Identification of Proteins Associated with the Formation of Oral Biofilms

ABSTRACT Objective: To identify proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms. Material and Methods: Biofilms composed of two bacterial species, S. gordonii and F. nucleatum, were cultured for 1, 4, 7, and 10 days. The presence of both species was confirmed via amplification of the srtA and radD genes using real-time PCR. The concentrations of proteins associated with the biofilms and individual species were quantified using Western blotting. Results: The protein profiles of S. gordonii and F. nucleatum from individual cultures determined using one-dimensional electrophoresis revealed proteins found in S. gordonii and in F. nucleatum. Ct and reciprocal Ct values were determined for the exposed S. gordonii and F. nucleatum biofilms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected in biofilms and F. nucleatum, whereas HSP40 protein was present only in biofilms after 7 and 10 days of formation. Conclusion: HSP40 was detected only in the formed biofilms; thus, HSP40 is an essential proteins for adhesion.

Macrophage Polarization Alters Postphagocytosis Survivability of the Commensal Streptococcus gordonii.

Oral streptococci are generally considered commensal organisms; however, they are becoming recognized as important associate pathogens during the development of periodontal disease as well as being associated with several systemic diseases, including as a causative agent of infective endocarditis. An important virulence determinant of these bacteria is an ability to evade destruction by phagocytic cells, yet how this subversion occurs is mostly unknown. Using Streptococcus gordonii as a model commensal oral streptococcus that is also associated with disease, we find that resistance to reactive oxygen species (ROS) with an active ability to damage phagosomes allows the bacterium to avoid destruction within macrophages. This ability to survive relies not only on the ROS resistance capabilities of the bacterium but also on ROS production by macrophages, with both being required for maximal survival of internalized bacteria. Importantly, we also show that this dependence on ROS production by macrophages for resistance has functional significance: S. gordonii intracellular survival increases when macrophages are polarized toward an activated (M1) profile, which is known to result in prolonged phagosomal ROS production compared to that of alternatively (M2) polarized macrophages. We additionally find evidence of the bacterium being capable of both delaying the maturation of and damaging phagosomes. Taken together, these results provide essential insights regarding the mechanisms through which normally commensal oral bacteria can contribute to both local and systemic inflammatory disease.

Streptococcus gordonii induces nitric oxide production through its lipoproteins stimulating Toll-like receptor 2 in murine macrophages.

Streptococcus gordonii, a Gram-positive commensal in the oral cavity, is an opportunistic pathogen that can cause endodontic and systemic infections resulting in infective endocarditis. Lipoteichoic acid (LTA) and lipoprotein are major virulence factors of Gram-positive bacteria that are preferentially recognized by Toll-like receptor 2 (TLR2) on immune cells. In the present study, we investigated the effect of S. gordonii LTA and lipoprotein on the production of the representative inflammatory mediator nitric oxide (NO) by the mouse macrophages. Heat-killed S. gordonii wild-type and an LTA-deficient mutant (ΔltaS) but not a lipoprotein-deficient mutant (Δlgt) induced NO production in mouse primary macrophages and the cell line, RAW 264.7. S. gordonii wild-type and ΔltaS also induced the expression of inducible NO synthase (iNOS) at the mRNA and protein levels. In contrast, the Δlgt mutant showed little effect under the same condition. Furthermore, S. gordonii wild-type and ΔltaS induced NF-κB activation, STAT1 phosphorylation, and IFN-ß expression, which are important for the induction of iNOS gene expression, with little activation by Δlgt. S. gordonii wild-type and ΔltaS showed an increased adherence and internalization to RAW 264.7 cells compared to Δlgt. In addition, S. gordonii wild-type and ΔltaS, but not Δlgt, substantially increased TLR2 activation while none of these induced NO production in TLR2-deficient macrophages. Triton X-114-extracted lipoproteins from S. gordonii were sufficient to induce NO production. Collectively, we suggest that lipoprotein is an essential cell wall component of S. gordonii to induce NO production in macrophages through TLR2 triggering NF-κB and STAT1 activation.

Differential interactions of Streptococcus gordonii and Staphylococcus aureus with cultured osteoblasts.

The impedance of normal osteoblast function by microorganisms is at least in part responsible for the failure of dental or orthopedic implants. Staphylococcus aureus is a major pathogen of bone, and exhibits high levels of adhesion and invasion of osteoblasts. In this article we show that the commensal oral bacterium Streptococcus gordonii also adheres to and is internalized by osteoblasts. Entry of S. gordonii cells had typical features of phagocytosis, similar to S. aureus, with membrane protrusions characterizing initial uptake, and closure of the osteoblast membrane leading to engulfment. The sensitivities of S. gordonii internalization to inhibitors cytochalasin D, colchicine and monensin indicated uptake through endocytosis, with requirement for actin accumulation. Internalization levels of S. gordonii were enhanced by expression of S. aureus fibronectin-binding protein A (FnBPA) on the S. gordonii cell surface. Lysosomal-associated membrane protein-1 phagosomal membrane marker accumulated with intracellular S. aureus and S. gordonii FnBPA, indicating trafficking of bacteria into the late endosomal/lysosomal compartment. Streptococcus gordonii cells did not survive intracellularly for more than 12 h, unless expressing FnBPA, whereas S. aureus showed extended survival times (>48 h). Both S. aureus and S. gordonii DL-1 elicited a rapid interleukin-8 response by osteoblasts, whereas S. gordonii FnBPA was slower. Only S. aureus elicited an interleukin-6 response. Hence, S. gordonii invades osteoblasts by a mechanism similar to that exhibited by S. aureus, and elicits a proinflammatory response that may promote bone resorption.

Toll-like receptor 2-mediated modulation of growth and functions of regulatory T cells by oral streptococci.

This study was designed to determine whether oral streptococci modulate the growth and functions of regulatory T cells. Heat-killed cells of wild-type strains of Streptococcus gordonii and Streptococcus mutans induced the Toll-like receptor 2 (TLR2) -mediated nuclear factor-κB (NF-κB) activation, but their lipoprotein-deficient strains did not. Stimulation with these streptococci resulted in a significant increase in the frequency of CD4(+) CD25(+) Foxp3(+) regulatory T cells in splenocytes derived from both TLR2(+/+) and TLR2(-/-) mice, but the level of increase in TLR2(+/+) splenocytes was stronger than that in TLR2(-/-) splenocytes. Both strains of S. gordonii enhanced the proliferation of CD4(+) CD25(+) Foxp3(+) regulatory T cells isolated from TLR2(+/+) mice at the same level as those from TLR2(-/-) mice in an interleukin-2-independent manner. However, wild-type and lipoprotein-deficient strains of both streptococci did not enhance the suppressive activity of the isolated regulatory T cells in vitro, but rather inhibited it. TLR ligands also inhibited the suppressive activity of the regulatory T cells. Inhibition of the suppressive activity was recovered by the addition of anti-IL-6 antibody. Pretreatment of antigen-presenting cells with the NF-κB inhibitor BAY11-7082 enhanced the suppressive activity of the regulatory T cells. These results suggested that interleukin-6 produced by antigen-presenting cells inhibits the suppressive activity of the regulatory T cells. Wild-type strain, but not lipoprotein-deficient strain, of S. gordonii reduced the frequency of CD4(+)  CD25(+)  Foxp3(+) regulatory T cells in the acute infection model, whereas both strains of S. gordonii increased it in the chronic infection model mice. Hence, this study suggests that oral streptococci are capable of modulating the growth and functions of regulatory T cells in vitro and in vivo.

Binding of Streptococcus gordonii to oral epithelial monolayers increases paracellular barrier function.

The junctional epithelium comprising the gingival attachment to the tooth acts as a barrier against pathogenic subgingival plaque microbes and their products. There is evidence that pathogenic Porphyromonas gingivalis has the potential to disrupt epithelial integrity, contributing to breakdown of the junctional epithelium characteristic of the immunopathological response of chronic periodontitis. The present study investigated the capacity of the oral commensal Streptococcus gordonii to increase epithelial barrier function to support epithelial integrity of healthy tissue. Oral epithelial barrier function was measured by permeability assay. Changes in expression of tight junction components were monitored by quantitative real-time RT-PCR and Western blot in an oral epithelial cell culture model following binding by S. gordonii strain FSS2. The data showed increased expression of genes encoding the tight junction components ZO-1, ZO-2, JAM-A, and occludin at a ratio of 100 bacterial colony forming units per epithelial cell. This was associated with increased expression at the protein level of ZO-1, ZO-2 and JAM-A. Reduction of permeability to fluorochrome-labelled dextran accompanied these changes. The data support the hypothesis that (some) commensal bacteria have a beneficial effect on oral epithelium.

Antimicrobial responses of primary gingival cells to Porphyromonas gingivalis.

BACKGROUND: Human beta-defensins (hBDs) and the C-C chemokine ligand 20 (CCL20) produced by gingival epithelial cells (GECs) and fibroblasts (HGFs) are antimicrobial peptides (AMPs) that play an important role in innate immunity. The aim of this study was to determine the differential immune response of GECs and HGFs to the oral commensal Streptococcus gordonii (SG) and the pathogen Porphyromonas gingivalis (PG). MATERIAL AND METHODS: In addition to the analysis of gingival biopsies, primary GECs and HGFs were exposed to SG and/or PG, and expression of various AMPs and pro-inflammatory mediators was studied by real-time PCR and ELISA. RESULTS: Gene expression of AMPs was detected in gingival connective tissue. Both SG and PG induced the mRNA-expression of hBD-2 and hBD-3 in GECs as well as HGFs after 24 h (p < 0.05). In HGFs, the commensal bacterium SG stimulated the mRNAs of hBD-3 and CCL20 after 24 h (p < 0.05), while not in GECs. In GECs, the inductive effect of PG on the mRNA-expression of hBD-2 was amplified when cells were first exposed to commensal SG (for 1 h) prior to stimulation with PG (SG-PG; p < 0.05). CONCLUSION: Our data indicate that cell-bacteria interactions and/or bacteria-bacteria cross-talk may have an impact on AMP-regulation in gingiva.

Interaction of oral bacteria with gingival epithelial cell multilayers.

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low-calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1ß and TNF-α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.

Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria.

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.

Synergistic BM-DC activation and immune induction by the oral vaccine vector Streptococcus gordonii and exogenous tumor necrosis factor.

Streptococcus gordonii, a potential mucosal vaccine delivery vector, is proficient at colonizing murine oral mucosa; however, it often fails to elicit significant antibody titers against its vaccine antigen payloads. The poor response may be due to an inability of S. gordonii to elicit cytokines needed to suppress mucosal tolerance; exogenously supplied cytokines, such as TNF, could overcome this effect. To test this, murine bone marrow-derived dendritic cells (BM-DCs) were stimulated with UV-killed S. gordonii PM14, that surface expresses a fragment of the immunodominant S1 subunit of pertussis toxin. Peptidoglycan (PGN), lipoteichoic acid (LTA), lipoprotein (LP), and DNA were also isolated from the bacteria, and used to stimulate BM-DCs. Stimulation with TNF, S. gordonii, PGN, LTA, or LP all resulted in increased surface expression of MHCII, CD80, and CD86, compared to unstimulated BM-DCs. Stimulation with S. gordonii elicited IL-6, IL-10, and IL-12p70 production from the BM-DCs, while stimulation with the bacterial components induced some or all of the three cytokines. When BM-DCs were simultaneously stimulated with S. gordonii and TNF, a marginal increase in surface marker upregulation was observed, and the two stimuli synergized to elicit substantially greater quantities of IL-6, IL-10, and IL-12p70. Synergy between TNF and the purified bacterial components was also observed. The effect of TNF was abolished when BM-DCs were obtained from mice deficient for either TNFR1 or TNFR2, and cytokine induction by S. gordonii was entirely dependent on functional MyD88. Synergistic IL-10 induction by S. gordonii and TNF was not observed in TLR-2(-/-) BM-DCs, and TNF was found to cause TLR-2 upregulation, providing at least a partial mechanism for the observed synergy. When S. gordonii and TNF were used to immunize mice, a more robust anti-S. gordonii IgG response was elicited as compared to immunization with S. gordonii alone. However, the addition of TNF did not result in stronger responses against the antigenic insert (S1 fragment) in immunized mice. These findings collectively demonstrate that TNF is able to prime BM-DCs to better respond to S. gordonii, through a mechanism at least partially involving TLR-2 upregulation.

Streptococcus cristatus attenuates Fusobacterium nucleatum-induced interleukin-8 expression in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells may be invaded by a polymicrobial intracellular flora, including pathogens together with commensals. Various oral pathogens can induce the production of interleukin-8, a potent neutrophil chemotractant, in oral epithelial cells. Evidence from the gut suggests that commensal species may modulate inflammatory responses to pathogens. The aim of this study was to examine the interleukin-8 responses of oral epithelial cells to an oral pro-inflammatory species, Fusobacterium nucleatum, in combination with an oral commensal, Streptococcus cristatus. MATERIAL AND METHODS: KB, TERT-2, TR146 and SCC15 cells were cocultured with F. nucleatum and S. cristatus, either alone or in combination, at 37 degrees C in 5% CO2 under various conditions. The mRNA expression of interleukin-8 was analyzed by reverse transcription-polymerase chain reaction and protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: F. nucleatum alone evoked a potent interleukin-8 response, whereas S. cristatus alone did not induce significant interleukin-8 expression in oral epithelial cells. When present together, S. cristatus attenuated the F. nucleatum-induced interleukin-8 production in the four oral epithelial cell lines to varying degrees. The inhibitory effect of S. cristatus was independent of its viability and its co-aggregation with F. nucleatum, was not related to soluble bacterial products and appeared to require bacterial contact with epithelial cells. Similar effects were seen with several other species of oral streptococci. CONCLUSION: Our data suggest that S. cristatus may exert immunomodulatory effects on the interleukin-8 response of oral epithelial cells to F. nucleatum challenge.