Cytokine induction by Gram-positive bacteria

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Gram-positive bacteria is by far less well understood than immune activation by Gram-negative bacteria. Our group has made available highly purified lipoteichoic acids (LTA) as a key Gram-positive immunostimulatory component. We have characterized the reasons for lower potency of LTA compared to Gram-negative lipopolysaccharide (LPS), identifying lack of IL-12/IFNgamma induction as a general characteristic of TLR2 agonists, and need for presentation of LTA on surfaces for enhanced immunostimulatory potency, as major aspects. Aspects of chemokine induction, where LTA is more potent than LPS, have been addressed. Furthermore, novel complement and plant defence activation, as well as CD36 as a new LTA receptor, were identified. The bacterial costimuli and modulators of LTA inducible responses are being investigated: LTA isolated from so far 16 bacterial species, although different in structure, behave remarkably similar while whole live and killed bacteria differ with regard to the pattern of induced responses. The purification and characterization of the respective components of the bacterial cell wall has begun.     

Fluoride uptake by Streptococcus mutans 6715.

The short-term kinetics of fluoride uptake by cells from 20- to 22-h cultures of Streptococcus mutans strain 6715 were studied using rapid filtration and centrifugation techniques. Saline-suspended organisms were diluted with fluoride-containing solutions buffered at four different pH values (2.0, 4.0, 5.5, and 8.2). Fluoride disappearance from the medium was inversely related to pH and to the duration of the exposure at any given pH. The uptake was rapid and extensive at the lower pH values and decreased as the pH increased. Media fluoride concentrations subsequently increased; i.e., fluoride was released from the cells. The presence of glucose, cyanide, or iodoacetate did not influence the results. However, preincubation of the cells in fluoride-free buffers, followed by the addition of fluoride, reduced fluoride uptake markedly. Cell-to-media pH gradients were determined by the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione. Fluoride uptake was found to be a function of the magnitude of the pH gradient (P less than 0.001). It is hypothesized that fluoride uptake occurs by the diffusion of hydrogen fluoride and the subsequent trapping of ionic fluoride.     

Conservation of cell wall peptidoglycan by strains of Streptococcus mutans and Streptococcus sanguis

Turnover of the cell wall peptidoglycan fraction of six different strains of Streptococcus mutans and eight different strains of Streptococcus sanguis was examined. Cells were grown in the presence of [3H]lysine and [14C]leucine for at least eight generations and then chased in growth medium lacking the two labels. At intervals during the chase, samples of cultures were removed, and the amounts of the two labeled precursors remaining in the peptidoglycan and protein fractions were quantitated. Similar experiments were done in which the pulse-labeling technique was used. In addition, cells were labeled in the presence of tetracycline or penicillin, chased with growth medium containing no inhibitor, and assayed at intervals during the chase for the amount of [3H]lysine present in peptidoglycan fractions. Studies of cultures of S. mutans strains FA-1, OMZ-61, OMZ-176, 6715, GS-5, and Ingbritt and of S. sanguis strains 10558, M-5, Wicky, DL-101, DL-1, 71X26, and 71X48 maintained in the exponential phase of growth in a chemically defined medium failed to show evidence of loss of insoluble peptidoglycan via turnover. Similarly, for the strains of S. mutans, insoluble peptidoglycan assembled during 2 h of benzylpenicillin or tetracycline treatment was also conserved during recovery from growth inhibition.     

Induction of Cytokines by Glucosyltransferases of Streptococcus mutans

Production of proinflammatory cytokines is implicated in the pathogenesis of viridans streptococcus-induced α-streptococcal shock syndrome and infective endocarditis. Streptococcus mutans, one of the opportunistic pathogens causing infective endocarditis, was reported previously to stimulate monocytes and epithelial and endothelial cells in vitro to produce various cytokines. We found that glucosyltransferases (GTFs) GtfC and GtfD of S. mutans stimulated predominantly the production of interleukin-6 (IL-6) from T cells cultured in vitro. The level of IL-6 but not of tumor necrosis factor alpha in blood was significantly elevated when rats were injected intravenously with S. mutans GS-5, whereas IL-6 was detected at a much lower level when rats were challenged with NHS1DD, an isogenic mutant defective in the expression of GTFs. The serum IL-6 level was elevated in patients with endocarditis caused by different species of viridans streptococci which express GTF homologues. Affinity column-purified GTFs reduced the levels of detectable IL-2 of T cells stimulated by another bacterial antigen, tetanus toxoid. These results suggested that GTFs might modulate the production of Th1-type cytokines and that GTFs of S. mutans play a significant role in stimulating the production of the proinflammatory cytokine IL-6 in vivo.     

Fractionation and properties of glucans produced by Streptococcus mutans.

Water-insoluble (ISG) and water-soluble (SG) fractions of glucans produced by cell-free glucosyltransferase of Streptococcus mutans AHT (serotype g) were isolated by centrifugation at 20,000 x g for 15 min. No further resolution of slightly sonicated ISG was observed with gel filtrations on any Bio-Gel beads, including A-50m. Bio-Gel P-100 filtration subdivided SG into two fractions with higher and lower molecular weights (designated SG-A and SG-B, respectively). SG-A was further resolved into two subfractions, SG-A-I and SG-A-II, by 10 to 40% and 50 to 80% ethanol precipitation, respectively. Relative amounts of ISG, SG-A-I, SG-A-II, and SG-B were 66.3:9.4:4.4:19.9. The molecular sizes of these fractions were >1.5 x 10(7), >==1.5 x 10(7), <==5 x 10(6) (>1 x 10(5)), and <==1 x 10(4) daltons, and their alpha-1,3 glucosidic linkage contents were approximately 35, 35, 16, and 4% for fractions ISG, SG-A-I, SG-A-II, and SG-B, respectively. Both ISG and SG-A-I were resistant to hydrolysis by dextranase and possessed the ability to aggregate with concanavalin A and to agglutinate S. mutans cells. SG-A-II had extremely low dextranase susceptibility and significant agglutinating activities, whereas SG-B showed high dextranase sensitivity and neither aggregating nor agglutinating activity. These results indicate that SG of S. mutans AHT consists of three types of glucans with distinctly different molecular sizes and chemical structures and strongly suggest that the ISG and SG-A-I fractions are different physical states of an inherently identical glucan. Preliminary observations suggest that the glucans produced by other S. mutans strains of several serotypes may be similarly classified.     

Penicillin-induced lysis of Streptococcus mutans

Treatment of Streptococcus mutans GS-5 cells with concentrations of penicillin G within a relatively narrow range resulted in substantial lysis. This penicillin-induced lysis was dependent upon cell density and pH of the lysis medium. Other oral streptococci (Streptococcus sobrinus, Streptococcus rattus, and Streptococcus cricetus) also demonstrated substantial levels of penicillin-induced lysis under appropriate conditions. Lesser degrees of lysis were seen in a related organism, Streptococcus ferus.     

Genetic Transformation of Streptococcus mutans

Three strains of Streptococcus mutans belonging to serotypes a, c, and f were transformed to streptomycin resistance by deoxyribonucleic acids derived from homologous and heterologous streptomycin-resistant strains of S. mutans and Streptococcus sanguis strain Challis. Homologous transformation of S. mutans was less efficient than heterologous transformation by deoxyribonucleic acids from other strains of S. mutans.     

变异链球菌生物膜结构观察

目的　建立变异链球菌生物膜模型 ,用激光共聚焦扫描显微镜 (CLSM)观察变异链球菌生物膜结构。方法　在盖玻片上分别形成 6、12、18、2 4、4 8、72h变异链球菌生物膜 ,将得到的各时段生物膜荧光染色后 ,用CLSM观察生物膜的断层扫描图像、生物膜厚度、每层红光绿光的面积 ,计算生物膜中细菌密度和活菌百分比 ,用软件处理扫描数据 ,得到生物膜的三维重建图像。结果　变异链球菌生物膜具有空间立体结构 ,形态多样 ,其中细菌密集 ,由死细菌和活细菌组成 ,还有丰富的基质和管道系统。 2 4h生物膜平均厚度最大 ,生物膜内层、中间层的细菌密度相对较大 ,而外层较低 ,72h内各时间段生物膜中活菌百分比由内往外逐渐增加。结论　变异链球菌生物膜有一定的厚度 ,具有三维立体空间结构 ,结构形态具有多样、不均质、开放的特点。     

Antigens of Streptococcus mutans I. Characterization of a Serotype-Specific Determinant from Streptococcus mutans

A membrane-associated glycerol teichoic acid antigen has been isolated from Streptococcus mutans AHT and a similar antigen has been demonstrated to be present in each of the other Bratthall serotype a organisms studied. Trichloroacetic acid-extracted material was resolved into two phosphorus-containing antigenic fractions (B and C) by agarose chromatography. Fraction B was preliminarily identified as a phospholipid moiety with a glycerol-to-phosphorus ratio of 2:1, and fraction C showed a ratio of 1:1 indicative of a glycerol teichoic acid. This latter fraction also was associated with glucose, galactose, alanine, and fatty acids. Diglycerol triphosphate, the compound characteristically released from 1-3 phosphodiester-linked glycerol teichoic acids by alkaline hydrolysis, was isolated and characterized. Alanine was identified as its alkaline-labile, ester-linked D-isomer. A glyceride was isolated containing a disaccharide of glucose and galactose attached to the 2-hydroxyl group of glycerol. Hapten inhibition analysis demonstrated that beta-galactosides were the greatest inhibitors of the precipitin reaction (>75%), whereas glucose and its derivatives inhibited to a much lesser extent (<30%). Comparative immunodiffusion and immuno-electrophoresis analyses demonstrated that all six Bratthall serotype a organisms tested contained this antigenic determinant and that it was absent in serotypes b, c, and d. It is suggested that the common antigenic determinant of this serotype within S. mutans may be a beta-galactoside associated with a glycerol teichoic acid and possibly other polymers.     

Active release of bound antibody by Streptococcus mutans.

Previous studies have shown that Streptococcus mutants is capable of releasing many surface protein antigens, particularly antigen P1. Antigen P1 is immunodominant and has been implicated in adherence of S. mutants to the acquired pellicles. The purpose of this study is to investigate the significance of release of this antigen by the cells. S. mutants NG8 (serotype c) was incubated with an anti-P1 rabbit immunoglobulin G (IgG) or a human colostral IgA which contains natural anti-P1 activity. Results indicated that the bound antibodies were released by the cells in a pH- and time-dependent manner. The optimal pH for release was between 6 and 8, and the release rate reached a plateau in 1 h at 37 degrees C. The release of bound antibodies was considered an active process, since heat-killed cells remained capable of antibody binding but failed to release the antibodies. The release was also dependent on the age of the culture, with early-exponential-phase cells releasing the maximum amount of bound IgG. The released IgG was isolated by polyethylene glycol precipitation and protein A-Sepharose column chromatography and found to be associated with antigen P1, indicating that the antibodies were released together with the antigen in the form of immune complexes. The binding of S. mutans by secretory IgA (SIgA) inhibited the adherence of the cells to salivary agglutinin-coated hydroxylapatite. However, when the SIgA-coated S. mutans was allowed to release the bound antibodies, the inhibitory effect of SIgA on adherence was abrogated. These results suggest that S. mutans is capable of shedding surface-bound antibodies in the form of antibody-antigen immune complexes. Such an action may be a strategy employed by the cells to counter the neutralizing effect of naturally occurring antibodies in the oral cavity.     

Insoluble glucan synthesis by Streptococcus mutans serotype c strains

Both dextransucrase and mutansynthetase activities have been purified from the culture fluids of Streptococcus mutans GS-5 (serotype c). Although homogeneous dextransucrase preparations normally synthesize little insoluble glucan, essentially all of the glucan synthesized by this enzyme in the presence of 1.5 M (NH4)2SO4 was water insoluble. Linkage analysis of the insoluble glucans indicated that the presence of NH4+ increased the portion of alpha-1,3-glucose linkages relative to alpha-1,6-glucose units in the product. Chromatofocusing of aggregated glucosyltransferase fractions synthesizing predominantly insoluble glucan yielded primarily dextransucrase activity separable from relatively low levels of mutansynthetase activity. The latter enzyme was detected only in 18-h assays and synthesized primer-dependent insoluble glucan, which was decreased in the presence of NH4+. In the absence of primer dextran T10, the addition of dextransucrase also stimulated insoluble glucan synthesis by mutansynthetase. Dextransucrase and mutansynthetase appear to be distinct enzymes, since the latter possesses a higher molecular weight (155,000 compared to 140,000), a much lower isoelectric point, and did not cross-react with antibody directed against dextransucrase. These results are discussed relative to the mechanism of insoluble glucan synthesis by S. mutans serotype c strains.     

Transfection of Streptococcus sanguis by phage deoxyribonucleic acid isolated from Streptococcus mutans.

Streptococcus sanguis ATCC 10556 cells were infected with free phage DNA of S, mutans strain PK 1. Two transformants were isolated which made colonies with large mucoid forms on mitis-salivarius agar. Both transformants had an increased ability to synthesize insoluble glucan and showed an adhesive nature on glass surfaces. These characteristics of the transformants bear a resemblance to S. mutans. These transformants had many physiological characteristics by which they could be recognized as S. sanguis. However, they resembled S. salivarius in forming a large amount of soluble fructan. Furthermore, the transformant cells did not produce ammonia from arginine, whereas their parent cells did.     

Streptococcus mutans clonal variation revealed by multilocus sequence typing

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.     

Platelet aggregation induced by serotype polysaccharides from Streptococcus mutans

Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I(2) (PGI(2)) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI(2). RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.     

Binding of Todd-Hewitt broth antigens by Streptococcus mutans.

Streptococcus mutans 10449, grown in chemically defined culture medium, was tested for its ability to bind 3H-labeled Todd-Hewitt broth components (greater than 12,000 Mr). Maximum adsorption of radioactivity occurred within 5 min at room temperature, and cell-bound material was not completely removed by extended washing with buffer. Heat-killed, arsenate-inhibited, and viable bacteria bound similar quantities. Only 0.09% of the radioactivity in the preparation of high Mr Todd-Hewitt broth components was removed by absorption with excess numbers of S. mutans 10449 cells. Binding followed saturation kinetics and was competitively inhibited by unlabeled medium components, both the dialyzable and nondialyzable fractions. Other oral streptococci were also found to bind these complex medium components. Rabbit antiserum elicited to the high-molecular-weight Todd-Hewitt broth components reacted with monkey cardiac muscle and with S. mutans coated with medium components. Absorption of the anti-Todd-Hewitt broth serum with homogenized heart removed antibodies that reacted with Todd-Hewitt broth-coated S. mutans. Therefore, the tissue-specific antigens of this beef heart infusion medium that adsorb to S. mutans can interfere with the detection and characterization of antigens shared by these bacteria and animal tissues.     

Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis

Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.     

Virulence of Streptococcus mutans: restoration of pathogenesis of a glucosyltransferase-defective mutant (C4)

Previous studies have shown that a mutant (designated C4) of Streptococcus mutans 6715 wild type (WT) is defective in glucosyltransferase (GTF)-synthesized insoluble glucan and is avirulent in gnotobiotic rats. This study investigated the factors which would render this mutant virulent in gnotobiotic rats. Microbial analysis of plaque from gnotobiotic rats (45 days old) infected with a mixture of C4 and virulent S. mutans PS-14 (approximately 15,000 C4 organisms to each S. mutans PS-14) yielded higher numbers of C4 organisms than S. mutans PS-14. These animals exhibited significantly lower caries scores than did gnotobiotic rats (age, 45 days) monoassociated with S. mutans PS-14. Similar mixed infection studies using C4 and an avirulent, aggregation-defective mutant of S. mutans 6715 WT (designated UAB 165) which exhibits GTF activity similar to that of the parent strain resulted in plaque consisting almost exclusively of UAB 165 and low caries activity. However, high levels of both C4 and UAB 165 in plaque and high caries activity were observed in gnotobiotic rats infected at weaning with C4 followed by UAB 165 3 days later. When dried S. mutans 6715 WT culture supernatant containing GTF activity was mixed with diet provided rats monoassociated with C4, significant caries activity was observed. Insoluble glucan supplemented in diet did not restore C4 to virulence; however, admixture of suboptimal GTF-rich supernatant with insoluble glucan and C4 resulted in high caries activity in gnotobiotic rats. These results suggest that in vivo restoration of pathogenesis of a GTF-defective mutant of S. mutans can be achieved either by complementation with a mutant defective in aggregation properties or by providing exogenous GTF and glucan from the parent S. mutans 6715 WT.     

Plasmid-mediated transformation of Streptococcus mutans.

Streptococcus mutans GS-5 was transformed to erythromycin resistance with streptococcal plasmid pVA736. Transformation frequencies were higher with plasmids reisolated from transformed GS-5 cells relative to plasmid originally derived from S. sanguis Challis.     

Inhibition of Streptococcus mutans by the lactoperoxidase antimicrobial system.

Inhibition of bacterial metabolism by the lactoperoxidase (LP)-hydrogen peroxide (H2O2)-thiocyanate system was studied with representatives of serotypes a through g of Streptococcus mutans. The aims were to determine whether the amount of H2O2 released from these catalase-negative bacteria is sufficient to activate the LP system and whether these oral bacteria are resistant to inhibition by the LP system, which is active in human saliva. When the washed, stationary-phase cells were incubated aerobically with LP, thiocyanate, and glucose (Glc), greater than 90% inhibition of Glc utilization and lactate production was obtained with strains that released large amounts of H2O2 (BHT, FA-1, OMZ-176); 20 to 50% inhibition was obtained with strains that released about half as much H2O2 (B-13, Ingbritt); and no inhibition was obtained with strains that released only small amounts of H2O2 (AHT, HS-6, GS-5, LM-7, OMZ-175, 6715-15). Inhibition was most effective at pH 5, whereas release of H2O2 and accumulation of the inhibitor (hypothiocyanite ion) were highest at pH 8. With H2O2-releasing cells from early stationary phase, preincubation with Glc abolished inhibition, though it did not influence H2O2 release. Cells harvested 24 h later were depleted of sulfhydryl compounds. Inhibition of these cells was abolished by preincubation with Glc and certain sulfhydryl or disulfide compounds (reduced or oxidized glutathione, cysteine or cystine). This preincubation increased cell sulfhydryl content but had no effect on H2O2 release. All strains were inhibited when incubated with LP, thiocyanate, and added (exogenous) H2O2. Smaller amounts of H2O2 were required to inhibit at pH 5, and larger amounts were required to inhibit cells preincubated with Glc or with Glc and the sulfhydryl or disulfide compounds. The results indicate that pH, amount of H2O2, cell sulfhydryl content, and stored-carbohydrate content determine susceptibility to inhibition.