Structural and genetic basis for the binding of a mouse monoclonal antibody to Flavobacterium psychrophilum lipopolysaccharide.

A mouse monoclonal antibody (mAb FL100A) previously prepared against Flavobacterium psychrophilum (Fp) CSF259-93 has now been examined for binding to lipopolysaccharides (LPS) of this strain and Fp 950106-1/1. The corresponding O-polysaccharides (O-PS) of these strains are formed by identical trisaccharide repeats composed of l-Rhamnose (l-Rha), 2-acetamido-2-deoxy-l-fucose (l-FucNAc) and 2-acetamido-4-R1-2,4-dideoxy-d-quinovose (d-Qui2NAc4NR1) where R1 represents a dihydroxyhexanamido moiety. The O-PS loci of these strains are also identical except for the gene (wzy1 or wzy2) that encodes the polysaccharide polymerase. Accordingly, adjacent O-PS repeats are joined through d-Qui2NAc4NR1 and l-Rha by wzy2-dependent α(1-2) linkages in Fp CSF259-93 versus wzy1-dependent ß(1-3) linkages in Fp 950106-1/1. mAb FL100A reacted strongly with Fp CSF259-93 O-PS and LPS but weakly or not at all with Fp 950106-1/1 LPS and O-PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5-times stronger to homologous Fp CSF259-93 LPS than to LPS from a strain with a different R-group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O-PS. Thus, Fp CSF259-93 O-PS formed a stable well-defined compact helix in which the R1 groups were displayed in a regular pattern on the helix exterior while unreactive Fp 950106-1/1 O-PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2-linked F. psychrophilum O-PS and LPS.

Effect of mechanically stimulated saliva on initial human dental biofilm formation.

This study evaluated the impact of mechanically stimulated saliva on initial bacterial colonization. Interaction between oral bacteria and both unstimulated and stimulated saliva was examined in vitro by laying labeled bacteria over SDS-PAGE-separated salivary proteins. The effects of chewing on in vivo biofilm, microbial composition, and spatial arrangement were examined in two human volunteers using an intraoral stent containing retrievable enamel chips. In vitro experiments showed that bacterial binding to proteins from stimulated saliva was lower than that to proteins from unstimulated saliva. Lack of binding activity was noted with Streptococcus mutans and Lactobacillus casei. Human Oral Microbe Identification Microarray (HOMIM) analyses revealed a consistent chewing-related increase in the binding of Streptococcus anginosus and Streptococcus gordonii. Immunofluorescence microscopy demonstrated the presence of multi-species colonies and cells bearing different serotypes of the coaggregation-mediating streptococcal cell-surface receptor polysaccharides (RPS). Differences in bacterial colonization were noted between the two volunteers, while the type 4 RPS-reactive serotype was absent in one volunteer. Cells reacting with antibody against Rothia or Haemophilus were prominent in the early biofilm. While analysis of the data obtained demonstrated inter-individual variations in both in vitro and in vivo bacterial binding patterns, stimulating saliva with multiple orosensory stimuli may modulate oral bacterial colonization of tooth surfaces.

Comparative Structural and Antigenic Characterization of Genetically Distinct Flavobacterium psychrophilum O-Polysaccharides.

Little is known about the underlying basis of serotype specificity among strains of Flavobacterium psychrophilum, the agent of rainbow trout fry syndrome and bacterial cold-water disease. The identification of different heat-stable O-serotypes among strains of this gram-negative pathogen does, however, suggest structural variations in the O-polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-rhamnose (L-Rha), 2-acetamido-2-deoxy-L-fucose (L-FucNAc) and 2-acetamido-4-R-2,4-dideoxy-D-quinovose (D-Qui2NAc4NR), where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of Fp CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of Fp 950106-1/1, which belongs to a different O-serotype, are identical except for wzy, which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the linkage of D-Qui2NAc4NR to L-Rha, which is α1-2 for Fp CSF259-93 versus ß1-3 for Fp 950106-1/1, is the only structural difference between O-PS from these strains. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti-F. psychrophilum antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of Fp CSF259-93 or Fp 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of F. psychrophilum O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.

Interbacterial Adhesion Networks within Early Oral Biofilms of Single Human Hosts.

Specific interbacterial adhesion, termed coaggregation, is well established for three early colonizers of the plaque biofilm: streptococci, actinomyces, and veillonellae. However, little is known about interactions of other early colonizers and about the extent of interactions within the bacterial community from a single host. To address these gaps, subject-specific culture collections from two individuals were established using an intraoral biofilm retrieval device. Molecular taxonomy (Human Oral Microbe Identification Microarray [HOMIM]) analysis of biofilm samples confirmed the integrity and completeness of the collections. HOMIM analysis verified the isolation of Streptococcus gordonii and S. anginosus from only one subject, as well as isolation of a previously uncultivated streptococcal phylotype from the other subject. Strains representative of clonal diversity within each collection were further characterized. Greater than 70% of these streptococcal strains from each subject coaggregated with at least one other coisolate. One-third of the strains carry a known coaggregation mediator: receptor polysaccharide (RPS). Almost all nonstreptococcal isolates coaggregated with other coisolates. Importantly, certain Rothia strains demonstrated more coaggregations with their coisolated bacteria than did any Streptococcus or Actinomyces strain, and certain Haemophilus isolates participated in twice as many. Confocal microscopy of undisturbed biofilms showed that Rothia and Haemophilus each occur in small multispecies microcolonies. However, in confluent high-biomass regions, Rothia occurred in islands whereas Haemophilus was distributed throughout. Together, the data demonstrate that coaggregation networks within an individual's oral microflora are extensive and that Rothia and Haemophilus can be important initiators of cell-cell interactions in the early biofilm.IMPORTANCE Extensive involvement of specific interbacterial adhesion in dental plaque biofilm formation has been postulated based on in vitro coaggregation between oral bacteria from culture collections that are not subject specific. In the present study, subject-specific culture collections were obtained from early plaque biofilm of two volunteers, and coaggregations within each culture collection were assayed. Coaggregations, several of which involved a coaggregation-mediating cell surface molecule known from well-studied streptococci, were widespread. Unexpectedly, the little-studied organisms Haemophilus and Rothia participated in the greatest numbers of interactions with community members; these two organisms showed different distributions within the undisturbed biofilm. The data show that coaggregation networks encompass most organisms within the biofilm community of each individual, and they indicate prominent participation of organisms such as Haemophilus and Rothia in early plaque biofilm formation.

Differential Utilization of Basic Proline-Rich Glycoproteins during Growth of Oral Bacteria in Saliva.

UNLABELLED: Although saliva is widely recognized as a primary source of carbon and nitrogen for growth of the dental plaque biofilm community, little is known about how different oral bacteria utilize specific salivary components. To address this question, 32 strains representing 16 genera commonly isolated from early plaque biofilms were compared for growth over two transfers in stimulated (by chewing Parafilm) whole saliva that was stabilized by heat treatment and dialysis. The cell densities, measured by quantitative PCR (qPCR), ranged from ∼1 × 10(6) to 1 × 10(7)/ml for strains of Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis and one strain of Streptococcus sanguinis Strains of Streptococcus mutans, Gemella haemolysans, and Granulicatella adiacens reached ∼1 × 10(5) to 1 × 10(6)/ml. In contrast, little or no growth was noted for three other strains of S. sanguinis, as well as for strains of Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus sobrinus, Actinomyces spp., Abiotrophia defectiva, and Rothia dentocariosa SDS-PAGE, lectin blotting, and two-dimensional gel electrophoresis of saliva from cultures of S. gordonii, S. oralis, and S. mitis revealed species-specific differences in the degradation of basic proline-rich glycoproteins (PRG). In contrast, saliva from cultures of other bacteria was indistinguishable from control saliva. Species-dependent differences in the utilization of individual host sugars were minor. Thus, differences in salivary glycan foraging between oral species may be important to cross-feeding and cooperation between organisms in dental plaque biofilm development. IMPORTANCE: Bacteria in the mouth use saliva for nutrition. How each of the many types of bacteria uses saliva is not clear. We show that a major protein in saliva, called PRG, is an important nutrition source for certain bacteria but not for others. PRG has many sugar molecules linked in chains, but the sugar is not available for bacteria until the chains are degraded. The bacteria that can grow by digesting this protein break the sugar chains into parts which not only support their own growth but could also be available to support the growth of those bacteria that cannot use the intact protein.

Cell Surface Glycoside Hydrolases of Streptococcus gordonii Promote Growth in Saliva.

UNLABELLED: The growth of the oral commensal Streptococcus gordonii in saliva may depend on a number of glycoside hydrolases (GHs), including three cell wall-anchored proteins that are homologs of pneumococcal ß-galactosidase (BgaA), ß-N-acetylglucosaminidase (StrH), and endo-ß-N-acetylglucosaminidase D (EndoD). In the present study, we introduced unmarked in-frame deletions into the corresponding genes of S. gordonii DL1, verified the presence (or absence) of the encoded proteins on the resulting mutant strains, and compared these strains with wild-type strain DL1 for growth and glycan foraging in saliva. The overnight growth of wild-type DL1 was reduced 3- to 10-fold by the deletion of any one or two genes and approximately 20-fold by the deletion of all three genes. The only notable change in the salivary proteome associated with this reduction of growth was a downward shift in the apparent molecular masses of basic proline-rich glycoproteins (PRG), which was accompanied by the loss of lectin binding sites for galactose-specific Erythrina cristagalli agglutinin (ECA) and mannose-specific Galanthus nivalis agglutinin (GNA). The binding of ECA to PRG was also abolished in saliva cultures of mutants that expressed cell surface BgaA alone or together with either StrH or EndoD. However, the subsequent loss of GNA binding was seen only in saliva cocultures of different mutants that together expressed all three cell surface GHs. The findings indicate that the growth of S. gordonii DL1 in saliva depends to a significant extent on the sequential actions of first BgaA and then StrH and EndoD on N-linked glycans of PRG. IMPORTANCE: The ability of oral bacteria to grow on salivary glycoproteins is critical for dental plaque biofilm development. Little is known, however, about how specific salivary components are attacked and utilized by different members of the biofilm community, such as Streptococcus gordonii. Streptococcus gordonii DL1 has three cell wall-anchored glycoside hydrolases that are predicted to act on host glycans. In the present study, we introduced unmarked in-frame deletions in the corresponding genes, verified the presence (or absence) of encoded proteins on the resulting mutant strains, and compared these strains with wild-type DL1 for growth and glycan foraging in saliva. The results indicate that the growth of S. gordonii DL1 depends to a significant extent on sequential action of these cell surface GHs on N-linked glycans of basic proline-rich salivary glycoproteins, which appears to be an essential first step in salivary glycan foraging.

Structures of Capsular Polysaccharide Serotypes 35F and 35C of Streptococcus pneumoniae Determined by Nuclear Magnetic Resonance and Their Relation to Other Cross-Reactive Serotypes.

UNLABELLED: The structures of Streptococcus pneumoniae capsular polysaccharides (CPSs) are essential for defining the antigenic as well as genetic relationships between CPS serotypes. The four serotypes that comprise CPS serogroup 35 (i.e., types 35F, 35A, 35B, and 35C) are known to cross-react with genetically related type 20, 29, 34, 42, or 47F. While the structures of CPS serotype 35A (CPS35A) and CPS35B are known, those of CPS35F and CPS35C are not. In the present study, the serotypes of CPS35F and CPS35C were characterized by high-resolution heteronuclear magnetic resonance (NMR) spectroscopy and glycosyl composition analyses to reveal the following repeat unit structures: [Formula: see text] where OAc indicates O-acetylated. Importantly, CPS35F, the immunizing serotype for the production of group 35 serum, more closely resembles CPS34 and CPS47F than other members of serogroup 35. Moreover, CPS35C is distinct from either CPS35F or CPS35B but closely related to CPS35A and identical to de-O-acetylated CPS42. The findings provide a comprehensive view of the structural and genetic relations that exist between the members of CPS serogroup 35 and other cross-reactive serotypes. IMPORTANCE: Cross-reactions of diagnostic rabbit antisera with Streptococcus pneumoniae capsular polysaccharide serotypes are generally limited to members of the same serogroup. Exceptions do, however, occur, most notably among a group of nonvaccine serotypes that includes the members of serogroup 35 (i.e., types 35F, 35A, 35B, and 35C) and other genetically related types. The presently determined structures of S. pneumoniae serotypes 35F and 35C complete the structural characterization of serogroup 35 and thereby provide the first comprehensive description of how different members of this serogroup are related to each other and to types 29, 34, 42, and 47F. The structural and genetic features of these serotypes suggest the existence of three distinct capsular polysaccharide subgroups that presumably emerged by immune selection in the human host.

Probing of microbial biofilm communities for coadhesion partners.

Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method's broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.

Chemical structures of Streptococcus pneumoniae capsular polysaccharide type 39 (CPS39), CPS47F, and CPS34 characterized by nuclear magnetic resonance spectroscopy and their relation to CPS10A.

Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized: [structure: see text] Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the α1-2 transfer of Gal pyranoside (Galp) to ribitol-5-phosphate in the synthesis of CPS10A, CPS47F, and CPS34 but with α1-1 transfer of Gal to ribitol-5-phosphate in the synthesis of CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e., wefM) of Streptococcus oralis that is associated with α1-1 transfer of Galp to ribitol-5-phosphate. These and other recent findings identify linkages from α-Galp to ribitol-5-phosphate and from this residue to adjacent Gal furanoside (Galf) as important sites of CPS structural and genetic diversity.

Genome Sequence of Salmonella enterica Serovar Typhi Oral Vaccine Strain Ty21a.

Attenuated Salmonella enterica serovar Typhi strain Ty21a is an important vaccine for controlling typhoid fever and serves as an oral vector for delivering heterologous antigens. The key attenuating features of this randomly mutated strain remain in question. Genome sequencing has revealed 679 single nucleotide polymorphisms (SNPs), and will help define alterations contributing to Ty21a safety and immunogenicity.

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10.

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had ß1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had ß1-3-linked Galf branches linked to GalNAc, only CPS10A had additional ß1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The ß1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent ß1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development.

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

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 (13)C chemical shifts at high resolution revealed the following repeat unit structure: →6)-ß-d-Galf-(1→6)-ß-d-GalpNAc-(1→3)-α-d-Galp-(1→P→6)-α-d-Galp-(1→3)-ß-L-Rhap-(1→4)-ß-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., ß-d-GalpNAc (1→3)-α-d-Galp) for coaggregation with other bacteria.

Dual function of a tip fimbrillin of Actinomyces in fimbrial assembly and receptor binding.

Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly.

The Actinomyces oris type 2 fimbrial shaft FimA mediates co-aggregation with oral streptococci, adherence to red blood cells and biofilm development.

Interbacterial interactions between oral streptococci and actinomyces and their adherence to tooth surface and the associated host cells are key early events that promote development of the complex oral biofilm referred to as dental plaque. These interactions depend largely on a lectin-like activity associated with the Actinomyces oris type 2 fimbria, a surface structure assembled by sortase (SrtC2)-dependent polymerization of the shaft and tip fimbrillins, FimA and FimB respectively. To dissect the function of specific fimbrillins in various adherence processes, we have developed a convenient new technology for generating unmarked deletion mutants of A. oris. Here, we show that the fimB mutant, which produced type 2 fimbriae composed only of FimA, like the wild type co-aggregated strongly with receptor-bearing streptococci, agglutinated with sialidase-treated red blood cells, and formed monospecies biofilm. In contrast, the fimA and srtC2 mutants lacked type 2 fimbriae and were non-adherent in each of these assays. Plasmid-based expression of the deleted gene in respective mutants restored adherence to wild-type levels. These findings uncover the importance of the lectin-like activity of the polymeric FimA shaft rather than the tip. The multivalent adhesive function of FimA makes it an ideal molecule for exploring novel intervention strategies to control plaque biofilm formation.

Structure and molecular characterization of Streptococcus pneumoniae capsular polysaccharide 10F by carbohydrate engineering in Streptococcus oralis.

Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfbeta1-6GalNAcbeta1-3Galalpha) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfbeta1-6GalNAcbeta1-3Galalpha rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a beta1-3 linkage between Galf and GalNAcbeta1-3Galalpha. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of beta1-6-linked Galf in recognition of adjacent GalNAcbeta1-3Galalpha in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.

Shear-enhanced oral microbial adhesion.

Shear-enhanced adhesion, although not observed for fimbria-mediated adhesion of oral Actinomyces spp., was noted for Hsa-mediated adhesion of Streptococcus gordonii to sialic acid-containing receptors, an interaction implicated in the pathogenesis of infective endocarditis.

Comparative structural and molecular characterization of ribitol-5-phosphate-containing Streptococcus oralis coaggregation receptor polysaccharides.

The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfbeta1-6GalNAcbeta1-3Galalpha1-2ribitol-5-PO(4)(-)-6Galfbeta1-3Galbeta1](n), thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is alpha1-2 in strain SK144 versus alpha1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque.

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.

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.