Streptococcus Cricetus Research Articles

The macrophage pattern recognition scavenger receptors SR-A and CD36 protect against microbial induced pregnancy loss.

Microbial products can act via stress-induced signaling cascades to link dysregulated endogenous microbiota to immune activation (e.g., macrophages) and pregnancy loss. Our previous studies demonstrated that mice deficient in the macrophage pattern recognition scavenger receptors, SR-A and CD36, are more susceptible to inflammatory complications including gut leakiness and experimental colitis. We hypothesized that bacterial penetration of the maternal mucosal surfaces and replication in embryonic fluids compromise the fetal status and can result in miscarriage. Eighty pregnant ICR and SR-A/CD36-deficient mice were injected via tail vein or intraperitoneally with commensal bacteria (Streptococcus cricetus and/or Actinobacillus sp.) or sham controls. Dams were monitored daily for physical distress, pain and abortion. Dams injected with single dose bacterial inoculum did not develop clinical symptoms. Day old pups injected with bacteria developed internal focal abscesses, lost weight but recovered after 1 week. Dams receiving a second bacterial inoculum delivered dead fetuses. However, SR-A/CD36-deficnet dams demonstrated 100% fetal death via aborted fetuses, and significant up-regulation of the proinflammatory markers (IL-6, serum Amyloid A) 24-74 h after single inoculum. These data indicate that macrophage scavenger receptors are required for the fetal protection against microbial attack and support that maternal transfer of innate immunity contributes to this protection.

Streptococcus cricetus antigen I/II (paaA) 遺伝子周辺配列の解析

Streptococcus cricetus antigen I/II (paaA) 遺伝子周辺配列の解析

Identification of another surface protein antigen I/II gene, paaB, and a putative transcriptional regulator gene, par, from Streptococcus cricetus.

The flanking region of the antigen I/II gene, paaA, in Streptococcus cricetus was examined using the gene-walking technique. In the region downstream of the paaA gene, another antigen I/II gene designated as paaB was found. The paaB gene was disrupted at the alanine-rich region (A region) by a novel insertion sequence element, ISScr1. ISScr1 is a member of the IS982 family and is composed of a 962-bp sequence and duplicated target DNA (the sequence 5'-TAGCTAAAT-3') resulting from its insertion. To clarify the structural divergence of the two antigen I/II proteins (PAaA and PAaB), computational analysis of the paaB gene was performed and the two structures were compared. The amino acid sequence homology indicated that PAaB resembled PAaA, but the middle region showed little similarity to that of PAaA. Phylogenetic analysis showed that PAaB was better classified in a major group with S. mutans PAc and S. gordonii SspA and SspB than with PAaA. The transcriptional expression of paaA and paaB was demonstrated by reverse transcription (RT)-PCR. In the region upstream of the paaA gene, three genes homologous to the genes located in the region upstream of the S. sobrinus antigen I/II gene (pag) were found. Of the three genes, ORF3 showed homology to the par gene encoding a transcriptional repressor for the pag gene in S. sobrinus. Therefore, ORF3 was designated the par gene of S. cricetus. Southern hybridization revealed that the par gene of S. cricetus was not found in other oral streptococci examined in this study.

Classification of oral streptococci by two-dimensional gel electrophoresis with direct activity stain for glycosyltransferases.

Thirty eight strains of oral streptococci were divided into six types by two-dimensional gel electrophoresis (2-DE) followed by glycosyltransferase (GLT) activity stain: Type 1, Streptococcus mutans; Type 2, Streptococcus rattus; Type 3, Streptococcus sobrinus and Streptococcus downei; Type 4, Streptococcus cricetus; Type 5, Streptococcus salivarius; and Type 6, Streptococcus sanguis, Streptococcus oralis and Streptococcus gordonii. In Types 1, 2 and 5, two glucosyltransferases synthesizing water-insoluble (GTF-I) and water-soluble glucans (GTF-S) and a fructosyltransferase (FTF) were detected. In Types 3 and 4, GTF-I and two GTF-Ss were detected. Only one GTF-S was detected in Type 6. The 2-DE patterns for these six types were clearly distinguished from each other based on the kind, number and location of GLTs in gel.

Genetic studies on reference strains of mutans streptococci

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.

Recommended conservation of the names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, and seven other names included in the Approved Lists of Bacterial Names. Request for an opinion.

With reference to the first Principle of the International Code of Nomenclature of Bacteria, which emphasizes stability of names, it is proposed that the original names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, Erwinia ananas, Eubacterium tarantellus, Lactobacillus sake, Nitrosococcus oceanus, Pseudomonas betle, Rickettsia canada and Streptomyces rangoon, all included in the Approved Lists of Bacterial Names, be conserved. Request for an Opinion.

Nucleotide sequencing and transcriptional analysis of two tandem genes encoding glucosyltransferase (water-soluble-glucan synthetase) in Streptococcus cricetus HS-6.

Two tandem genes encoding glucosyltransferase synthesizing water-soluble glucan (GTF-S) were cloned from the lambda gene library of Streptococcus cricetus HS-6 (serotype a) using anti-GTF-S antibody, and the nucleotide sequences were analyzed. The two genes (ORF1 and ORF2) were identified as streptococcal glucosyltransferases based on the following evidence: [1] the deduced amino acid sequences of their products have an active site for catalytic action and C-terminal repeated units for dextran binding, and [2] a homology search revealed that the ORF1 and ORF2 products are homologous to the GtfS protein (77.4%) of S. downei Mfe28 and GtfT protein (83.8%) of S. sobrinus OMZ176, respectively, which are both known to have GTF-S activity. Therefore, ORF1 and ORF2 might be designated gtfS and gtfT of S. cricetus, respectively. A Northern blotting and RNase protection assay suggested that the gtfS and gtfT of S. cricetus are transcribed as a single bicistronic mRNA as well as separate monocistronic mRNAs. Primer extension analysis indicated multiple transcriptional start points for each gene.

Dextran structural factors which affect binding to the lectin of Streptococcus cricetus

Oral bacteria, such as Streptococcus cricetus and S. sobrinus, are aggregated by high molecular weight fractions of the linear α-(1→6) dextran produced by Leuconostoc mesenteroides. The linkage specificity of this interaction with a cell surface lectin of the streptococci is high. As a further probe of factors affecting recognition, we prepared partially methylated samples of dextran of average MW of 2 × 10 6 and 10 4. 13C NMR spectroscopy proved to be a convenient method for determining both site and degree of monomethylation and dimethylation of the polymer: relative reactivity of the OH groups was established as 02: 04: 03 = 4.3: 1.7: 1.0. A methylation DS of only 0.17 caused almost complete loss of recognition by the lectin. A structural feature of dextran oligomers is suggested to explain this result.

Effect of Porphyromonas gingivalis ATCC 33277 vesicle on adherence of Streptococcus mutans OMZ 70 to the experimental pellicle.

The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.

Fluoride inhibits the glucan-binding lectin ofStreptococcus sobrinus

The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.

On mechanisms of oral microbial adhesion.

Streptococcus cricetus HS-6 grown with or without added manganous ions in their growth medium, i.e. with or without GBLs respectively, show distinct differences in the pH dependence of their zeta potentials and their adhesion to hexadecane confirming that non-specific surface properties can be a corollary of the presence of specific adhesins. It was suggested that at certain pH values, the GBLs are shielded by other cell surface components. The delay in glucan induced aggregation at pH 4 but not at pH 7 and 9 was taken as support for this suggestion. Furthermore, the pH dependence of the glucan induced aggregation of S. cricetus HS-6 is suggested to demonstrate clearly the electrostatic nature of the highly specific interaction between glucans and lectins and therewith provides support for our suggestion that the same fundamental forces are responsible for specific and non-specific interactions

Comparison of aminopeptidase activities in four strains of mutans group oral streptococci

In this study, native cells of Streptococcus mutans VA-29R and Streptococcus rattus FA-1 displayed significantly higher aminopeptidase activity than did cells of Streptococcus cricetus AHT or Streptococcus sobrinus 6715 toward the nitroanilide derivatives of leucine, alanine, methionine, arginine, and lysine. These differences in cellular aminopeptidase activity led us to investigate the subcellular localization of the aminopeptidase in these mutans group streptococci. Following conversion of native cells to protoplasts by treatment with lysozyme, most of the aminopeptidase activity detected in the native-cell preparations remained associated with the intact protoplasts. After lysis of protoplasts and differential centrifugation, most of the total cellular aminopeptidase activity was recovered with the cytoplasmic fraction. Membrane-associated aminopeptidases represented only minor activities in these mutans group streptococci. Although the four strains showed no differences with respect to a predominant cytoplasmic localization for the aminopeptidase activities, the levels of activity in the cytoplasmic fractions from S. cricetus AHT and S. sobrinus 6715 were significantly lower than those measurable in the corresponding fractions from S. mutans VA-29R and S. rattus FA-1. These results support the conclusion that the differences in aminopeptidase activity expressed by these streptococci reflect quantitative differences rather than differences in enzyme subcellular localization.

Reduced nicotinamide adenine dinucleotide oxidase involvement in defense against oxygen toxicity of Streptococcus mutans.

The growth inhibition of the Streptococcus mutans group, including Streptococcus mutans, Streptococcus cricetus, Streptococcus rattus and Streptococcus sobrinus, on glucose by oxygen and the properties of reduced nicotinamide adenine dinucleotide (NADH) oxidase induced by oxygen, using a representative oxygen-tolerant strain, were examined. The growth response to oxygen varied among strains and correlated with the level of NADH oxidase activity in the cell extract. The induced synthesis of NADH oxidase as well as superoxide dismutase was affected by oxygen tension and energy sources. The induced NADH oxidase involved at least two types, major H2O-forming NADH oxidase and minor H2O2-forming NADH oxidase activity. In the presence of a scavenger of H2O2, pyruvate, the growth inhibition by oxygen in an oxygen-sensitive strain (GS5) was protected but not in another oxygen-sensitive strain (MT8148). The high level of induced H2O-forming NADH oxidase activity protected against oxygen toxicity.

Chelating agents inhibit activity and prevent expression of streptococcal glucan-binding lectins.

Several of the cariogenic mutans streptococci produce cell wall-associated glucan-binding lectins (GBLs). The lectins bind alpha-1,6-linked glucans and have no affinity for other polysaccharides or anomeric linkages. When citrate or lactate was included in the growth medium, expression of the activities of the GBLs of Streptococcus cricetus and S. sobrinus was prevented. Furthermore, chelating agents, including citrate, lactate, EDTA, and acetylacetone, were able to reversibly inhibit glucan-induced aggregation of GBL+ streptococci. In addition, the chelating agents prevented sucrose-dependent streptococcal adhesion to glass surfaces and dispersed preformed adherent masses of the streptococci. Neither citrate nor other chelating agents modified the activities of glucosyltransferases. Expression of the lectin could only be achieved by the addition of manganous ion to the growth medium. Chloramphenicol and other metabolic inhibitors prevented synthesis of GBL in cells obtained from manganese-deficient medium and shifted to manganous ion-sufficient medium. The GBL may be a manganoprotein, the manganese of which may be perturbed, but not removed, by chelating agents. During synthesis of the GBL, manganous ion may be required in order for the protein to achieve an active conformation. Citrate or other chelating agents may have promise as anticaries agents.

Influence of the culture medium on the synthesis of α- d-glucans by Streptococcus cricetus AHT

Three different α- d-glucosyltransferases (GTFs) were separated from culture filtrates of Streptococcus cricetus strain AHT grown in a complex. standard medium in batch culture or under defined conditions of growth in the chemostat. Two of the enzymes (GTF-s1) converted sucrose into branched soluble dextrans, and the third (GTF-I) produced a relatively linear, water-insoluble, predominantly (1→3)-linked α- d-glucan. When the organism was grown in comlex medium by the removal of the fraction of high molecular weigth, only GTF-S1 and GTF-S2 were released, and GTF-I was detected. The water-insoluble glucan fraction obtained by incubating the cell-free filtrate with sucrose contained from 17 to 25% of (1→3)-glucosidic linkages, and accounted for up to 78 and 4% of the total glucans derived from growth in standard and modified medium, respectively. The soluble glucans produced in the same reaction were fractionated with ethanol to give, from both media, two distinct dextrans comprising ( 1) a highly branched dextran similar to the S1-dextran product of GTF-S1 and ([it2 ) a dextran containing fewer branch linkages and up to 86% of (1→6)-α- d-glucosidic linkages. A GTF responsible for the synthesis of the latter dextran was not separated. The structures of the glucan fractions and the products of the separated GTF were examined by enzymic degradation and methylation analysis.

Immunological relationships between glucosyltransferases synthesizing insoluble glucan from Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei

The Mr values and isoelectric points of glucosyltransferases synthesizing insoluble glucan (GTF-Is) were determined, and the immunological relationships between them studied. The GTF-I enzymes were from Streptococcus cricetus (mutans group serotype a), Streptococcus sobrinus (mutans group serotypes d and g) and Streptococcus downei (mutans group serotype h). By double immunodiffusion tests, the GTF-I enzymes from the three species possessed a common antigenic determinant; in addition, the GTF-I enzymes of serotypes d, g and h shared a further determinant. The S. sobrinus serotypes d and g GTF-I enzymes were immunologically identical. The GTF-I enzymes of S. sobrinus serotypes d and g, and of S. downei, had an Mr of 161,000 and isoelectric points of 4.8-4.9, while S. cricetus GTF-I had a lower Mr (150,000) and a higher isoelectric point (5.2). This suggests that the S. cricetus GTF-I enzyme may lack a sequence of amino acids which include the determinant shared by S. sobrinus and S. downei GTF-I enzymes. Antibodies specific to the determinant shared by all four serotypes inhibited the homologous and heterologous enzymes by 94-100%.

Effects of Propolis on Dental Caries in Rats

Propolis, the resinous hive product collected by bees, is important in the defense of the hive. The effects of propolis on growth and glucosyltransferase activity of Streptococcus sobrinus 6715, Streptococcus mutans PS14 and Streptococcus cricetus OMZ61 in vitro, and on dental caries in rats infected with S. sobrinus 6715 were investigated. Propolis had antimicrobial activity against S. sobrinus, S. mutans and S. cricetus, and inhibited both water-insoluble glucan synthesis and glucosyltransferase activity. In rats inoculated with S. sobrinus, about half of their fissures were carious, while dental caries was significantly less (p = 0.01) in rats given propolis. Dental caries was markedly decreased by the multiple actions of propolis which had antimicrobial activity, inhibited water-insoluble glucan synthesis, and inhibited glucosyltransferase activity. No toxic effects of propolis on the growth of rats were observed under experimental conditions in this study. These results suggest that propolis can control dental caries in the rat model system.

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.

Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus

A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.

Streptococcus cricetus and Streptococcus rattus bind to different segments of collagen molecules

Strains of Streptococcus cricetus and Streptococcus rattus exhibited striking differences in their ability to bind to different types of collagen. For example, S. cricetus AHT bound in highest numbers to hydroxyapatite (HA) treated with human type V collagen, while rat type I collagen was ineffective. In contrast, human type V collagen was least effective in promoting attachment of S. rattus LB-1, while treatment with rat or human type I collagen was effective. Adsorption of both species to human type I collagen-treated HA showed a high correlation with a Langmuir model. Estimates of adsorption parameters indicated there were greater numbers of binding sites with higher affinity for S. rattus LB-1 than for S. cricetus AHT. Treatment of HA with either the alpha 1 (1) or alpha 2 (1) polypeptide chains of collagen was effective in promoting adhesion of S. rattus LB-1 cells. In contrast, the alpha 2 (1) chain was more effective than the alpha 1 (1), chain for S. cricetus AHT. These observations indicate that S. cricetus AHT and S. rattus LB-1 cells bind to different segments of collagen molecules. Adhesion of both species was also promoted by collagen-rich fractions of human dentin.