A possible mechanism for the cellular coaggregation between Actinomyces viscosus ATCC 19246 and Streptococcus sanguis ATCC 10557

The cells of Actinomyces viscosus ATCC 19246 (Av19246) and Streptococcus sanguis ATCC 10557 (Ss10557) coaggregated immediately after mixing in 40 mM-Tris/HCl buffer. Optimal conditions were pH 7.5 in the presence of Ca2+ at 0.1 mM or higher. Na2 EDTA and its analogues, Na2MgEDTA and Na2MnEDTA at 7.5 mM inhibited the coaggregation. Trypsin and heat treatment impaired the reactive site on Av19246 cells, but not on Ss10557 cells. The coaggregates, once formed, dissociated gradually during extended incubation at 37 degrees C; this was prevented by addition of sufficient Ca2+. The disaggregation appears to be a spontaneous denaturation of the proteinaceous reactive site on Av19246 cell surface. Thus, the coaggregation involves the interaction of a lectin-like substance on the surface of Av19246 with a carbohydrate site on Ss10557. Native Ss10557 cell walls possessed reactivity with Av19246 cells but 5% (w/v) TCA-extracted cell wall residues did not. A carbohydrate moiety extracted from Ss10557 exhibited a high potency in blocking coaggregation, and coaggregates were dissociated upon addition of the carbohydrate. Lactose, galactose and N-acetyl-D-galactosamine (the latter two are major constituents of the antigen extract) also significantly inhibited the coaggregation, but the other antigen components, glucose and rhamnose, did not. Relative inhibitory activity, expressed as molar potency, of carbohydrate antigen, lactose, galactose and N-acetyl-D-galactosamine respectively, was approximately 26 X 10(3):16:4:1. Ss10557 cells and cell walls reacted only with a Ricinus communis (castor bean) agglutinin-120 but not with Glycine max (soybean) agglutinin, Arachis hypogaea (peanut) agglutinin or Phaseolus vulgaris agglutinin (phytohaemagglutinin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Plaque formation in vitro by Actinomyces viscosus in the presence of Streptococcus sanguis or Streptococcus mutans.

Actinomyces viscosus, growing on a tooth in the presence of sucrose, slowly produced a loosely-attached plaque, the pH being 6.1 after 120 h. When the tooth was inoculated simultaneously with A. viscosus and either Streptococcus sanguis or Streptococcus mutans, firmly-adherent plaque was quickly formed and the pH fell below 5 after 33 h with the former Streptococcus and 24 h with the latter. A. viscosus disappeared from each mixed plaque by 120 h.     

Detection and localization of a lectin on Actinomyces viscosus T14V by monoclonal antibodies

A cell-associated lectin activity that mediates lactose-inhibitable adherence of Actinomyces viscosus T14V has been localized to a specific population of fimbriae by the use of monoclonal antibodies. Nine monoclonal antibodies were produced that reacted with only 1 of 2 immunoelectrophoretically distinct fimbrial components on T14V. The fibrillar morphology of this component was revealed by the immunoelectronmicroscopic examination of bacteria incubated with the monoclonal antibodies. The lectin activity associated with these structures was detected when isolated fimbriae were cross-linked with monoclonal antibodies to form immune complexes with agglutination activity for neuraminidase-treated human erythrocytes, a reaction that was inhibited by lactose. Although the 9 monoclonal antibodies differed in their fine specificities, they reacted only with strains of A. viscosus and A. naeslundii that exhibited lactose-inhibitable adherence. These findings indicate that the lectin activity common to these bacteria resides on fimbriae that are antigenically related to those of T14V.     

Cell-surface proteins of Streptococcus sanguis associated with cell hydrophobicity and coaggregation properties

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

Dominant low responsiveness in the IgG response of mice to the complex protein antigen type 1 fimbriae from Actinomyces viscosus T14V.

Type 1 fimbriae from Actinomyces viscosus T14V, composed of a complex protein of Mr 65,000, mediate the adherence of A. viscosus T14V to the host, whereas type 1 fimbriae-specific antibodies inhibit adherence. Genetic control of the serum IgG response to type 1 fimbriae was evaluated in a series of inbred, hybrid, recombinant inbred, and back-cross mice. Mice were given i.p. injections of 10(8) A. viscosus T14V cells in saline on days 0 and 14, and IgG anti-type 1 fimbriae in sera obtained on day 26 were measured by ELISA. Segregation analysis of the responses of (BALB/cJ x A/J)F1 x A/J backcross mice suggested polygenic control. Linkage analysis in (BALB/cJ x A/J)F1 x A/J backcross and SWXL recombinant inbred mice suggested control by genes linked with H-2 and with Ly-17 and Akp-1. In several F1 hybrid strains derived from H-2-disparate high and low responder parental strains, low responsiveness was dominant. The F1 derived from the H-2-identical high and low responder strains CBA/J and C3H/HeJ was a low responder, suggesting that dominant low responsiveness was mediated by non-H-2-linked genes. A three-gene model is proposed for regulation of the type 1 fimbriae response, including an MHC-linked gene, a gene linked with Ly-17 and Akp-1 on the telomeric portion of chromosome 1, and a background gene whose linkage is unknown.     

Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation

Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.     

Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.

Microorganisms in dental plaque live in constant association with saliva. The role of saliva in the adherence of bacteria to the teeth and the antibacterial properties of saliva have been well investigated; less interest has been shown in the possible role of saliva as a substrate for oral microorganisms. In this study it was shown that saliva can serve as a growth medium for oral Streptococcus spp. and Actinomyces viscosus. The cell production of these organisms on saliva was carbohydrate limited. The doubling times for growth on glucose-supplemented saliva (4 to 5 mmol/liter) ranged from 1.6 to 4.0 h. The availability of carbohydrate sources for the oral microflora is discussed in relation to microbial growth in the oral cavity.     

Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 x 10(3) to 1.2 x 10(4) for the different 125I-labelled mAbs and was approximately 7 x 10(4) for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 x 10(4) molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.     

Complete structure of the polysaccharide from Streptococcus sanguis J22

The cell wall polysaccharides of certain oral streptococci such as Streptococcus sanguis strains 34 and J22, although immunologically distinct, act as receptors for the fimbrial lectins of Actinomyces viscosus T14V. We report the complete covalent structure of the polysaccharide from S. sanguis J22 which is composed of a heptasaccharide subunit linked by phosphodiester bonds. The repeating subunit, which contains alpha-GalNAc, alpha-rhamnose, beta-rhamnose, beta-glucose, and beta-galactose all in the pyranoside form and beta-galactofuranose, is compared with the previously published structure of the polysaccharide from strain 34. The structure has been determined almost exclusively by high-resolution nuclear magnetic resonance methods. The 1H and 13C NMR spectra of the polysaccharides from both strains 34 and J22 have been completely assigned. The stereochemistry of pyranosides was assigned from JH-H values determined from phase-sensitive COSY spectra, and acetamido sugars were assigned by correlation of the resonances of the amide 1H with the sugar ring protons. The 13C spectra were assigned by 1H-detected multiple-quantum correlation (HMQC) spectra, and the assignments were confirmed by 1H-detected multiple-bond correlation (HMBC) spectra. The positions of the glycosidic linkages were assigned by detection of three-bond 1H-13C correlation across the glycosidic linkage in the HMBC spectra. The positions of the phosphodiester linkages were determined by splittings observed in the 13C resonances due to 31P coupling and also by 1H-detected 31P correlation spectroscopy.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

A neuraminidase from Streptococcus sanguis that can release O-acetylated sialic acids

The naturally occurring sialic acids can have different types of N- and O-substitutions, resulting in more than 20 known isomers and compounds. Most methods for the detailed study of these various sialic acids require that the molecules be first released from their alpha-glycosidic linkage. When mild acid hydrolysis is used for this purpose, significant destruction of O-substituent groups occur. On the other hand, the presence of O-substituent groups renders the sialic acid molecule partially or completely resistant to the action of the currently known neuraminidase. To circumvent this problem, we searched for a neuraminidase whose activity is not affected by O-substitution. We reasoned that because Streptococcus sanguis from the human oral cavity is continually exposed to O-substituted sialic acids, its extracellular neuraminidase might not be blocked by O-substitution. We therefore purified this enzyme 3100-fold (56% yield) using ammonium sulfate precipitation, N-(p-aminophenyl)oxamic acid-agarose affinity chromatography, and chromatography on quaternary aminoethyl (QAE)-Sephadex, sulfopropyl (SP)-Sephadex, and Sephacryl S-200. The purified preparation is free of other significant glycosidase activities and proteolytic activities. It is capable of quantitatively releasing all the O-acetylated sialic acids that we studied with the single exception of the 4-O-acetylated sialic acid of equine submaxillary mucin. The activity of the enzyme is also not restricted by the type pf sialic acid linkage or the nature of the underlying oligosaccharide. However, it has maximal activity on gangliosides only in the presence of detergents. The general properties of this enzyme are described and its substrate specificities are contrasted with those of the commonly used neuraminidase from Vibrio cholerae.     

Structure of type 3Gn coaggregation receptor polysaccharide from Streptococcus cristatus LS4

The presence of a novel coaggregation receptor polysaccharide (RPS) on the dental plaque isolate Streptococcus cristatus LS4 was suggested by this strain’s antigenic and coaggregation properties. Examination of RPS isolated from strain LS4 by a combination of 2-dimensional and pseudo 3-dimensional single quantum heteronuclear NMR methods that included detection of ¹³C chemical shifts at high resolution revealed the following repeat unit structure:→6)-β-d-Galf-(1→6)-β-d-GalpNAc-(1→3)-α-d-Galp-(1→P→6)-α-d-Galp-(1→3)-β-L-Rhap-(1→4)-β-d-Glcp-(1→.The identification of this polysaccharide as RPS3Gn, a new structural type, was established by the α-d-Galp-containing epitope of RPS serotype 3 and Gn recognition motif (i.e., β-d-GalpNAc (1→3)-α-d-Galp) for coaggregation with other bacteria.     

Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl alpha-D-glucopyranoside.

Dextransucrase from Streptococcus sanguis ATCC 10558 was photolabeled using p-azidophenyl alpha-D-glucopyranoside with an apparent rate constant of inactivation of 1.40 min-1. The dissociation constant for this compound, which acts as an acceptor molecule in the enzymatic reaction, is 90 microM. Apparently two acceptor binding sites exist on dextransucrase as shown by (i.) photolabeling the enzyme with p-azidophenyl-alpha-D-[5,6-3H]glucopyranoside and (ii.) fluorescence titration experiments.     

Isolation and analysis of sacculi from Streptococcus sanguis.

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.     

Transformation of Streptococcus sanguis Challis with Streptococcus lactis plasmid DNA.

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), and a potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin were evaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizing ability to a mutant deficient in Enzyme IIlac, Factor IIIlac, and phospho-beta-galactosidase of the lactose phosphoenolpyruvate-phosphotransferase system. Plasmid pDB101 was capable of high-efficiency transformation of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains. However, when 20 pLM2001 Lac+ transformants were analyzed by a variety of techniques for the presence of plasmids, none could be detected. In addition, attempts to cure the Lac+ transformants by treatment with acriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transformants had acquired a phospho-beta-galactosidase characteristic of that normally produced by S. lactis and not S. sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in the transformants due to their integration into the host chromosome. The efficient transformation into and expression of pLM2001 and pDB101 genes in S. sanguis provides a model system which could allow the development of a system for cloning genes from dairy starter cultures into S. sanguis to examine factors affecting their expression and regulation.