Molecular and antigenic characterization of a Streptococcus oralis coaggregation receptor polysaccharide by carbohydrate engineering in Streptococcus gordonii

The coaggregation receptor polysaccharides (RPS) of Streptococcus oralis and related species are recognized by lectin-like adhesins on other members of the oral biofilm community and by RPS-specific antibodies. The former interactions involve beta-GalNAc or beta-Gal containing host-like motifs in the oligosaccharide repeating units of these polysaccharides, whereas the latter involves features of these molecules that are immunogenic. In the present investigation, the molecular and corresponding structural basis for the serotype specificity of S. oralis ATCC 10557 RPS was determined by engineering the production of this polysaccharide in transformable Streptococcus gordonii 38. This involved the systematic replacement of genes in the rps cluster of strain 38 with different but related genes from S. oralis 10557 and structural characterization of the resulting polysaccharides. The results identify four unique genes in the rps cluster of strain 10557. These include wefI for an alpha-Gal transferase, wefJ for a GalNAc-1-phosphotransferase that has a unique acceptor specificity, wefK for an acetyl transferase that acts at two positions in the hexasaccharide repeating unit, and a novel wzy associated with the beta1-3 linkage between these units. The serotype specificity of engineered polysaccharides correlated with the wefI-dependent presence of alpha-Gal in these molecules rather than with partial O-acetylation or with the linkage between repeating units. The findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and antigenicity.     

Carbohydrate engineering of the recognition motifs in streptococcal co-aggregation receptor polysaccharides

The cell wall polysaccharides of certain oral streptococci function as receptors for the lectin-like surface adhesins on other members of the oral biofilm community. Recognition of these receptor polysaccharides (RPS) depends on the presence of a host-like motif, either GalNAcbeta1-3Gal (Gn) or Galbeta1-3GalNAc (G), within the oligosaccharide repeating units of different RPS structural types. Type 2Gn RPS of Streptococcus gordonii 38 and type 2G RPS of Streptococcus oralis J22 are composed of heptasaccharide repeats that are identical except for their host-like motifs. In the current investigation, the genes for the glycosyltransferases that synthesize these motifs were identified by high-resolution nuclear magnetic resonance (NMR) analysis of genetically altered polysaccharides. RPS production was switched from type 2Gn to 2G by replacing wefC and wefD in the type 2Gn gene cluster of S. gordonii 38 with wefF and wefG from the type 2G cluster of S. oralis J22. Disruption of either wefC or wefF abolished cell surface RPS production. In contrast, disruption of wefD in the type 2Gn cluster or wefG in the type 2G cluster eliminated beta-GalNAc from the Gn motif or beta-Gal from the G motif, resulting in mutant polysaccharides with hexa- rather than heptasaccharide subunits. The mutant polysaccharides reacted like wild-type RPS with rabbit antibodies against type 2Gn or 2G RPS but were inactive as co-aggregation receptors. Additional mutant polysaccharides with GalNAcbeta1-3GalNAc or Galbeta1-3Gal recognition motifs were engineered by replacing wefC in the type 2Gn cluster with wefF or wefF in the type 2G cluster with wefC respectively. The reactions of these genetically modified polysaccharides as antigens and receptors provide further insight into the structural basis of RPS function.     

Relationship between the ability of oral streptococci to interact with platelet glycoprotein Ibalpha and with the salivary low-molecular-weight mucin, MG2

The oral streptococci Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis are common aetiological agents of infective endocarditis, and their ability to adhere to and induce the aggregation of platelets is thought to be a virulence trait. The platelet glycoprotein GPIbalpha has been implicated as the adhesion receptor for S. sanguinis and S. gordonii, but it is not known if this is the case for S. oralis and other species. The aim of this study was to determine the GPIbalpha-interactive capability of a range of oral streptococci and to determine the relationship between this capability and their ability to interact with the salivary constituents that they would encounter in their normal habitat. All platelet-adhesive S. sanguinis strains and most S. gordonii strains adhered in a GPIbalpha-dependent manner, but strains of S. oralis, Streptococcus cristatus, Streptococcus parasanguinis and Streptococcus mitis had no direct affinity for platelets. Those strains that were able to bind GPIbalpha also bound to the low-molecular-weight submandibular salivary mucin, MG2, and this interaction was sialic acid-dependent. The data suggest that S. sanguinis and S. gordonii may be efficient colonizers of platelet vegetations because of their adaptation to recognize sialylated salivary mucins. In contrast, S. oralis does not interact with platelets and so is likely to colonize vegetations through an as yet unidentified mechanism.     

Genetic loci for coaggregation receptor polysaccharide biosynthesis in Streptococcus gordonii 38

The cell wall polysaccharide of Streptococcus gordonii 38 functions as a coaggregation receptor for surface adhesins on other members of the oral biofilm community. The structure of this receptor polysaccharide (RPS) is defined by a heptasaccharide repeat that includes a GalNAcbeta1-->3Gal-containing recognition motif. The same RPS has now been identified from S. gordonii AT, a partially sequenced strain. PCR primers designed from sequences in the genomic database of strain AT were used to identify and partially characterize the S. gordonii 38 RPS gene cluster. This cluster includes genes for seven putative glycosyltransferases, a polysaccharide polymerase (Wzy), an oligosaccharide repeating unit transporter (Wzx), and a galactofuranose mutase, the enzyme that promotes synthesis of UDP-Galf, one of five predicted RPS precursors. Genes outside this region were identified for the other four nucleotide-linked sugar precursors of RPS biosynthesis, namely, those for formation of UDP-Glc, UDP-Gal, UDP-GalNAc, and dTDP-Rha. Two genes for putative galactose 4-epimerases were identified. The first, designated galE1, was identified as a pseudogene in the galactose operon, and the second, designated galE2, was transcribed with three of the four genes for dTDP-Rha biosynthesis (i.e., rmlA, rmlC, and rmlB). Insertional inactivation of galE2 abolished (i) RPS production, (ii) growth on galactose, and (iii) both UDP-Gal and UDP-GalNAc 4-epimerase activities in cell extracts. Repair of the galE1 pseudogene in this galE2 mutant restored growth on galactose but not RPS production. Cell extracts containing functional GalE1 but not GalE2 contained UDP-Gal 4-epimerase but not UDP-GalNAc 4-epimerase activity. Thus, provision of both UDP-Gal and UDP-GalNAc for RPS production by S. gordonii 38 depends on the dual specificity of the epimerase encoded by galE2.     

Coaggregation-mediated interactions of streptococci and actinomyces detected in initial human dental plaque

Streptococci and actinomyces that initiate colonization of the tooth surface frequently coaggregate with each other as well as with other oral bacteria. These observations have led to the hypothesis that interbacterial adhesion influences spatiotemporal development of plaque. To assess the role of such interactions in oral biofilm formation in vivo, antibodies directed against bacterial surface components that mediate coaggregation interactions were used as direct immunofluorescent probes in conjunction with laser confocal microscopy to determine the distribution and spatial arrangement of bacteria within intact human plaque formed on retrievable enamel chips. In intrageneric coaggregation, streptococci such as Streptococcus gordonii DL1 recognize receptor polysaccharides (RPS) borne on other streptococci such as Streptococcus oralis 34. To define potentially interactive subsets of streptococci in the developing plaque, an antibody against RPS (anti-RPS) was used together with an antibody against S. gordonii DL1 (anti-DL1). These antibodies reacted primarily with single cells in 4-h-old plaque and with mixed-species microcolonies in 8-h-old plaque. Anti-RPS-reactive bacteria frequently formed microcolonies with anti-DL1-reactive bacteria and with other bacteria distinguished by general nucleic acid stains. In intergeneric coaggregation between streptococci and actinomyces, type 2 fimbriae of actinomyces recognize RPS on the streptococci. Cells reactive with antibody against type 2 fimbriae of Actinomyces naeslundii T14V (anti-type-2) were much less frequent than either subset of streptococci. However, bacteria reactive with anti-type-2 were seen in intimate association with anti-RPS-reactive cells. These results are the first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo. Further, these results demonstrate the spatiotemporal development and prevalence of mixed-species communities in early dental plaque.     

Expression of green fluorescent protein in Streptococcus gordonii DL1 and its use as a species-specific marker in coadhesion with Streptococcus oralis 34 in saliva-conditioned biofilms in vitro

Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter, PhppA, which is situated upstream of the chromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp ('gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-'gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the PhppA-'gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement of S. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.     

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.     

Mode of action of a lysostaphin-like bacteriolytic agent produced by Streptococcus zooepidemicus 4881.

Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall. Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449. Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall. The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S. pyogenes, Streptococcus gordonii, Streptococcus oralis, S. mutans, and Streptococcus rattus has been evaluated.     

Novel model for multispecies biofilms that uses rigid gas-permeable lenses

Oral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novel in vitro model system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprising Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguinis; S. gordonii, Actinomyces naeslundii, and Fusobacterium nucleatum; or S. gordonii, F. nucleatum, and Porphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.     

The effect of a novel anti-adherent compound on the adherence of oral streptococci to hydroxyapatite

The effect of the copolymer M239, 144 with and without chlorhexidine on the adherence of oral streptococci to saliva-coated hydroxyapatite was investigated. At 1% w/v M239, 144 reduced the adherence of Streptococcus sanguis NCTC 7863 by 94%. It had a moderate effect on the adherence of other Strep. sanguis strains and a Streptococcus gordonii strain but had no effect on the adherence of Streptococcus oralis or Streptococcus mutans. Chlorhexidine did not influence the anti-adhesive properties of 1% w/v M239, 144.     

In vitro evaluation of the effect of nicotine, cotinine, and caffeine on oral microorganisms

The aim of this in vitro study was to evaluate the effects of nicotine, cotinine, and caffeine on the viability of some oral bacterial species. It also evaluated the ability of these bacteria to metabolize those substances. Single-species biofilms of Streptococcus gordonii, Porphyromonas gingivalis, or Fusobacterium nucleatum and dual-species biofilms of S. gordonii -- F. nucleatum and F. nucleatum -- P. gingivalis were grown on hydroxyapatite discs. Seven species were studied as planktonic cells, including Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes, Actinomyces naeslundii, and the species mentioned above. The viability of planktonic cells and biofilms was analyzed by susceptibility tests and time-kill assays, respectively, against different concentrations of nicotine, cotinine, and caffeine. High-performance liquid chromatography was performed to quantify nicotine, cotinine, and caffeine concentrations in the culture media after the assays. Susceptibility tests and viability assays showed that nicotine, cotinine, and caffeine cannot reduce or stimulate bacterial growth. High-performance liquid chromatography results showed that nicotine, cotinine, and caffeine concentrations were not altered after bacteria exposure. These findings indicate that nicotine, cotinine, and caffeine, in the concentrations used, cannot affect significantly the growth of these oral bacterial strains. Moreover, these species do not seem to metabolize these substances.     

Platelet aggregation by oral streptococci

One proposed mechanism in the pathogenesis of infective endocarditis is the direct aggregation of platelets by the bacteria causing the disease. Some, but not all, strains of Streptococcus sanguis have been reported to aggregate platelets but the taxonomy of this and related taxa has changed recently. The ability to aggregate platelets by 24 genetically grouped laboratory stock strains was studied along with 8 recent isolates from cases of endocarditis. Strains belonging to S. sanguis could aggregate platelets, but not S. gordonii, "S. parasanguis", S. mitis, S. oralis or related taxa. Also, preliminary data indicate that certain biotypes of S. sanguis lack the ability to aggregate platelets. Of the recent clinical isolates, only 4 aggregated platelets and each of these showed phenotypes typical of S. sanguis. These data suggest that the ability to aggregate platelets is not essential for an organism to be able to cause endocarditis, although it may be a significant virulence factor.     

Adherence and internalization of Streptococcus gordonii by epithelial cells involves beta1 integrin recognition by SspA and SspB (antigen I/II family) polypeptides

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II- mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II- mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.     

Autoinducer 2: a concentration-dependent signal for mutualistic bacterial biofilm growth

4,5-Dihydroxy-2,3-pentanedione (DPD), a product of the LuxS enzyme in the catabolism of S-ribosylhomocysteine, spontaneously cyclizes to form autoinducer 2 (AI-2). AI-2 is proposed to be a universal signal molecule mediating interspecies communication among bacteria. We show that mutualistic and abundant biofilm growth in flowing saliva of two human oral commensal bacteria, Actinomyces naeslundii T14V and Streptococcus oralis 34, is dependent upon production of AI-2 by S. oralis 34. A luxS mutant of S. oralis 34 was constructed which did not produce AI-2. Unlike wild-type dual-species biofilms, A. naeslundii T14V and an S. oralis 34 luxS mutant did not exhibit mutualism and generated only sparse biofilms which contained a 10-fold lower biomass of each species. Restoration of AI-2 levels by genetic or chemical (synthetic AI-2 in the form of DPD) complementation re-established the mutualistic growth and high biomass characteristic for the wild-type dual-species biofilm. Furthermore, an optimal concentration of DPD was determined, above and below which biofilm formation was suppressed. The optimal concentration was 100-fold lower than the detection limit of the currently accepted AI-2 assay. Thus, AI-2 acts as an interspecies signal and its concentration is critical for mutualism between two species of oral bacteria grown under conditions that are representative of the human oral cavity.     

Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study

Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. These streptococci all coaggregated strongly with both A. viscosus and A. naesludii strains, whereas S. oralis C104 interacted preferentially with certain strains of the latter species. Receptor polysaccharide was isolated from S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The 1H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by 1H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments (1H and 13C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages with the following structure. [----6)Galf(beta 1----3)Galp(beta 1----6)Galf(beta 1----6)GalpNAc(beta 1----3) Galp(alpha 1----1)ribitol(5----PO4-]n     

Genotypic heterogeneity of Streptococcus oralis and distinct aciduric subpopulations in human dental plaque

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (chi(2) = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.     

Streptococcus gordonii Hsa environmentally constrains competitive binding by Streptococcus sanguinis to saliva-coated hydroxyapatite

Competition between pioneer colonizing bacteria may determine polymicrobial succession during dental plaque development, but the ecological constraints are poorly understood. For example, more Streptococcus sanguinis than Streptococcus gordonii organisms are consistently isolated from the same intraoral sites, yet S. gordonii fails to be excluded and survives as a species over time. To explain this observation, we hypothesized that S. gordonii could compete with S. sanguinis to adhere to saliva-coated hydroxyapatite (sHA), an in vitro model of the tooth surface. Both species bound similarly to sHA, yet 10- to 50-fold excess S. gordonii DL1 reduced binding of S. sanguinis SK36 by 85 to >95%. S. sanguinis, by contrast, did not significantly compete with S. gordonii to adhere. S. gordonii competed with S. sanguinis more effectively than other species of oral streptococci and depended upon the salivary film on HA. Next, putative S. gordonii adhesins were analyzed for contributions to interspecies competitive binding. Like wild-type S. gordonii, isogenic mutants with mutations in antigen I/II polypeptides (sspAB), amylase-binding proteins (abpAB), and Csh adhesins (cshAB) competed effectively against S. sanguinis. By contrast, an hsa-deficient mutant of S. gordonii showed significantly reduced binding and competitive capabilities, while these properties were restored in an hsa-complemented strain. Thus, Hsa confers a selective advantage to S. gordonii over S. sanguinis in competitive binding to sHA. Hsa expression may, therefore, serve as an environmental constraint against S. sanguinis, enabling S. gordonii to persist within the oral cavity, despite the greater natural prevalence of S. sanguinis in plaque and saliva.     

Co-ordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species

It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.