J O Cisar

National Institutes of Health, Maryland, United States

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Publications (81)270.33 Total impact

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    ABSTRACT: 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 co-adhesive 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 co-adhering 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 A. naeslundii or RPS-bearing S. 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.
    Applied and environmental microbiology. 08/2014;
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    ABSTRACT: Background Coaggregations of viridans group streptococci with other members of the oral microbial community play an important role in dental plaque biofilm formation. These interactions generally depend on lectin-like recognition of specific host-like motifs within the hexa- and heptasaccharide repeating units of different streptococcal receptor polysaccharides (RPS). To date, seven structural types of RPS (1Gn, 2Gn, 2G, 3Gn, 3G, 4Gn, and 5Gn) have been identified from over 25 different streptococcal strains. The repeating units of different RPS types invariably contain either GalNAcβ1-3Gal (Gn) or Galβ1-3GalNAc (G) disaccharide motifs, which are receptors of Actinomyces spp. type 2 fimbriae. Streptococci that bear Gn types of RPS also coaggregate with strains of Streptococcus gordonii and S. sanguinis that bear GalNAc-binding adhesins. Highlight Insight into the genetic basis of RPS structure and function was gained from comparative characterization of gene clusters that are involved in RPS production that occurs in different streptococci by carbohydrate engineering, which involves gene swapping between strains to obtain polysaccharides with altered structural and biological properties. Conclusion Production of genetically modified polysaccharides on bacterial cell surfaces (i.e., carbohydrate engineering) appears to be useful for a wide range of potential applications for basic research on polysaccharide structure and function as well as in the possible production of new or improved vaccines.
    Journal of Oral Biosciences 08/2014;
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    ABSTRACT: Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling antigenic as well as genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 were extended to include genetically related S. pneumoniae CPS34, 39 and 47F. High-resolution hetero-nuclear NMR spectroscopy confirmed the published structure of CPS34 and in conjunction with glycosyl composition analyses, revealed repeat unit structures of the other serotypes, which have not been previously characterized: jb;JB.01731-14v1/FU1F1FU1 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 Galp to ribitol-5-phosphate in synthesis of CPS10A, CPS47F and CPS34 but α1-1 transfer of Gal to ribitol-5-phosphate in CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e. wefM) of S. oralis 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 Galf as important sites of CPS structural and genetic diversity.
    Journal of bacteriology. 07/2014;
  • Jinghua Yang, Yasuo Yoshida, John O Cisar
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    ABSTRACT: Interbacterial adhesion between streptococci and actinomyces promotes early dental plaque biofilm development. Recognition of coaggregation receptor polysaccharides (RPS) on strains of S. sanguinis, S. gordonii and S. oralis by Actinomyces spp. type 2 fimbriae is the principal mechanism of these interactions. Previous studies of genetic loci for synthesis of RPS (rps) and RPS precursors (rml, galE1 and galE2) in S. gordonii 38 and S. oralis 34 revealed differences between these strains. To determine whether these differences are strain- or species-specific, we identified and compared loci for polysaccharide biosynthesis in additional strains of these species and in several strains of the previously unstudied species, S. sanguinis. Genes for synthesis of RPS precursors distinguished the rps loci of different streptococci. Thus, rml genes for synthesis of TDP-L-Rha were in rps loci of S. oralis strains but at other loci in S. gordonii and S. sanguinis. Genes for two distinct galactose epimerases were also distributed differently. Thus, galE1 for epimerization of UDP-Glc and UDP-Gal was in galactose operons of S. gordonii and S. sanguinis strains but surprisingly, this gene was not present in S. oralis. Moreover, galE2 for epimerization of both UDP-Glc and UDP-Gal and UDP-GlcNAc and UDP-GalNAc was at a different locus in each species, including rps operons of S. sanguinis. The findings provide insight into cell surface properties that distinguish different RPS-producing streptococci and open an approach for identifying these bacteria based on the arrangement of genes for synthesis of polysaccharide precursors. This article is protected by copyright. All rights reserved.
    Molecular oral microbiology. 11/2013;
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    ABSTRACT: 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.
    Genome announcements. 01/2013; 1(4).
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    ABSTRACT: 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 β1-6-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 β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-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 β1-3-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 β1-6-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.
    Journal of Biological Chemistry 08/2011; 286(41):35813-22. · 4.65 Impact Factor
  • Jinghua Yang, John O Cisar, C Allen Bush
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    ABSTRACT: 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)-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.
    Carbohydrate research 08/2011; 346(11):1342-6. · 2.03 Impact Factor
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    ABSTRACT: 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.
    Journal of bacteriology 07/2011; 193(13):3197-206. · 3.94 Impact Factor
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    ABSTRACT: 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.
    Molecular Microbiology 06/2011; 81(5):1205-20. · 5.03 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 07/2010; 285(31):24217-27. · 4.65 Impact Factor
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    ABSTRACT: Summary 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.
    Molecular Microbiology 06/2010; · 5.03 Impact Factor
  • S. RUHL, A. EIDT, U. REISCHL, J.O. CISAR
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    ABSTRACT: The formation of early oral biofilm communities involves specific coadhesive interactions between different bacterial species. The most common mechanism is binding of proteinaceous lectin-like adhesins on one bacterium to complementary carbohydrate-containing receptors on another species. A well studied example is the interaction of a Gal/GalNAc-reactive lectin on type 2-fimbriated Actinomyces naeslundii with receptor polysaccharides carried by certain strains of viridans group streptococci. Actinomyces also interact with other oral species. However, these interactions have not been systematically explored. Objective: To identify additional coaggregating partner strains for A. naeslundii from samples of overnight dental plaque. Methods: Dental biofilms were collected from labial surfaces of upper incisors of two individuals using sterile cotton swabs. Bacteria were cultured on agar plates and colony lifts were probed by overlay with fluorescein-labeled Actinomyces naeslundii WVU45. Bound bacteria were detected by a fluorescence scanner. Positive colonies were streaked on plates and reprobed as above. Partner strains were identified by comparing 16 S rDNA sequences with the NCBI GenBank using BLAST. Isolates were further characterized by standard coaggregation assays as well as by dot-blot experiments to determine the sensitivity or resistance of coadhesion to protein denaturation by formaldehyde. Results: Thirteen strains with putative carbohydrate-containing receptors for A. naeslundii were identified by bacteria overlay. Twelve of these were positive in standard coaggregation assays with A. naeslundii. All partner strains were identified as either Streptococcus spp. (3) or Rothia spp. (10) Additional partner strains with formaldehyde-sensitive putative adhesins for A. naeslundii WVU45 were also identified. Most of these belonged to the species Streptococcus and Neisseria. Conclusion: Novel coaggregation partners (i.e. Rothia spp. and Neisseria spp.) of A. naeslundii were isolated from samples of dental plaque using the bacteria overlay technique. The approach described has a number of promising applications for studying adhesive inter-species interactions between members of the oral microbiome.
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: Certain systemic pathogens can be detected within oral microbial biofilms. The oral cavity therefore may serve as a reservoir for respiratory or gastrointestinal infectious agents. One example of the latter is the stomach pathogen Helicobacter pylori that on its route of infection or transmission traverses the oral cavity. Objective: To isolate and identify bacterial strains from dental plaque that interact coadhesively with H. pylori. Methods: Supragingival interproximal plaque as well as samples from the dorsum of the tongue were obtained from dentally healthy subjects and plated on Columbia agar. Bacterial colonies were transferred by colony lift to nitrocellulose membranes and overlaid using fluorescein-labeled H. pylori J99A as a probe. Following removal of unattached bacteria by washing, H. pylori-reactive colonies were detected by a fluorescence scanner. Positive colonies were subcultured twice and reevaluated to ensure coadhesive activity. Isolated strains were identified by 16S rDNA sequencing and matching within the NCBI GenBank using BLAST. Strains were further tested to determine the mechanism of interbacterial binding by using formaldehyde treatment to inactivate putative proteinaceous adhesin activities of each coadhesion partner strain. Results: Approximately 20 strains were identified that interacted with H. pylori, including strains of Actinomyces, Streptococcus, Granulicatella, and Abiotrophia species. General mechanisms of coadhesion likely involve proteins on the oral bacteria and formaldehyde-stable components, possibly carbohydrates, on H. pylori. Conclusion: Oral biofilm bacteria can be isolated and identified that coadhesively interact with H. pylori supporting the hypothesis that this pathogen may colonize the oral biofilm microbiota.
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: 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.
    Applied and Environmental Microbiology 12/2009; 76(4):1294-7. · 3.95 Impact Factor
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    ABSTRACT: Seventy-one strains of viridans streptococci, classified as Streptococcus sanguis, S. gordonii, S. oralis, S. mitis or S. anginosus by a revised taxonomic scheme, were characterized and compared by their specific adhesive properties. The frequency of bacterial adhesion to saliva-coated hydroxyapatite (SH A) was greater among strains of S. sanguis, S. gordonii and S. oralis than among those of S. mitis and S. anginosus. Similarly, the expression of sialic acid reactive adhesins, detected by neuraminidase sensitive bacterial haemagglutination, was noted more frequently with strains of S. sanguis (19 of 21), S. gordonii (14 of 16) and S. oralis (8 of 11) than those of S. mitis (2 of 12) and S. anginosus (0 of 11). Most strains of S. gordonii (14 of 16) and S. oralis (7 of 11) also aggregated acidic proline rich protein-coated latex beads, but this activity was observed rarely with strains of S. sanguis (2 of 21), S. mitis (1 of 12) and S. anginosus (0 of 11). Strains of S. anginosus (6 of 11) participated in lactose resistant coaggregations with actinomyces in coaggregation groups A (e.g. Actinomyces viscosus T14V-J1) and B (e.g. A. naeslundii WVU45). Lactose resistant coaggregations were also observed between strains of S. gordonii (9 of 16) and actinomyces in coaggregation group A. Lactose sensitive coaggregations occurred between actinomyces and each of 11 S. oralis strains but less frequently with strains of S. sanguis (6 of 21), S. gordonii (3 of 16), S. mitis (3 of 12) and S. anginosus (1 of 11). Certain streptococcal strains with receptors for the lactose sensitive lectins of actinomyces, including 9 of 11 S. oralis, also coaggregated frequently with strains of either S. sanguis (10 of 21) or S. gordonii (9 of 16). Further studies with representatives of these latter three streptococcal species suggested that the streptococci with receptors for the GalNAc sensitive lectins of S. sanguis and S. gordonii were those with GalNAcpi→3Gal- rather than Galpl→3GalNAc-containing cell wall polysaccharides.
    Microbial Ecology in Health and Disease 07/2009; 7(3):125-137.
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    ABSTRACT: 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.
    Journal of bacteriology 02/2009; 191(6):1891-900. · 3.94 Impact Factor
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    ABSTRACT: 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.
    Journal of bacteriology 10/2008; 190(24):8145-54. · 3.94 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 06/2008; 283(18):12654-64. · 4.65 Impact Factor
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    ABSTRACT: Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb-rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.
    Vaccine 09/2007; 25(33):6167-75. · 3.49 Impact Factor
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    ABSTRACT: The type 1 fimbriae of Actinomyces naeslundii T14V mediate adhesion of this gram-positive species to the tooth surface. The present findings show that the locus for type 1 fimbria production in this strain includes three genes, fimQ for a minor fimbrial subunit that appears to be an adhesin, fimP for the major structural subunit, and srtC1 for a type 1 fimbria-specific sortase involved in the assembly of these structures.
    Infection and Immunity 09/2007; 75(8):4181-5. · 4.07 Impact Factor

Publication Stats

2k Citations
270.33 Total Impact Points

Institutions

  • 1995–2014
    • National Institutes of Health
      • • Laboratory of Cell and Developmental Biology
      • • Branch of Oral Infection and Immunity
      • • Laboratory of Molecular Microbiology
      Maryland, United States
  • 2010–2011
    • University of Texas Health Science Center at Houston
      • Department of Microbiology and Molecular Genetics
      Houston, TX, United States
  • 1994–2009
    • University of Maryland, Baltimore County
      • Department of Chemistry and Biochemistry
      Baltimore, Maryland, United States
  • 1991–2008
    • University of Maryland, Baltimore
      Baltimore, Maryland, United States
  • 2007
    • U.S. Department of Health and Human Services
      Washington, Washington, D.C., United States
    • The National Institute of Diabetes and Digestive and Kidney Diseases
      Maryland, United States
  • 1998
    • U.S. Food and Drug Administration
      • Division of Viral Products
      Washington, Washington, D.C., United States
  • 1990
    • Illinois Institute of Technology
      • Division of Chemistry
      Chicago, IL, United States