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Infection and Immunity, April 1999, p. 1837-1843, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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,1 M. Rangarajan,2 D. M. Williams,1 and M. A. Curtis2

Department of Oral Pathology1 and MRC Molecular Pathogenesis Group, Department of Oral Microbiology,2 St. Bartholomew's and Royal London School of Medicine and Dentistry, London E1 2AD, United Kingdom

Received 3 February 1998/Returned for modification 11 May 1998/Accepted 8 December 1998

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 beta 1 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 beta 1 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 alpha 5beta 1 integrin (fibronectin receptor) from HGF. No effect was observed on the alpha Vbeta 3 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 alpha 5beta 1 integrin. Exact colocalization of RI with fibronectin and its alpha 5beta 1 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 alpha 5beta 1 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.

* Corresponding author. Mailing address: Department of Oral Pathology, St. Bartholomew's and Royal London School of Medicine and Dentistry, Turner St., London, E1 2AD, United Kingdom. Phone: 44 171 295 7154 or 44 171 295 7130. Fax: 44 171 295 7153. E-mail: m.a.scragg{at}mds.qmw.ac.uk.

Infection and Immunity, April 1999, p. 1837-1843, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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