Treponema denticola does not induce production of common innate immune mediators from primary gingival epithelial cells.

It has been hypothesized that the neutrophil chemoattractant interleukin-8 (IL-8) forms a gradient in the oral cavity, with the highest concentration of IL-8 produced closest to the bacterial biofilm. In periodontitis, this gradient is disrupted, impairing neutrophil chemotaxis to diseased sites. Treponema denticola is prominently associated with periodontal disease, yet little is known about its ability to modulate the production of inflammatory mediators by epithelial cells. Others have shown that dentilisin, the major outer membrane protease of T. denticola, degrades IL-8 in vitro. We now provide evidence that T. denticola also fails to induce IL-8 production from primary gingival epithelial cells (PGEC). The lack of IL-8 production is not explained by IL-8 degradation, because a protease mutant that does not degrade IL-8 does not induce IL-8 production with these stimuli either. The lack of innate immune mediator production may be a more global phenomenon because T. denticola fails to induce IL-6 or intercellular adhesion molecule 1 production from PGEC. T. denticola also fails to induce transcription of IL-8 and human beta-defensin-2 messenger RNA. The lack of immune mediator production is not explained by the failure of T. denticola to interact with Toll-like receptor 2 (TLR-2), as T. denticola stimulates nuclear factor-kappaB nuclear translocation in TLR-2-transfected HEK293 cells. Not only can T. denticola degrade the IL-8 present in the periodontal lesion, but this organism also fails to induce IL-8 production by PGEC. The lack of an epithelial cell response to T. denticola may contribute to the pathogenesis of periodontitis by failing to trigger chemotaxis of neutrophils into the periodontal pocket.

Resistance to human beta-defensins is common among oral treponemes.

BACKGROUND/AIMS: Oral treponemes are implicated in the pathogenesis of periodontal disease. We have previously shown that Treponema denticola ATCC type strains and strain GM-1 are resistant to killing by human beta-defensins (hbetaD)-1 and -2. We hypothesize that resistance to beta-defensins is a common feature of oral treponemes, which allows colonization and persistence in the oral cavity. In this study, we tested additional isolates of T. denticola, as well as six other species of treponemes, for resistance to hbetaD-1, -2 and -3. We also examined the four ATCC strains of T. denticola and strain GM-1 for resistance to hbetaD-3. METHODS: Resistance was determined by motility and Alamar Blue assays for metabolic activity. RESULTS: All T. denticola strains tested were resistant to hbetaD-1, -2 and -3, with the exception of strain Ambigua, which was sensitive to hbetaD-2 and -3. All other treponemes except Treponema vincentii were resistant to hbetaD-1. Treponema pectinovorum was sensitive to hbetaD-2, while T. vincentii, T. pectinovorum and Treponema maltophilum were sensitive to hbetaD-3. Escherichia coli was used as a control organism and was killed by all three defensins. CONCLUSION: Resistance to the constitutively expressed hbetaD-1 may assist treponemes in initial colonization of epithelial surfaces, while resistance to the inducible hbetaD-2 and -3 would allow some treponemes to survive in active periodontal lesions.

Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion.

Actinobacillus actinomycetemcomitans is an important pathogen implicated in juvenile and adult periodontal diseases. An important virulence factor of A. actinomycetemcomitans is the ability to invade human oral epithelial cells. A clinical isolate, A. actinomycetemcomitans SUNY 465, has previously been shown to enter epithelial cells by an actin-dependent mechanism. The internalized bacteria are surrounded by an actin halo upon entry. These data are consistent with the mode of entry associated with many enteric pathogens. We tested the effects of cytochalasin D, an inhibitor of the actin microfilament network, on bacterial entry to determine whether this mode of entry was common to other A. actinomycetemcomitans clinical isolates. Cytochalasin D was added prior to infection. A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065 exhibited enhanced ability to enter epithelial cells in the presence of cytochalasin D. Immunofluorescent labeling of bacteria and host cell actin confirmed that actin was not being mobilized by the entry of A. actinomycetemcomitans SUNY 523. Inhibitors of receptor-mediated endocytosis inhibited invasion of A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065. Microtubule effectors did not inhibit invasion of A. actinomycetemcomitans. A. actinomycetemcomitans SUNY 523, but not A. actinomycetemcomitans 4065, was deficient in exit from epithelial cells as determined by the absence of organisms in the assay medium. These data suggest that A. actinomycetemcomitans strains utilize at least two distinct mechanisms for entry into epithelial cells, and that A. actinomycetemcomitans SUNY 523 may be defective in exit and cell-to-cell spread.