Targeted Disruption of Fibronectin-Integrin Interactions in Human Gingival Fibroblasts by the RI Protease of Porphyromonas gingivalis W50

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 gingivalisproteases on human gingival fibroblast (HGF) integrins and their fibronectin matrix. Culture supernatant from the virulent strain W50 caused considerably greater loss of the β1 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 Nα-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 β1 integrin expression. However purified RI, an α/β 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 α5β1 integrin (fibronectin receptor) from HGF. No effect was observed on the αVβ3 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. gingivaliswhole cells indicated localization of RI on the fibroblasts in a clear, linear pattern typical of that seen with fibronectin and α5β1 integrin. Exact colocalization of RI with fibronectin and its α5β1 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 α5β1integrin 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.

Microbiota at teeth and implants in partially edentulous patients. A 10‐year retrospective study

“…They disturb the protease-protease inhibitor balance in infected tissues (Laugisch et al 2012) and degrade the extracellular matrix (Houle et al 2003). Gingipains cleave b 1 and a 5 integrins, they degrade fibronectin (Scragg et al 1999), and following that, they disturb adhesion of fibroblasts to surfaces (Baba et al 2001). Tannerella forsythia was found in higher loads at implants in comparison with teeth, especially in smokers.…”

Local and Systemic Effects of Porphyromonas gingivalis Infection

“…Increased fibronectin and its fragments are observed in gingival crevicular fluid (GCF) from patients with periodontitis but not from healthy subjects [ 80 ]. Similarly, fibronectin degradation is observed when purified arginine gingipains are incubated with human gingival fibroblast cells [ 108 ]. Moreover, purified RgpA, RgpB, or Kgp gingipains can cleave isolated human serum fibronectin [ 80 ].…”

Mechanisms of Porphyromonas gingivalis to translocate over the oral mucosa and other tissue barriers

“…The gingipains secreted by P. gingivalis are also known to degrade molecules of the ECM, such as fibronectin and tenascin-C. This was found via fragmentation of fibronectin and tenascin-C by purified proteases, observed in a SDS-PAGE gel and via immunofluorescence staining [ 27 , 28 ]. Using a complex three-dimensional models of engineered human oral mucosa (EHOM) it was illustrated that P. gingivalis is able to penetrate into the tissue by degradation of components of the basement membrane [ 21 ].…”