Use of the photoaffinity cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid to characterize salivary-glycoprotein-bacterial interactions.

The present study has utilized the iodinatable cross-linking agent N-hydroxysuccinimidyl-4-azidosalicylic acid (ASA) to examine the specific interaction between the proline-rich glycoprotein (PRG) of human parotid saliva and Streptococcus sanguis G9B. The binding of 125I-ASA-PRG to Streptococcus sanguis G9B displayed saturation kinetics, reversibility and was inhibited by unlabelled PRG. Inhibition studies with other glycoproteins and saccharides indicated that binding was mediated by a bacterial adhesin with specificity towards N-acetylneuraminic acid, galactose, and N-acetylgalactosamine. After cross-linking, the 125I-ASA-PRG-adhesin complex could be extracted with SDS and separated from uncoupled 125I-ASA-PRG by gel filtration on Sepharose CL-6B. Approx. 1% of the 125I-ASA-PRG was cross-linked to the bacterial surface. Examination of the 125I-ASA-PRG-adhesin complex by SDS/polyacrylamide-gel electrophoresis/fluorography on 5% -(w/v)-polyacrylamide gels revealed that PRG was bound to two bacterial components. These findings support our previous suggestion that human salivary glycoproteins can specifically interact with oral streptococci and that these interactions occur between the glycoprotein's carbohydrate units and lectin(s) on the bacterial cell surface.     

Trypsin-susceptible cell surface characteristics of Streptococcus sanguis

The adherence of Streptococcus sanguis to saliva-coated hydroxylapatite was markedly reduced by treatment of the cells with trypsin. In Scatchard plots of adherence data, protease-treated S. sanguis did not exhibit the characteristic positive slopes, suggesting that trypsin prevented cooperative interactions between the cells and artificial pellicle. Trypsin also reduced the tendency of S. sanguis to bind to hexadecane and to octyl-Sepharose. When sodium dodecyl sulfate was used to elute S. sanguis from columns of octyl-Sepharose, it was observed that the elution profiles of trypsin-treated cells were more complex than those of control cells. Water and salts were incapable of removing the cells from octyl-Sepharose. The results suggest that adherence to saliva-coated hydroxylapatite, binding to hexadecane and to octyl-Sepharose depend on trypsin-susceptible cell surface molecules.     

Genetic transformation of the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis by electroporation

Electroporation methods for introduction of plasmid DNA into the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis were developed. Electroporation of the strictly anaerobic B. fibrisolvens was carried out in an anaerobic glovebox with a buffer of 10% (v/v) glycerol and 1 mM MgCl₂ in distilled water. Streptococcus bovis electroporation could be carried out aerobically with a buffer of 10% (v/v) glycerol in distilled water. The Escherichia coli/Bacillus subtilis shuttle vector pBS42 could be transformed into B. fibrisolvens strain H17c, selecting for chloramphenicol resistance. The Streptococcus sanguis/E. coli shuttle vector pVA838 could replicate and express erythromycin resistance in Strep. bovis. Both vectors were stable in each organism in the absence of antibiotic selection. While the efficiency was low (<10²/μg DNA), the results demonstrate a means to introduce cloned genes into these organisms.     

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

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

对氨基苯甲酸对牙龄卟啉单胞菌胰酶样蛋白酶活性的影响

目的：测定不同浓度PABA对牙龄卟啉单胞菌胰酶样蛋白酶（TLP）活性的影响,以探讨血链球菌在龈下菌微生态平衡中的作用。方法：在1/2浓度的BH培养基中加入不同浓度的PABA和一定浊度的P.gingivalis,行常规培养。测定培养物上清液和菌细胞中TLP活性及蛋白质含量,计算TLP的比活。结果：PABA对P.gingivalis TLP的比活产生促进作用。结论：PABA影响P.gingivalis TLP的活性提示血链球菌可能对龈下菌斑的微生态平衡具调节作用。     

In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins

AIMS: The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro. METHODS AND RESULTS: The lectins used were extracted from Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL). Fluorescence microscopy was applied to visualize the ability of fluorescein isothiocyanate-labelled lectins to attach to the pellicle and revealed a similar capability for both lectins. Streptococcal adherence assays were performed using saliva-coated microtitre plates. BSL inhibited more than 75% of Streptococcus sanguis, Streptococcus mitis, Streptococcus sobrinus and Streptococcus mutans adherence, achieving 92% to the latter. BTL only obtained statistically significant results on S. mitis and S. sobrinus, whose adherence was decreased by 32.5% and 54.4%, respectively. CONCLUSION: Algal lectins are able to inhibit streptococcal adherence. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed application of lectins in antiadhesion therapeutics.     

对氨基苯甲酸对牙龈卟啉单胞菌生长的影响

目的：测量不同浓度PABA对P.gingivalis生长的影响,以探讨血链球菌在龈下菌斑微生态平衡中的作用。方法：用1/2浓度的BHI培养基为实验培养基,分别加入不同浓度的PABA,对P.gingivalis行厌氧培养后测定培养物的A值。结果：PABA对P.gingivalis的生长有一定影响。结论：血链球菌产生的PABA影响P.ginivalis的生长,该结果提示血链球菌对龄下菌斑的微生态平衡具有调节作用。     

苹果多酚作为微生态调节剂对窝沟致龋菌调节作用的研究

目的初步探讨苹果多酚作微生态调节剂抑制变形链球菌,扶持血链球菌生长的实验研究,为龋病的防治提供一定的依据。方法采用抑菌环实验观察苹果多酚对变形链球菌和血链球菌生长的影响,检测其对变形链球菌产酸的变化,并通过扫描电镜观察对变形链球菌在玻片粘附情况的影响。结果苹果多酚能够明显抑制变形链球菌的生长,阻止其对玻片的粘附,降低其产酸能力,但对血链球菌生长没有明显的抑制作用。对变形链球菌与血链球菌的影响比较差异有统计学意义。结论苹果多酚具有调整窝沟致龋菌的作用,进一步研究很可能是一良好的窝沟菌群的微生态调节剂。     

Adherence of streptococci to surface-modified glass

Four types of surface-modified glass were prepared. Aminopropyl glass was prepared by alkylsilylation of glass slides with gamma-aminopropyltriethoxysilane. This glass carries primary amino groups which may be protonated at pH 7.2. Owing to the presence of both positively charged ions and hydrophobic ethoxyl groups, the glass is considered to be amphipathic. Three other types of surface-modified glass slides were prepared from aminopropyl glass by forming Schiff's bases with three aldehydes: glucose, glyoxylic acid and hexanal. The aldehyde-treated slides were subsequently reduced using sodium borohydride. Thus, the surface of the glass was rendered hydrophilic, ampholytic or hydrophobic, respectively. The adherence of two Streptococcus sanguis strains and two Streptococcus mutans strains to the surface-modified glass slides was studied. Different strains showed differences in adherence to these slides depending on their physico-chemical surface properties. For S. sanguis ATCC 10556, hydrophobic bonds seemed to be most important, while in S. mutans OMZ 176, ionic interactions made the highest contribution to adhesion. Hydrogen bonds seemed to contribute least to adherence.     

Competition between oral Streptococcus species in the chemostat under alternating conditions of glucose limitation and excess

Abstract Oral Streptococcus species experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. To investigate the competitiveness of various streptococcal species under fluctuating carbohydrate supply, 2-membered chemostat cultures were run.
Under continuous limitation of glucose or sucrose, all 6 Streptococcus mutans test strains were outcompeted by Streptococcus sanguis P4A7 or Streptococcus milleri B448. This indicated that S. mutans had a lower affinity for glucose and sucrose than S. sanguis and S. milleri .
Mixed cultures were then subjected to hourly pulses with glucose. Under these conditions S. mutans Ny344 competed successfully with S. milleri B448, but still lost the competition against S. sanguis P4A7. The streptococci responded to pulses by taking up glucose at the maximum rate almost instantaneously. S. sanguis P4A7 had the highest rate of glucose uptake while the q _max value of S. mutans Ny344 was higher than that of S. milleri B448. This suggested a causal relationship between q _max and competitiveness.     

天然药物对血链球菌生长和产酸影响的体外研究

目的：通过研究不同天然药物对血链球菌生长及产酸的影响,为今后筛选出能有效调理口腔菌群生态平衡的药物奠定基础。方法：选用变形链球菌SB179作为实验菌株,测定川芎,血藤,五倍子等11种天然药物的最低抑菌浓度MIC,再以低于MIC的4个浓度配制含药的TPY液体培养基,调定其初始pH为7．4,接种血链球菌,厌氧培养后测定其终末pH,结果：当药物浓度低于或等于8．000mg/ml时,各天然药物对血链球菌的生长均有一定的抑制作用,且以五倍子作用较强,槟榔,茶多酚,蜂房,大黄,三七,血藤,白芷对血链球菌的产酸具有一定的抑制能力,而黄芩,川芎,五倍子和儿茶没有明显的抑制作用,结论：槟榔,茶多酚,蜂房,大黄,三七,血藤和白芷对血链球菌的生长和产酸都有一定的抑制作用.     

血链球菌拮抗可疑牙周病原菌机理探讨Ⅲ过氧化氢产生及影响因素

本研究采用过氧化物酶法,定性和定量分析不同条件下血链球菌产生的过氧化氢,结果显示,过氧化酶法则定过氧化氢的最小浓度为３ｐｐｍ,过氧化氢产生的量随血链球菌生长速度提高而增加,在对数生长期产量最多,２４小时后速度减慢。血链球菌产生过氧化氢的最高值为０．０２％。在厌氧条件下,血链球菌不产生或很少产生过氧化氢。     

Stimulatory effect of water stress on plant regeneration in aromatic Indica rice varieties

Frequency of regeneration of fertile plants from cell suspensions was significantly increased using water stress treatments in two commercially cultivated Indian aromatic rice varieties, Basmati 385 and Pusa Basmati 1. The water stress treatments included the use of 1.0% (w/v) agarose instead of 0.5% (w/v) for medium solidification, inclusion of mannitol (0.1, 0.2 and 0.4 M) in regeneration medium, or 24 h partial desiccation of calli. When the agarose concentration of the regeneration medium was increased from 0.5% to 1.0% (w/v), the frequency of shoot formation in Pusa Basmati 1 from cell suspension-derived calli increased by over eightfold, to 86%. Mannitol, at 0.1 to 0.2 M concentration, stimulated the frequency of shoot regeneration in Pusa Basmati 1 by fivefold but had no effect in Basmati 385. Mannitol at 0.4 M concentration completely inhibited shoot regeneration but promoted embryogenesis. These calli regenerated shoots with greater frequencies when transferred to mannitol-free medium. Partial desiccation of rice calli resulted in an up to threefold increase in the shoot regeneration frequency. Best regeneration frequencies (54–98%) were obtained when 24 hdesiccated calli were grown on regeneration medium with 1.0% (w/v) agarose. A similar stimulatory effect of water stress on plant regeneration was observed in another Indica rice variety, IR43, and a Japonica rice variety, Taipei 309.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -napthaleneacetic acid On leave from Department of Genetic, Haryana Agricultural University, Hisar, India     

Autolysis in strains of viridans streptococci.

Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.     

A method for evaluating the cariogenicity of oral bacteria using radio-labelled synthetic hydroxyapatite

R. YAMAGUCHI, M. SATO, H. TSUCHIYA, K. YAMAMOTO, Y. DOI AND F. IWAKU. 1996. Radioactive hydroxyapatite was synthesized using ⁴⁵Ca to evaluate the cariogenicity of oral streptococci. Discs prepared from it were suspended in media containing sucrose, then inoculated with Streptococcus mutans or Streptococcus sanguis . The radioactivity in the supernatant fluid was measured at specified time intervals. Released ⁴⁵Ca in the supernatant fluids markedly increased in both species during the experimental period, while Strep, mutans showed much higher decalcification than Strep, sanguis . The present method would be useful for semi-quantitative evaluation of bacterial decalcification ability.     

Streptococcus sanguis modulates type II collagen-induced arthritis in DBA/1J mice

Native type II collagen is tolerogenic when given orally or i.p. to DBA/1J mice and induces autoimmune arthritis when given s.c. in CFA. The tolerogenic epitope is contained in cyanogen bromide fragment 11 (CB11) and is structurally mimicked by PGEQGPK within the platelet aggregation-associated protein (PAAP) on Streptococcus sanguis. To learn whether S. sanguis modulates transmucosally the Ag-specific development of autoimmune arthritis, DBA/1J pups were given live S. sanguis, CB11, or type II collagen intragastrically. Feeding S. sanguis at 6 days postpartum delayed the onset of arthritis, and reduced the rate, final severity, and percentage of affected limbs. Next, PAAP(+) S. sanguis and type II collagen were tested for T cell cross-reactivity. T cells primed with the tolerogenic epitope of type II collagen proliferated more when incubated with PAAP(+) S. sanguis than with PAAP(-) Streptococcus gordonii or type II collagen, suggesting an Ag-specific transmucosal tolerogenic effect. In neonatal mice, therefore, bacterial surface Ags that mimic self can transmucosally stimulate Ag-specific inhibitory T cells. In adult mice immunized with type II collagen, these Ag-specific inhibitory T cells manifest later as attenuated arthritis. The PAAP(+) S. sanguis appear to activate adult memory, rather than naive, type II collagen-specific T cells, suggesting that systemic challenge with commensal self-mimicking microorganisms may perpetuate existing autoimmunity, but not initiate autorecognition.     

The effect of ionophores on growth and glycosyltransferase production of Streptococcus sanguis

Abstract Batch cultures of Streptococcus sanguis NCTC7865 were grown in complex medium in the presence and absence of the ionophores gramicidin, valinomycin and nigericin, to study their effects on growth and glycosyltransferase production. Growth of S. sanguis was markedly inhibited by nigericin or gramicidin, whereas valinomycin had no significant effect. The presence of ionophores caused only slight decreases in glucosyltransferase activity. Fructosyltransferase activity was however reduced by at least 90%. The results indicate that ΔpH rather than ΔΨ is essential for maintaining normal growth in S. sanguis . However, both ΔpH and ΔΨ are necessary for fructosyltransferase synthesis and secretion, but are not apparently involved in the synthesis and secretion of glucosyltransferase.     

DNA base sequence homologies among strains of Streptococcus sanguis.

DNA was isolated from 19 strains and substrains of Streptococcus sanguis and analysed for guanine plus cytosine (GC) contents and base sequence homologies. Three groups could be discerned: group 1 strains had 40-8 to 42-8 mol % GC; group 2, 42-7 to 44-0 mol % GC; group 3, 43-8 to 46-4 mol % GC. DNA homologies between groups 1 and 3 were 40 to 60% at 67 degrees C and 40% at 72 degrees C. The homologies of group 2 towards groups 1 and 3 were much lower. Strains in groups 1 and 3 hydrolysed arginine and aesculin and fermented inulin; group 2 strains did not. Groups 1 and 3 could be considered subspecies of S. sanguis. Group 2 should not be considered S. sanguis.     

Heterologous expression of an endoglucanase gene (endA) from the ruminai anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis

Abstract The heterologous expression of a cloned endoglucanase gene ( endA ) from the ruminai bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DLL The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.     

Influence of immobilization parameters on growth and lactic acid production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> co-immobilized in calcium alginate gel beads

Streptococcus thermophilusand Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1–1.5M) and alginate (1–2<><>, w/v) concentrations. Highest lactic acid production was 35 g l₁ when both bacteria were in high viscosity beads (1<><>, w/v alginate) hardened in 0.1 M CaCl₂ .The gel bead composition affected size and distribution of entrapped lactic acid bacteria.