Aggregatibacter actinomycetemcomitans Omp29 Is Associated with Bacterial Entry to Gingival Epithelial Cells by F-Actin Rearrangement

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29+/OmpA− E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29−/OmpA− E. coli. While the entry of Aa and Omp29+/OmpA− E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway

Omp29 induces actin rearrangement and FAK phosphorylation in OBA9.

<p>(A–C) The confluent OBA9 cells in the tissue culture flask were incubated for 1 hour in medium alone (A) or in the presence of Omp16 (10 µg/ml) (B) or Omp29 (10 µg/ml) (C). In order to stain F-actin, OBA9 cells removed from culture plate by trypsin-EDTA were permeabilized and stained with FITC-Phalloidin. (A) The solid histogram # shows the non-stained OBA9 and the open histogram in gray line * indicates the FITC-Phalloidin staining of OBA9. (B and C) The open histogram * indicates the FITC-Phalloidin stained OBA9 incubated with medium alone and the solid histograms display the staining of OBA9 stimulated with Omp16 (B) or Omp29 (C), respectively. (D and E) After stimulation of OBA9 cells with Omp16 (10 µg/ml) (D) or Omp29 (10 µg/ml) (E) for the time periods shown in the figure, whole cellular proteins dissolved in lysis buffer were immuno-precipitated with anti-total FAK antibody bound to GammaBind Plus Sepharose beads. The proteins pulled down with the beads were blotted onto NC membrane and reacted with anti-total FAK antibody or anti-phospho FAK antibody using Western-blot method.</p

Induction of Omp29 surface expression in OmpA-deficient <i>E. coli</i>.

<p>(A) Using Western blot analysis, anti-Omp29 serum IgG was reacted to the purified Omp29, whole <i>Aa</i> Y4, <i>E. coli</i> Bre51 and <i>omp34</i> gene-transfected <i>E. coli</i> Bre51. The heat-modifiable property of Omp29 resulted in two distinct migration patterns at 29 kD and 34 kD. (B–D) The formalin-fixed whole <i>Aa</i> Y4 (B), formalin-fixed whole <i>E. coli</i> Bre51 (C) or <i>E. coli</i> 3826 (D) was reacted with anti-Omp29-specific IgG and control serum IgG at various concentrations as indicated on the x-axis. The antibody binding to each fixed bacterial antigen was detected with goat anti-mouse IgG-biotin (×10,000), followed by HRP-conjugated avidin (×40,000). Colorimetric reactions were developed with o-Phenylenediamine in appropriate buffer. The optical density (OD) of each well of ELISA plate was measured at 490 nm. The results are expressed as mean OD ± SD of triplicate wells.</p

Bacterial Omp29 up-regulates F-actin rearrangement in OBA9, which results in induction of <i>Aa</i> Y4 entry into OBA9.

<p>(A and B) <i>Aa</i> Y4 harvested at mid-log growth was incubated with confluent OBA9 cells (5×10⁵ bacteria/100 µl/well) in the presence or absence of cytochalasin D (F-actin rearrangement inhibitor) by the indicated concentration in the figures. The number of bacteria entered or adherent to OBA9 cells was shown (A, bacterial entry; B; bacterial attachment). *, Significantly lower than control medium alone by Student's <i>t</i> test (<i>P</i><0.05). (C–F) After pretreatment of sub confluent OBA9 cells with or without anti-Omp29 IgG (10 µg/ml) or control non-immunized IgG (10 µg/ml) for 15 min, OBA9 cells were co-cultured with <i>Aa</i> Y4 (1×10⁷ bacteria/2 ml/dish) in the antibiotics-free K-SFM for 1 hr. F-actin (green) and <i>Aa</i> Y4 (red) were observed using immunofluorescence microscopy in OBA9 cells. White arrow indicates co-localization (yellow) of F-actin and <i>Aa</i> Y4. (C) medium alone, (D) <i>Aa</i> Y4, (E) anti-Omp29 IgG and <i>Aa</i> Y4, and (F) control IgG and <i>Aa</i> Y4. Scale bar: 10 µm.</p

Influence of Rho GTPases inhibitor and PI-3-kinase inhibitor on bacterial entry and attachment to OBA9.

<p><i>Aa</i> harvested at mid-log growth phase was incubated with confluent OBA9 cells (5×10⁵ bacteria/100 µl/well) in the presence or absence of Wortmannin or EDIN by the indicated concentration in the figures. The number of bacteria that entered or adherent to OBA9 cells is shown (A, bacterial entry; B, bacterial attachment). *, Significantly lower than control medium alone by Student's <i>t</i> test (<i>P</i><0.05).</p

Anti-Omp29 IgG antibody inhibits bacterial entry, but not adhesion, to OBA9.

<p>Live <i>Aa</i> Y4, Omp29⁻/OmpA⁻<i>E. coli</i> strain Bre51 or Omp29⁺/OmpA⁻<i>E. coli</i> strain 3826 harvested at the mid-log phase was incubated with confluent OBA9 in a 96-well plate (5.5×10⁵ bacteria/100 µl/well) in the presence of anti-Omp29 IgG antibody or control serum IgG. For <i>E. coli</i> strains, a dose of anti-Omp29 serum IgG (10 µg/ml) or control serum IgG (10 µg/ml) was applied to each well. After 4 hours of incubation, entry (A) or adhesion (B) of <i>Aa</i> Y4, or entry (C) or adhesion (D) of Omp29⁻/OmpA⁻<i>E. coli</i> strain Bre51 or Omp29⁺/OmpA⁻<i>E. coli</i> strain 3826 to OBA9 was measured. Each column represents mean CFU ± SD of three wells. One representative result from at least 3 different experiments is shown. *, Significantly lower than <i>Aa</i> Y4 incubated in medium alone (without antibody) or control IgG by Student's <i>t</i> test (<i>P</i><0.05). #, Significantly higher than <i>E. coli</i> Bre51 entry or adhesion to OBA9 in the absence of antibody by Student <i>t</i> test (<i>P</i><0.05). †, Significantly lower than the entry of <i>E. coli</i> 3826 incubated in medium alone (without antibody) or control IgG by Student <i>t</i> test (<i>P</i><0.05).</p

Kinetics of <i>Aa</i> or <i>S. sanguis</i> entry and adhesion to OBA9.

<p>(A and B) <i>Aa</i> or <i>S. sanguis</i> growing in middle of logarithmic phase in broth culture was applied to confluent OBA9 cells in 96-well plates (5.5×10⁵ bacteria/100 µl/well). The multiplicity of infection (MOI) value used in these entry and adhesion assays were about 55 (5.5×10⁵ bacteria/1.0×10⁴ epithelial cells/well). The co-culture of bacteria and OBA9 cells was incubated at various times, and bacterial entry and attachment to OBA9 cells were evaluated by the protocol described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018287#s2" target="_blank">Materials and Methods</a>. The number of bacteria which entered (A) or adhered (B) to OBA9 was expressed as a colony forming unit (CFU). Each point represents mean ± SD of CFU from three wells.</p