell surface component. The S-1 antibody was isolated from antisera to normal (N) and pleomorphic (O) cells by a similar adsorption-elution procedure. The S-1 antibody isolated from antisera to N cells reacted in gel diffusion in identify with the S-1 antibody to O cells, indicating that the antigen which binds S-1 antibody (Ag-1) may not be radically altered when cells become pleomorphic. The S-1 antibodies directed against both N and O cells had restricted heterogeneity, indicating that for both types of cell Ag-1 may have a simple repeating structure. However, N cells were agglutinated to a greater extent by S-1 antibody than O cells. In addition the distribution of the bound S-1 antibody became altered as the cells became pleomorphic. Utilizing the technique of indirect immunofluorescence we observed that the S-1 antibody was distributed evenly on the surface of N cells. As the cells became pleomorphic, the antibody appeared to bind preferentially at the cell poles (capping). Later, as the cells became more grossly deformed, additional bands of immunofluorescence appeared to bisect the cells. Electron microscopic analysis indicated that the bound antibody was not associated with septal notches. The results suggest that the arrangement rather than the immunological properties of Ag-1 became altered as cells became pleomorphic.

Chemical Composition of the Washed Cells of Streptococcus sanguis (804) and Streptococcus mutans (B-14)

Washed cells of Streptococcus saguis (804) and Streptococcus mutans (B14) were analyzed for water, ash, protein, carbohydrate, lipid, and nucleic acid content. Lipids were analyzed by gas chromatography. Ash was subjected to elemental analysis. These data were compared with the published values for the composition of dental plaque.

Cell-bound glucosyltransferase activity of Streptococcus sanguis strain 804

Cell-bound glucosyltransferase (GT) activity of washed cells of Streptococcus sanguis, strain 804, represents about 12 per cent of the total GT activity of glucose-grown cultures of this organism. During culture, cell-associated GT activity could be detected prior to its being found extracellularly. Sonication of the washed cells harvested at the stationary phase indicated that predominant proportions of GT and β-fructofuranosidase (FH) activities were associated with the cell debris fraction. Extracellular fibrillar material, demonstrated by electron microscopic examination, was synthesized by washed cells incubated with sucrose and chemical analysis indicated glucan formation. Incubation with sucrose also promoted the osmotic diffusion of proteins from the intracellular phase. Sodium fluoride inhibited cell metabolism without effecting the formation of glucan or the release of proteins into the supernatant fluid. Maltose in equimolar concentration with sucrose considerably reduced the synthesis of insoluble glucans. Washed cells incubated with sucrose did not release any GT.

Glycogen accumulation by pleomorphic cells of [formula omitted

Cells of Streptococcus sanguis undergo gross morphological changes when cultured in the presence of oxygen. These cells accumulate large amounts of an intracellular polysaccharide. The chemical nature and location of this polysaccharide were investigated and we showed that it is an intracellular polymer composed primarily of glucose in a linkage of the glycogen-amylopectin type. Glycogen accumulation appeared to coincide temporally with the onset of pleomorphism while its disappearance coincided temporally with cell death.