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Biofilms are subjected to many environmental pressures that can influence community structure and physiology. In the oral cavity, and many other environments, biofilms are exposed to forces generated by fluid flow; however, our understanding of how oral biofilms respond to these forces remains limited. In this study, we developed a linear rocker model of fluid flow to study the impact of shear forces on Streptococcus gordonii and dental plaque‐derived multispecies biofilms. We observed that as shear forces increased, S. gordonii biofilm biomass decreased. Reduced biomass was largely independent of overall bacterial growth. Transcriptome analysis of S. gordonii biofilms exposed to moderate levels of shear stress uncovered numerous genes with differential expression under shear. We also evaluated an ex vivo plaque biofilm exposed to fluid shear forces. Like S. gordonii, the plaque biofilm displayed decreased biomass as shear forces increased. Examination of plaque community composition revealed decreased diversity and compositional changes in the plaque biofilm exposed to shear. These studies help to elucidate the impact of fluid shear on oral bacteria and may be extended to other bacterial biofilm systems. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis activates protease-activated receptors (PARs) on oral keratinocytes, resulting in downstream signalling for an innate immune response. Activation depends on P. gingivalis gingipains, but could be confounded by lipopolysaccharide signalling through Toll-like receptors. We therefore hypothesized that P. gingivalis cleaves oral keratinocyte PARs in an Arg-(Rgp) or Lys- (Kgp) gingipain-specific manner to upregulate pro-inflammatory cytokines. Immortalized human oral keratinocytes (TERT-2) were incubated with wild-type P. gingivalis (ATCC 33277) or strains from a panel of isogenic gingipain deletion mutants: Kgp-deficient (KDP 129); Rgp-deficient (KDP 133); or Kgp-and Rgp-deficient (KDP 136). After incubation with P. gingivalis, keratinocytes were probed with specific antibodies against the N-terminus of PAR-1 and PAR-2. Using flow cytometry and immunofluorescence, receptor cleavage was marked by loss of specific antibody binding to the respective PARs. TERT-2 cells constitutively expressed high levels of PAR-1 and PAR-2, and lower levels of PAR-3. P. gingivalis ATCC 33277 cleaved PAR-1 and PAR-2 in a dose-dependent manner, while the receptors were unaffected by the protease-negative double mutant (KDP 136) at all m.o.i, tested. The single Kgp-negative mutant preferentially cleaved PAR-l, whereas the Rgp-negative mutant cleaved PAR-2. Wild-type or Kgp-negative mutant cleavage of PAR-1 upregulated expression of IL-1 [alpha], IL-1 [beta], IL-6 and TNF-[alpha]; the Rgp-negative mutant did not modulate these cytokines. Selective cleavage of PAR-1 on oral epithelial cells by P. gingivalis Rgp therefore upregulates expression of pro-inflammatory cytokines. DOI 10.1099/mic.0.029132-0

Extracellular DNA (eDNA) is produced by several bacterial species and appears to contribute to biofilm development and cell-cell adhesion. We present data showing that the oral commensals Streptococcus sanguinis and Streptococcus gordonii release DNA in a process induced by pyruvate oxidase-dependent production of hydrogen peroxide ([H.sub.2][O.sub.2]). Surprisingly, S. sanguinis and S. gordonii cell integrity appears unaffected by conditions that cause autolysis in other eDNA-producing bacteria. Exogenous [H.sub.2][O.sub.2] causes release of DNA from S. sanguinis and S. gordonii but does not result in obvious lysis of cells. Under DNA-releasing conditions, cell walls appear functionally intact and ribosomes are retained over time. During DNA release, intracellular RNA and ATP are not coreleased. Hence, the release mechanism appears to be highly specific for DNA. Release of DNA without detectable autolysis is suggested to be an adaptation to the competitive oral biofilm environment, where autolysis could create open spaces for competitors to invade. Since eDNA promotes cell-to-cell adhesion, release appears to support oral biofilm formation and facilitates exchange of genetic material among competent strains. doi: 10.1128/JB.00906-09

The degradation of complex substrates, like salivary mucins, requires an arsenal of glycosidases and proteases to sequentially degrade the oligosaccharides and polypeptide backbone. The mucin MUC5B is a complex oligomeric glycoprotein, heterogeneous in molecular mass (14-40x[10.sup.6] Da), with a diverse repertoire of oligosaccharides, differing in composition and charge. The aim of this study was to investigate whether proteolytic degradation of the mucin polypeptide backbone could be identified and if cooperation of dental biofilm bacteria was required. Cooperative bacteria-mediated proteolysis of MUC5B was determined by comparing individual species and mixed consortia of strains isolated from supragingival plaque, and freshly harvested supragingival plaque. Proteolytic activity was analysed using fluorescent labelled substrate and by visualizing mucin degradation by SDS-PAGE. Dental plaque degraded the polypeptide backbone of the salivary MUC5B mucin. The mucin was also degraded by a specific consortium of isolated species from supragingival plaque, although individual species and other consortia did not. Certain bacteria in supragingival dental plaque therefore cooperate as a consortium to proteolyse human salivary MUC5B and hydrolyse glycosides.

Surface adhesion plays an essential part in the survival of the commensal organism Streptococcus gordonii in the oral cavity as well as during opportunistic infections such as endocarditis. At least two types of cell surface protein involved in adhesion are found on the surface of Gram-positive bacteria: those anchored via an LPXTG motif by the enzyme sortase A (SrtA) and those associated with the cell surface by, as yet, unknown mechanisms. In [srtA.sup.-] mutants, LPXTG-containing proteins have been shown to be released rather than cross-linked to the cell wall. We have therefore used 2D gel electrophoresis of released proteins from an [srtA.sup.-] mutant as well as the wild-type strain, followed by peptide identification by MS, to identify a set of novel proteins predicted to be present on the surface of S. gordonii DLI. This includes two large LPXTG-linked proteins (SGO_0707 and SGO_1487), which both contain tandemly repeated sequences similar to those present in known fibrillar adhesins. A 5'-nucleotidase and a protein with a putative collagen-binding domain, both containing LPXTG motifs, were also identified. Anchorless proteins with known chaperone, stress response and elongation factor functions, apparently responsible for bacterial binding to keratinocytes and saliva-coated surfaces in the absence of the LPXTGlinked adhesins, were also associated with the cell surface. These data reveal a range of proteins to be present on the S. gordonii DL1 cell surface, the expression of which plays an important role in adhesion to epithelia and which represent likely candidates for novel virulence factors in S. gordonii.

The putative two-component system BfrAB is involved in Streptococcus gordonii biofilm development. Here, we provide evidence that BfrAB regulates the expression of bfrCD and bfrEFG, which encode two ATP-binding cassette (ABC) transporters, and bfrH, which encodes a CAAX amino-terminal protease family protein. BfrC and BfrE are ATP-binding proteins, and BfrD, BfrF and BfrG are homologous membrane-spanning polypeptides. Similarly, [BfrAB.sub.ss], the BfrAB homologous system in Streptococcus sanguinis, controls the expression of two bfrCD-homologous operons ([bfrCD.sub.ss] and [bfrXY.sub.ss]), a bfrH-homologous gene ([bfrH[1.sub.ss]) and another CAAX amino-terminal protease family protein gene (bfrH[2.sub.ss]). Furthermore, we demonstrate that the purified BfrA DNA-binding domain from S. gordonii binds to the promoter regions of bfrCD, bfrEFG, bfrH, [bfrCD.sub.ss], [bfrXY.sub.ss] and bfrH[1.sub.ss] in vitro. Finally, we show that the BfrA DNA-binding domain recognizes a conserved DNA motif with a consensus sequence of TTTCTTTAGAAATA-FITTAGAATT. These data suggest, therefore, that S. gordonii BfrAB controls biofilm formation by regulating multiple ABC-transporter systems.

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide ([H.sub.2][O.sub.2]) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of [H.sub.2][O.sub.2]. Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the [H.sub.2][O.sub.2]- dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.

Sortase A (SrtA) is required for cell-wall anchoring of LPXTG-containing Gram-positive surface proteins. It was hypothesized, therefore, that disruption of the srtA gene would alter surface anchoring and functions of target LPXTG motif-bearing SspA and SspB proteins of Streptococcus gordonii. Mutant strains in srtA (V288[srtA.sup.-], DL1[srtA.sup.-]) were constructed in S. gordonii V288 (wtV288) and DL1 (wtDL1). When compared to wtV288, the V288[srtA.sup.-] mutant showed decreased biofilm formation on polystyrene, and reduced binding to immobilized purified salivary agglutinin (BIAcore analysis). The wtV288 and V288[srtA.sup.-] strains were similar in ultrastructure, but immunogold-labelled SspA/SspB surface expression was reduced on the V288[srtA.sup.-] mutant. DL1[srtA.sup.-] was also complemented to obtain DL1[srtA.sup.+]. From the wild-type strains (wtV288, wtDL1), [srtA.sup.-] mutants (V288[srtA.sup.-], DL1[srtA.sup.-]), and the complemented mutant (DL1[srtA.sup.+]), cytoplasmic, cell-wall and released extracellular protein fractions were isolated. Each fraction was analysed by SDS-PAGE and immunoblotting with anti-P1. Spent medium from [srtA.sup.-] mutant cells contained over-represented proteins, including SspA/SspB (P1 antigen). Mutants showed less P1 on the cell surface than wild-types, as estimated using whole-cell ELISA, and no P1 appeared in the cytoplasmic fractions. Expression of several adhesin genes (sspA/B, cshA/B, fbpA) was generally upregulated in the mutants (V288[srtA.sup.-], DL1[srtA.sup.-]), but restored to wild-type levels in DL1[srtA.sup.+]. These data therefore imply that in addition to its role in processing LPXTGcontaining adhesins, sortase A has the novel function of contributing to transcriptional regulation of adhesin gene expression.

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.

Streptocoecus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferuse systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and ofigochitosaecharides induced genes in one of the three heta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.

Abstract Background Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. Results To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Δenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Conclusion Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

Abstract Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1.

Abstract Background Primary HIV infection can develop from exposure to HIV in the oral cavity. In previous studies, we have documented rapid and extensive binding of HIV virions in seminal plasma to intact mucosal surfaces of the palatine tonsil and also found that virions readily penetrated beneath the tissue surfaces. As one approach to understand the molecular interactions that support HIV virion binding to human mucosal surfaces, we have examined the distribution of the primary HIV receptor CD4, the alternate HIV receptors heparan sulfate proteoglycan (HS) and galactosyl ceramide (GalCer) and the co-receptors CXCR4 and CCR5 in palatine tonsil. Results Only HS was widely expressed on the surface of stratified squamous epithelium. In contrast, HS, GalCer, CXCR4 and CCR5 were all expressed on the reticulated epithelium lining the tonsillar crypts. We have observed extensive variability, both across tissue sections from any tonsil and between tonsils, in the distribution of epithelial cells expressing either CXCR4 or CCR5 in the basal and suprabasal layers of stratified epithelium. The general expression patterns of CXCR4, CCR5 and HS were similar in palatine tonsil from children and adults (age range 3–20). We have also noted the presence of small clusters of lymphocytes, including CD4+ T cells within stratified epithelium and located precisely at the mucosal surfaces. CD4+ T cells in these locations would be immediately accessible to HIV virions. Conclusion In total, the likelihood of oral HIV transmission will be determined by macro and micro tissue architecture, cell surface expression patterns of key molecules that may bind HIV and the specific properties of the infectious inoculum.

Dental school deans surveyed expressed greater need for clinical than basic sciences faculty, especially those with independent research skills and trained in two clinical or one clinical and one basic science field, since these faculty are in short supply. (MSE)