Arg-Gingipain Is Responsible for the Degradation of Cell Adhesion Molecules of Human Gingival Fibroblasts and Their Death Induced by Porphyromonas gingivalis

Abstract

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are two major cysteine proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis, which has been shown to act as major pathogen in the development and progression of periodontal diseases. These enzymes are also important for this organism to proliferate and survive in periodontal pockets. Here we show that Rgp is responsible for the disruption of fibronectin-integrin interactions in human gingival fibroblasts by P. gingivalis. Fibroblasts incubated with the culture supernatant of P. gingivalis showed a time-dependent loss of the adhesion activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that fibronectin and integrin subunits alpha2, beta1 and beta3 in the fibroblast culture largely disappeared with the treatment. The detached cells became committed to death by disruption of contacts between adhesion molecules. In contrast, the culture supernatants from the Rgp-deficient mutants produced no significant changes in either cell adhesion or viability. Prior treatment of the culture supernatant of P. gingivalis with an Rgp inhibitor, but not a Kgp inhibitor, strongly inhibited the detachment of fibroblasts followed by cell death. These results suggest that Rgp disrupts the integrin-fibronectin interactions in fibroblasts, thereby contributing to the damage of periodontal tissues in periodontal diseases caused by P. gingivalis.

Inhibitory effects of Porphyromonas gingivalis fimbriae on interactions between extracellular matrix proteins and cellular integrins

“…It has been reported that the arginine-specific protease (gingipain) complex of P. gingivalis degrades vitronectin, fibronectin, and a5b1-integrin, which in turn blocks the interaction of integrin-ligand with human fibroblasts [11,32]. Since the presence of serum proteins interferes with the proteolytic activity of P. gingivalis [11], this degradation was not markedly observed in the presence of 10% FCS (data not shown).…”

Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C

“…It has been published before that P. gingivalis bacterial extracts eliminate fibronectin from the pericellular matrix of cultured fibroblasts [15, 14]. However, actual fragmentation of fibronectin by gingipains has not been demonstrated yet with the purified proteins.…”

“…The important role of gingipains in tissue destruction during periodontitis has long been recognized, and several lines of evidence indicate that these bacterial proteases specifically attack the interaction of gingival and periodontal cells with their extracellular matrix [7, 14, 15, 43–45]. The gingipain concentration in periodontal pockets of patients has been measured to be in the order of 1 μM [5], which is 10–100 times higher than what was used in the present or previous studies to digest relevant ECM substrates in vitro .…”

“…It mediates adhesion, spreading and motility of fibroblasts by interacting with specific cellular receptors, most notably integrins [13]. Interestingly, P. gingivalis appears to use gingipains to specifically attack the interaction of fibronectin with its receptors on fibroblast surfaces [14, 15]. Incubation of cultured gingival fibroblasts with P. gingivalis culture supernatants or purified gingipains lead to a rapid loss of fibronectin and α5β1 integrin from cell surfaces, and to cell detachment.…”

Porphyromonas gingivalis Gingipains: The Molecular Teeth of a Microbial Vampire

“…Scragg et al [47] have reported that the RgpA complex was much more effective at degrading the fibronectin matrix and the fibronectin receptor (α5β1 integrin) on human gingival fibroblasts than the Arg proteinase without the adhesins. Furthermore, Baba et al [48] demonstrated that the degradation of the fibronectin receptor on human gingival fibroblasts by RgpA results in apoptosis. Incubation of endothelial cells with RgpA or Kgp resulted in a loss of cell to cell adhesion and eventual cell death [49].…”