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Specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus or Actinomyces naeslundii.

Coaggregation reactions between actinomycete and streptococcal cells occurred frequently when human strains of Actinomyces viscosus or A. naeslundii were mixed with human isolates of Streptococcus sanguis or S. mitis, but were infrequent with other oral actinomycetes and streptococci. Two groups of actinomycetes and four groups of streptococci were defined by the patterns of their coaggregation reactions and by the ability of beta-linked galactosides (i.e., lactose) to reverse these reactions. Coaggregations occurred by one of the following three kinds to cell-cell interactions: (i) coaggregation that was blocked by heating the streptococcus but not the actinomycete and was not reversed by lactose; (ii) coaggregation that was blocked by heating the actinomycete but not the streptococcus and was reversed by lactose; and (iii) coaggregation that was blocked only by heating both cell types. The latter reaction was a combination of the first two since lactose reversed coaggregation between heated streptococci and unheated actinomycetes but did not reverse coaggregations between unheated streptococci and heated actinomycetes. Cells that could be heat inactivated also were inactivated by amino group acetylation or protease digestion, whereas cells that were unaffected by heat were not inactivated by these treatments. Coaggregation reactions of each kind were Ca2+ dependent and insensitive to dextranase treatment. These findings are consistent with the hypothesis that human strains of A. viscosus and A. naeslundii coaggregate with strains of S. sanguis and S. mitis by a system of specific cell surface interactions between protein or glycoprotein receptors on one cell type and carbohydrates on the other type.

Acetylation↗

Use of specifically labeled sucrose for comparison of extracellular glucan and fructan metabolism by oral streptococci.

Sucrose labeled in the fructosyl ((3)H) and glucosyl ((14)C) moieties was used to quantitate extracellular polysaccharide production and degradation by cariogenic and noncariogenic oral streptococci. All of the strains produced glucan and fructan. Streptococcus salivarius produced primarily fructan, whereas S. mutans and S. sanguis produced more glucan than fructan. The cariogenic streptococci could degrade the fructan produced by noncariogenic strains. Although the soluble glucans from all of the strains were sensitive to dextranase, the insoluble glucan from S. mutans could be distinguished from the S. sanguis insoluble glucan by its greater resistance to this enzyme.

Carbon Isotopes↗

Glucans synthesized in situ in experimental salivary pellicle function as specific binding sites for Streptococcus mutans.

Many researchers have suggested that the role of glucan-mediated interactions in the adherence of Streptococcus mutans is restricted to accumulation of this cariogenic bacterium following its sucrose (i.e., glucan)-independent binding to saliva-coated tooth surfaces. However, the presence of enzymatically active glucosyltransferase in salivary pellicle suggests that glucans could also promote the initial adherence of S. mutans to the teeth. In the present study, the commonly used hydroxyapatite adherence assay was modified to include the incorporation of glucosyltransferase and the synthesis of glucans in situ on saliva-coated hydroxyapatite beads. Several laboratory strains and clinical isolates of S. mutans were examined for their ability to adhere to experimental pellicles, either with or without the prior formation of glucans in situ. Results showed that most strains of S. mutans bound stereospecifically to glucans synthesized in pellicle. Inhibition studies with various polysaccharides and fungal dextranase indicated that alpha 1,6-linked glucose residues were of primary importance in the glucan binding observed. Scanning electron microscopic analysis showed direct binding of S. mutans to hydroxyapatite surface-associated polysaccharide and revealed no evidence of trapping or cell-to-cell binding. S. mutans strains also attached to host-derived structures in experimental pellicles, and the data suggest that the bacterial adhesins which recognize salivary binding sites were distinct from glucan-binding adhesins. Furthermore, glucans formed in experimental pellicles appeared to mask the host-derived components. These results support the concept that glucans synthesized in salivary pellicle can promote the selective adherence of the cariogenic streptococci which colonize human teeth.

Bacterial Adhesion↗

Mechanism of adherence of Streptococcus mutans to smooth surfaces. II. Nature of the binding site and the adsorption of dextran-levan synthetase enzymes on the cell-wall surface of the streptococcus.

The mechanism of adsorption of the Streptococcus mutans enzymes responsible for the synthesis of insoluble dextran-levan to the S. mutans cell-wall binding sites has been studied. Certain characteristics of these binding sites are presented. The adsorption of these enzymes to the cell surface occurred rapidly without the addition of a source of energy and over a pH range of 3 to 11. The adsorption was inhibited by soluble dextran, probably due to the strong affinity of the polymer to the enzyme. All other polymers and sugars studied showed little or no inhibition. The adsorption was also inhibited by antibody globulin to the a-d immunologically specific group antigen surface polysaccharide of S. mutans and by anti-dextran globulin. The inhibition by anti-a-d globulin is considered to be due to a restriction of access of enzyme to the binding site of the enzyme which may be located in close proximity to the group antigen. On the other hand, anti-dextran globulin appeared to directly inhibit the adsorption by covering the binding site. Dextranase destroyed the binding site and released glucose from the S. mutans cells. These data indicate that S. mutans grown in media containing glucose possesses a small amount of dextran on the cell surface, and that this dextran is, or is a part of, the binding site for enzymes which synthesize the insoluble dextran-levan polymer. Trypsin inhibited the synthesis of insoluble polysaccharide and the adherence of cells. It is not clear in this case that destruction of the binding sites occurred. These data present a partial explanation of the processes which may be concerned in the formation of dental plaque on the smooth surfaces of teeth.

Adsorption↗

Influence of antibody on the structure of glucans.

Glucosyltransferase (GTF) plays an essential role in the formation of the biofilm known as dental plaque and in the pathogenesis of dental caries. Mutans streptococci produce at least three distinct GTFs (GtfB, C and D), each of which forms a glucan polymer from sucrose. Glucan is a major constituent of plaque biofilm. GTF adsorbed to a surface forms glucans that differ in structure from those formed by the same enzyme in solution. In the present study, activities of GtfB and GtfC in solution or adsorbed on a surface were inhibited in the presence of a polyclonal antiserum (DS-1) to a mixture of GTFs and by immunoglobulin G (IgG) prepared from DS-1; in contrast, enzyme activity was enhanced by normal rabbit serum (NRS) and IgG from NRS. GtfD activity on a surface was enhanced by both antiserum DS-1 and NRS, and IgG prepared from either serum; GtfD activity in solution was slightly inhibited by each of the sera. The structure of GtfB and GtfC glucans formed in the presence of antiserum differed from that of controls based on linkage analyses, and on their susceptibilities to the glucanohydrolases mutanase (alpha-1,3 hydrolase) and dextranase (alpha-1,6 hydrolase); soluble products from the enzymatic digestion also differed. The results show that the effects of antibody on enzyme activity are more complex than simple inhibition or enhancement and that the presence of antibody may influence glucan structure, which clearly could impact plaque formation. The results have implications for the formation and properties of biofilms formed in other environments.

Adsorption↗

Adherence of glucan-positive and glucan-negative streptococcal strains to normal and damaged heart valves.

The adherence of 18 strains of streptococci to sections of normal canine and human aortic, mitral, and tricuspid valves and to canine interatrial septum was compared in an in vitro system. Quantitative measurements of adherence ratios were performed by two independent methods. Adherence ratios for Streptococcus mutans, S. sanguis, S. bovis, and Group D streptococci were higher (0.0058-0.0101) than for the other streptococcal strains studied (0.0025-0.0041). With the exception of Group D streptococci, adherence ratios for each bacterial strain were similar with the aortic, mitral, and tricuspid valve sections. Adherence ratios with normal human and canine valve leaflets were similar, but adherence ratios with interatrial septum were lower than with normal valve sections. Adherence ratios for glucan-positive and glucan-negative strains of streptococci with normal and with damaged aortic valve leaflets were also compared. The adherence ratios of the glucan-positive streptococci (S. mutans, S. sanguis, and S. bovis) and one glucan-negative enterococcal strain (KG-3) were approximately five times higher with damaged aortic valves (0.039-0.051) than with normal aortic valves (0.009-0.010). For glucan-positive strains, adherence ratios with normal aortic leaflets were similar when bacteria were grown in media which contains or lacks sucrose. In striking contrast, growth of the glucan-positive strains in medium which lacks sucrose, with resultant deficiency of glucan production, decreased the adherence ratios with damaged aortic valve leaflets to those found with normal aortic leaflets. Treatment of glucan-positive strains with dextranase resulted in a decrease in their adherence ratios to levels seen with bacteria grown in medium lacking sucrose, but the higher adherence ratios could be restored in the presence of exogenous dextran.It is concluded that glucan production is one quantitatively important factor that contributes to the greater adherence of glucan-positive streptococci to damaged rather than to normal aortic heart valve leaflets. However, glucan production is not the only factor that determines preferential adherence of streptococci to damaged heart valves, because glucan-negative strains may also show some degree of increased adherence to damaged valves. Thus, bacterial glucan production is one of the factors that could contribute to the pathogenesis of bacterial endocarditis.

Animals↗

Multicomponent nature of the glucosyltransferase system of Streptococcus mutans.

The glucosyltransferases of S mutans 6715 were resolved into two major fractions. One fraction synthesized water-soluble glucans and the other made water-insoluble glucans. Each fraction was found by polyacrylamide gel electrophoresis to be composed of several catalytically active species, apparently glycoprotein in nature. Treatment of the glucosyltransferases with dextranase in the absence of sucrose caused an interconversion of enzyme forms concomitant with a time-dependent loss of enzyme activity, but did not appear to remove significant amounts of the carbohydrate associated with the enzymes. Comparison of enzyme activity patterns on polyacrylamide gels of the five different S mutans serotypes further emphasizes the complexity of the glucosyltransferase system from this group of microorganisms.

Chemical Phenomena↗

Label-free high-throughput functional lytic assays.

Refractive index-sensitive resonant waveguide grating biosensors are used to assay the label-free enzymatic degradation of biomolecules. These assays provide a robust means of screening for functional lytic modulators. The biomolecular substrates in this study were covalently immobilized through amine groups. Using the Corning Epic System, the digestion signatures for multiple protein substrates on the biosensors are measured. Label-free digestion profiles for these proteins were substrate specific. Similarly, the authors find that the label-free digestion is protease specific. Enzyme-substrate pairs were used to evaluate high- throughput biosensors as tools for screening functional modulators. The lytic inhibitor properties for several proteases and dextranase are determined. The authors find that the IC50 values for the protease inhibitors agree with the reported values for several known inhibitors. The Z' values, using biosensor-based functional lytic screens, were routinely greater than 0.5, making this label-free application feasible for high-throughput screening.

Animals↗

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.

Agglutination↗

New method for the selection of multicopy transformants of Pichia pastoris, using 3-amino-1,2,4 triazol.

The methylotrophic yeast Pichia pastoris has been successfully used for the expression of many heterologous proteins. The level of expression of some of these proteins depends on the copy number of the gene inserted into the yeast genome. Several methods have been reported in the past few years for the isolation of multicopy transformants. One of these methods used an expression vector that contains the bacterial kanamycin-resistance gene Tn903kanr, which confers resistance to G418. Here, we report a different selection method in a mutant strain of P. pastoris (his3-) based on the resistance to 3-amino-1,2,4 triazol, with a vector containing the HIS3 gene from Saccharomyces cerevisiae. Using this selection method, we isolated here P. pastoris transformants containing several copies of the dextranase gene (dex) from Penicillium minioluteum.

Amitrole↗

Induction of antibody response by antigen conjugates and E. coli lipopolysaccharide in mice tolerant to dextran B-512.

Three groups of mice were made tolerant to dextran (Dex) with one of three schedules: a) an immunogenic dose of 10 micrograms three times a week for 26 weeks; b) an immunogenic dose of 50 micrograms three times a week for two weeks, and c) a single tolerogenic dose of 10 mg. Prior to tolerance induction, continuous administration of 10 micrograms Dex induced a predominantly IgM antibody response. The tolerant mice were then challenged with immunogenic doses of the dextran conjugates Dex-Salmonella typhi (Dex-S. typhi) and Dex-sheep red blood cells (Dex-SRBC), and with E. coli lipopolysaccharide (LPS). IgM and IgG responses were compared in each group. Immunogenic dextran alone did not induce a response in the 1 tolerant mice. The response induced by Dex-S. typhi or Dex-SRBC was low but LPS induced an elevated anti-Dex SFC in the three groups of resistant mice. The antibody responses were not affected by pretreatment of antigen with dextranase. Loss of tolerance was more pronounced in mice made tolerant by continuous stimulation as compared to mice made tolerant by a single large dose of antigen. Thus, LPS is able to induce specific responses from B cells of tolerant animals. Furthermore, the level of response was dependent on the toleration schedule.

Animals↗

Analysis of the mechanism of recognition in the complement alternative pathway using C3b-bound low molecular weight polysaccharides.

The human complement (C) system recognizes bacterial, fungal and viral activators of the alternative pathway following covalent attachment of the protein C3b to carbohydrates (CHO) on the surface of the organisms. Recognition first manifests itself as a 3- to 10-fold reduction in the affinity of C3b for factor H, a regulatory protein of C. This report describes the use of a fluorimetric assay which is sensitive to the C3b-H interaction to study the characteristics of recognition. Fluid phase C3b covalently bound to CHO (C3b-CHO) was prepared by activating C3 in the presence of the small homopolymers dextran (alpha 1-6 polyglucose) or inulin (beta 1-2 polyfructose). In particulate form both polysaccharides are activators of C. The conjugates exhibited increased resistance to inactivation in the factor H-dependent assays compared to C3b not bound to CHO and to C3b bound to mono- or disaccharides. The dextran-induced restriction of inactivation was partially reversed by treatment of the conjugate with dextranase. C3b-CHO conjugates failed to bind to factor H-Sepharose and when introduced into serum behaved as though C3b was attached to particulate activators of C, suggesting that the fluorimetric assay accurately reports recognition. The results suggest that the recognition site which induces a reduction in the affinity of C3b for factor H is distinct from the thioester site of C3b and can recognize structural features of polysaccharides including size, sialic acid content, and possibly aspects of three-dimensional oligosaccharide structure.

Binding Sites↗

The action of selected agents on the accumulation of 18F by Streptococcus mutans.

The action of certain substances known to induce cellular alterations, or encounted in the oral cavity, on the accumulation of 18F by Streptococcus mutans GS-5 has been investigated. A 62-67% inhibition in the number of 18F atoms bound per mg dry weight of cells could be induced by a 15 min pretreatment with 2.7 X 10(-4) M cetyltrimethylammoniumbromide, 1 X 10(-1) M acetic anhydride, or 7 X 10(-2) M HCl. Plate counts indicated that alteration of the cellular composition rather than viability was responsible for this diminution in 18F accumulation. Prior exposure for 15 min of this organism to 1 M HCHO or 0.1 M NaOH did not alter 18F accumulation. Of the common salts encountered in the oral cavity, CaCl2 enhanced 18F binding. Pretreatment of the assay cells for 15-160 min with 0.1-10 mg/ml of trypsin, pronase, protease, alpha-glucosidase, dextranase, or lactoferrin had no significant effect on the accumulation of 18F. However, pre-exposure of cells for 60 min to 1-10 mg/ml of either amylase or lipase induced a 40-67% inhibition in the binding of 18F, while lysozyme enhanced the binding of 18F by the cells. It would appear then that the binding of 18F by S. mutans may be altered by certain substances encountered in the oral cavity.

Acetic Anhydrides↗

Properties of enzymes immobilized by the diazotized m-diaminobenzene method.

Some properties of a number of enzymes immobilized by the diazotized m-diaminobenzene (dDAB) method are described. The pH-activity profiles of beta-D-glucosidase, glucoamylase, peroxidase, uricase, and D-glucose oxidase were virtually unchanged on immobilization while those of catalase and dextranase were significantly altered. beta-D-Glucosidase, glucoamylase, and glucose oxidase were found to be more susceptible to denaturation on lyophilization when immobilized than in the native state; however, sorbitol had a marked protective effect in every case examined. Sorbitol was also found to exert a stabilizing effect when lyophilized immobilized preparations were stored. Immobilization marginally improved the stabilities of a number of enzymes to heating at 60 degrees at pH 8.0. The usefulness for continuous reaction of a column of glucoamylase attached to celite was established. The reuse of the solid supports was demonstrated.

Aniline Compounds↗

Variation in N-linked oligosaccharide structures on heterologous proteins secreted by the methylotrophic yeast Pichia pastoris.

We report the characterization of N-linked oligosaccharides on six foreign glycoproteins secreted from the methylotrophic yeast Pichia pastoris. These proteins included: a bacterial enzyme, Bacillus licheniformis alpha-amylase; three fungal enzymes, Saccharomyces cerevisiae invertase, Penicillium minioluteum dextranase, and Mucor pusillus aspartic protease; and two higher eukaryotic proteins, Boophilus microplus (tick) gut antigen and bovine enterokinase catalytic subunit. The carbohydrates on these proteins were observed to vary in size, with Man8GlcNAc2 and Man9GlcNAc2 structures being the most frequently observed species. Substantial amounts of shorter oligomannoside structures were present only on invertase, and longer structures (up to Man18GlcNAc2) were common on aspartic protease and enterokinase. Phosphorylated oligosaccharides were observed on one protein, aspartic protease. Unlike oligosaccharides on glycoproteins secreted from S. cerevisiae, no terminal alpha1,3-linked mannosylation was observed on any of the six P. pastoris-secreted proteins. Changing the growth and induction medium from a minimal salt-based medium to a molasses-based medium had little effect on the size of the oligomannosides. From these results, it is apparent that most foreign proteins secreted from P. pastoris are not subjected to the extensive mannosylation (hyperglycosylation) that commonly occurs in proteins secreted from S. cerevisiae.

Bacterial Proteins↗

Dextran/glucan binding by Streptococcus mutans: the role of molecular size and binding site in agglutination.

1) S. mutans strains of serotypes a, d and g were strongly agglutinated with soluble glucans and dextran T2000. Homologous glucan did not in all cases produce agglutination. 2) The quantity of low molecular weight dextrans bound (T20 and T70) does not correspond to the agglutination induced by glucan or T2000. 3) The agglutination and binding of high molecular weight glucan by B13 cells was sensitive to heat, trypsin, dextranase, EDTA, SDS and urea, whereas no inhibition of binding of T20 and T70 was seen. 4) Pretreatment of B13 cells with anti-d, or anti-glucan sera, or Con A, RCA I, or RCA II completely inhibited agglutination by T2000 and caused a significant reduction of the binding of glucan. No reduction in the binding of T20 and T70 occurred. 5) An agglutination-negative mutant was agglutinated by sucrose but not by T2000 or high molecular weight glucan. It bound normal levels of T20 and T70. 6) The results indicate that B13 cells possess multiple glucan binding sites and that the site responsible for agglutination consists of both polysaccharide and protein. 7) Inhibition studies on agglutination and adherence using B13 cells indicate that the two processes involve different mechanisms.

Antibodies, Bacterial↗

Enzymatic basis for protection of fish embryos by the fertilization envelope.

The mechanism by which the fertilization envelope (FE) is able to protect the embryo of fish until hatching is almost unknown, except for its function as a physical barrier. FE extract from activated or fertilized eggs of the fish Salmo gairdneri was demonstrated to contain enzyme activities using an agar plate enzyme assay. The enzymes apparently active were carboxymethylcellulase (cellulase; EC 3.2.1.4), laminaranase (endo-1,3(4)-beta-glucanase; EC 3.2.1.6), carboxymethylchitinase (chitinase; EC 3.2.1.14), xylanase (endo-1,4-beta-xylanase; EC 3.2.1.8), mannanase (mannan 1,2-(1,3)-alpha-mannosidase; EC 3.2.1.77), dextranase (EC 3.2.1.11), a protease and lysozyme (EC 3.2.1.17). The FE extract exerted an antifungal or fungicidal action on the fungus Saprolegnia parasitica, whereas an extract from the vitelline envelopes (VE) has no apparent enzyme activity nor antifungal or fungicidal action. Enzymes acquired by the FE through the cortical reaction may have an important defensive role, protecting the embryo against invaders or pathogens.

Animals↗

Production and use of glucosyltransferases from Leuconostoc mesenteroides NRRL B-1299 for the synthesis of oligosaccharides containing alpha-(1-->2) linkages.

Glucosyltransferase activities, produced by batch culture of Leuconostoc mesenteroides NRRL B-1299, were recovered both in the culture supernatant (SGT) and associated with the insoluble part of the culture (IGT). A total glucosyltransferase activity of 3.5 U/mL was measured in batch culture. The enzymes from the supernatant were purified 313 times using aqueous two-phase partition between dextran and PEG phases, yielding a preparation with 18.8 U/mg protein. It was shown that both SGT and IGT preparations catalyze acceptor reactions and transfer the glucose unit from sucrose onto maltose to produce glucooligosaccharides. Some of the glucooligosaccharides synthesized (Ln series) contain alpha-(1-->6) osidic linkages and a maltose residue at the reducing end. They were completely hydrolyzed by glucoamylase and dextranase. The other glucooligosaccharides synthesized (Bn series) resisted the action of these enzymes. The tetrasaccharide of this series has been characterized by 13C NMR. Its structure was determined as 2-O-alpha-D-glucosylpanose. The oligosaccharides synthesized by the maltose acceptor reaction with the SGT and IGT preparations only differed in the relative amounts in which they were produced. The difference may arise from diffusional limitations appearing when the insoluble catalyst is used. Under the assay conditions, the glucanase resistant oligosaccharide yield was 35% with both glucosyltransferase preparations.

Carbohydrate Conformation↗