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. 1999 Apr;67(4):1837-43.
doi: 10.1128/IAI.67.4.1837-1843.1999.

Targeted disruption of fibronectin-integrin interactions in human gingival fibroblasts by the RI protease of Porphyromonas gingivalis W50

M A Scragg  1 , S J Cannon, M Rangarajan, D M Williams, M A Curtis
Affiliations

Targeted disruption of fibronectin-integrin interactions in human gingival fibroblasts by the RI protease of Porphyromonas gingivalis W50

M A Scragg et al. Infect Immun. 1999 Apr.

Abstract

Cell surface integrins mediate interactions between cells and their extracellular matrix and are frequently exploited by a range of bacterial pathogens to facilitate adherence and/or invasion. In this study we examined the effects of Porphyromonas gingivalis proteases on human gingival fibroblast (HGF) integrins and their fibronectin matrix. Culture supernatant from the virulent strain W50 caused considerably greater loss of the beta1 integrin subunit from HGF in vitro than did that of the beige-pigmented strain W50/BE1. Prior treatment of the W50 culture supernatant with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) blocked its effects on cultured cells, indicating that this process is proteolytically mediated. Purified arginine-specific proteases from P. gingivalis W50 were able to mimic the effects of the whole-culture supernatant on loss of beta1 integrin expression. However purified RI, an alpha/beta heterodimer in which the catalytic chain is associated with an adhesin chain, was 12 times more active than RIA, the catalytic monomer, in causing loss of the alpha5beta1 integrin (fibronectin receptor) from HGF. No effect was observed on the alphaVbeta3 integrin (vitronectin receptor). The sites of action of RI and RIA were investigated in cells exposed to proteases pretreated with TLCK to inactivate the catalytic component. Use of both monoclonal antibody 1A1, which recognizes only the adhesin chain of RI, and a rabbit antibody against P. gingivalis whole cells indicated localization of RI on the fibroblasts in a clear, linear pattern typical of that seen with fibronectin and alpha5beta1 integrin. Exact colocalization of RI with fibronectin and its alpha5beta1 receptor was confirmed by double labeling and multiple-exposure photomicroscopy. In contrast, RIA bound to fibroblasts in a weak, patchy manner, showing only fine linear or granular staining. It is concluded that the adhesin component of RI targets the P. gingivalis arginine-protease to sites of fibronectin deposition on HGF, contributing to the rapid loss of both fibronectin and its main alpha5beta1 integrin receptor. Given the importance of integrin-ligand interactions in fibroblast function, their targeted disruption by RI may represent a novel mechanism of damage in periodontal disease.

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Figures

FIG. 1
FIG. 1
β1 staining in HGF incubated for 1 h with culture supernatant from strains W50 (●) and W50/BE1 (○). The maximum concentration of each supernatant used was a 1/2 dilution in DMEM. The arginine-specific enzyme activities of doubling dilutions of the culture supernatants are shown on a log2 scale. Integrin staining was graded as described in Materials and Methods. The results represent the mean integrin grade ± standard error (SE) from five separate experiments for W50 and three for W50/BE1. Integrin staining in control cultures incubated in DMEM is indicated by the dotted line.
FIG. 2
FIG. 2
β1 integrin staining in HGF incubated for 1 h with RI (solid line) and RIA (dashed line) in DMEM without supplements. β1 integrin staining, as described previously, is plotted against arginine-specific enzyme activity on a log2 scale. The results represent the mean integrin grade ± standard error (SE) from five separate experiments.
FIG. 3
FIG. 3
HGF incubated for 1 h with TLCK-treated RI (A and C) or TLCK-treated RIA (B and D) and stained with antibody 1A1 (A and B), which recognizes only the adhesin component of RI, or PgWC antibody against whole-cell components (C and D). The nuclear staining seen in panels A and B was the result of nonspecific staining which occurred with certain batches of biotin-conjugated anti-mouse secondary antibodies. Magnification, ×750; bar = 10 μm.
FIG. 4
FIG. 4
HGF incubated for 1 h with TLCK-treated RI (A to I) or TLCK-treated RIA (J to L). The first column shows cells stained with the PgWC antibody, and the second column shows the same cells double labelled with mouse antibodies against β1 integrin (B), αV integrin (E), and fibronectin (H and K). The final column shows cells multiply exposed to both labels. RI-treated cells showed a comparable linear distribution of both P. gingivalis components and β1 integrin along the cytoplasm (C), whereas the peripheral distribution of αV integrin mainly around the tips of the cell differed from the more central staining with PgWC (F). Exact colocalization of fibronectin and P. gingivalis components in RI-treated cells is indicated by the yellow, generally linear, cytoplasmic staining (I), whereas the weaker staining of P. gingivalis components in RIA-treated cells resulted in a predominantly green staining due to the fibronectin (L). Identical exposure periods were used for panels I and L. The patterns of staining with α5 and β3 integrin subunits (not shown) resembled those illustrated for β1 and αV respectively. Magnification, ×750; bar = 10 μm.
FIG. 5
FIG. 5
HGF stained to demonstrate fibronectin after incubation for 1 h with RI (A) or RIA (B), both at 0.2 U of arginine-specific enzyme activity per ml, compared with control cells in DMEM (C). RI-treated cells show only remnants of fine granular cytoplasmic staining and often an indistinct nucleus, in contrast with the clear fibronectin network associated with RIA-treated and control cells. Following removal of cells, the fibronectin matrix was totally disrupted after exposure to RI at 0.2 U/ml (D), whereas a clear fibronectin network was retained in the presence of a similar concentration of RIA (E) and in the DMEM control (F). Magnification, ×750; bar = 10 μm.
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References

    1. Aduse-Opoku J, Muir J, Slaney J M, Rangarajan M, Curtis M A. Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50. Infect Immun. 1995;63:4744–4754. - PMC - PubMed
    1. Birkedal-Hansen H, Taylor R E, Zambon J J, Barwa P K, Neiders M E. Characterization of collagenolytic activity from strains of Bacteroides gingivalis. J Periodontal Res. 1988;23:258–264. - PubMed
    1. Birkedal-Hansen H, Wells B R, Lin H Y, Caulfield P W, Taylor R E. Activation of keratinocyte-mediated collagen (type I) breakdown by suspected human periodontopathogen. Evidence of a novel mechanism of connective tissue breakdown. J Periodontal Res. 1984;19:645–650. - PubMed
    1. Bretscher M S. Endocytosis and recycling of the fibronectin receptor in CHO cells. EMBO J. 1989;8:1341–1348. - PMC - PubMed
    1. Collinson L M, Rangarajan M, Curtis M A. Altered expression and modification of proteases from an avirulent mutant of Porphyromonas gingivalis W50 (W50/BE1) Microbiology. 1998;144:2487–2496. - PubMed

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