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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
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 N
-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
1 integrin expression. However purified RI, an
/
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
5
1 integrin
(fibronectin receptor) from HGF. No effect was observed on the
V
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
5
1 integrin. Exact colocalization of RI
with fibronectin and its
5
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
5
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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