Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis

Abstract

Arginine-specific cysteine proteinase (Arg-gingipain [RGP]), a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB ؉ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.Porphyromonas (Bacteroides) gingivalis, an oral anaerobic bacterium, has been isolated frequently from subgingival lesions in patients with adult periodontal disease, implying that the microorganism is one of the most important pathogens for the disease (25,31,36). P. gingivalis possesses several potential virulence factors (13). Among them, secretory proteinases have received much attention because they can degrade host tissue and cause inflammation (9, 27). Many proteinases have been isolated from culture supernatants, vesicles, membrane fractions, and cell extracts of P. gingivalis (for a review, see reference 5), but it has recently been reported that the proteinases with trypsin-like activity are derived from either Arggingipain (RGP) or Lys-gingipain (24). In a previous study (7), we reported the isolation of RGP (formerly, argingipain) from the culture supernatant of P. gingivalis and its unusual catalytic features related to periodontopathogenicity of the organism (e.g., the abilities to degrade periodontal tissue components, to evade inactivation by internal proteinase inhibitors such as cystatins and ...

Identification of bistable populations of Porphyromonas gingivalis that differ in epithelial cell invasion

“…Here we present evidence for the existence of highly invasive and poorly invasive subpopulations in cultures of two strains of P. gingivalis. We demonstrate differences in gene expression between the two subtypes and show that the surface-associated protease enzyme complex Arg-gingipain (encoded by rgpA : B) (Nakayama et al, 1995(Nakayama et al, , 1996bCurtis et al, 2001) influences invasion and is a key feature of the bistable phenotype that we observe.…”

“…The organism produces a range of virulence factors, including fimbriae (implicated in attachment and colonization; Nakayama et al, 1996a;Yoshimura et al, 2009), proteases known as gingipains (implicated in tissue destruction; Pike et al, 1996;Kadowaki et al, 1998;Curtis et al, 2001) and lipopolysaccharide (Darveau et al, 2004). In addition, there have been a number of reports demonstrating the ability of the organism to invade and survive within epithelial cells (Lamont et al, 1992(Lamont et al, , 1995Duncan et al, 1993;Madianos et al, 1996).…”

Effects of egg yolk antibody against Porphyromonas gingivalis gingipains in periodontitis patients

“…Gingipains play a major role in the progression of human periodontal disease, especially in host colonization, inactivation of host defenses, tissue destruction, and host immune system modulation (8)(9)(10)(11)(12)(13)(14). In addition, gingipains play a role in bacterial housekeeping (15), including amino acid uptake from host proteins, heme acquisition from erythrocytes (16), and fimbriae maturation (17). We postulated that the inhibition of gingipains may reduce the pathogenic nature of P.…”

Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities

“…Proteases, haemagglutinins, fimbriae and superoxide dismutase (Nakayama et al, 1996 ;Lamont & Jenkin-son, 1998 ;Curtis et al, 1999) are among the many virulence factors produced by this organism. Several physiologically important proteins, including collagens (Birkedal-Hansen et al, 1988 ;Kato et al, 1992), fibronectin (Smalley et al, 1988 ;Uitto et al, 1989), plasma protease inhibitors (Carlsson et al, 1984), immunoglobulins (Kilian, 1981 ;Sundqvist et al, 1985 ;Sato et al, 1987) and complement factors (Sundqvist et al, 1985 ;Schenkein & Berry, 1988 ;Wingrove et al, 1992), have been shown to be degraded by proteases from P. gingivalis and it is plausible that these proteases may play a major role in evading host defence mechanisms in addition to causing destruction of the tissues supporting the teeth.…”