Altered Gingipain Maturation in vimA- and vimE -Defective Isogenic Mutants of Porphyromonas gingivalis

Abstract

We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA-and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membraneassociated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA-and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic periodontitis. Furthermore, accumulating data suggest that this organism is also associated with systemic diseases and complications including atherosclerosis, preterm births, and low-birth-weight babies (15). While several virulence factors are implicated in the pathogenicity of P. gingivalis, the proteolytic abilities of this organism have been considered to play the most significant role in its virulence (7,11,14,15,17). The major proteases, called gingipains, consist of an arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp. These proteases, which are both extracellular and cell associated, are required for growth of the organism and other housekeeping functions, which include processing enzymes for various cell surface proteins, including individual proteases (24). Moreover, the multiple pathogenic effects of the gingipains, such as degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increase in vascular permeability and blood clotting prevention, are well documented (14).We have reported previously that the vimA and vimE genes can modulate the phenotypic expression of the gin...

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

“…VimA is a putative membrane protein that appears to play a role in virulence regulation in P. gingivalis via gingipain biogenesis (Abaibou et al, 2001;Olango et al, 2003;Vanterpool et al, 2005b). If vimA is part of a pathway(s) that is involved in the maturation and/or activation of the gingipains, then it is likely that VimA could interact with the gingipain.…”

“…Furthermore, we cannot rule out the possibility that the VimA protein may regulate the function of these proteins via a common mechanism shared with the gingipains. Carbohydrates biogenesis was altered in a P. gingivalis vimA-defective mutant (Vanterpool et al, 2005b). The presence of sialidase in many organisms suggests that sialidase activity may be important for colonization and/or pathogenicity.…”

“…We have shown that the vimA gene can modulate the phenotypic expression of the gingipains in P. gingivalis (Abaibou et al, 2001;Olango et al, 2003;Vanterpool et al, 2005b). The vimA gene is part of the bcp-recA-vimA transcriptional unit (Fig.…”

“…We have previously shown that inactivation of the vimA gene alters the maturation of the gingipains (Olango et al, 2003;Vanterpool et al, 2005b; Fig. 1).…”

Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis

“…If gingipain activation occurs by an autoproteolytic mechanism, questions are raised on how this process is regulated and the involvement of specific bacterial host factors. In fact, in previous reports, we have demonstrated the secretion of the inactive proenzyme gingipain species in vimA-, vimE-and vimF-defective mutants (150,213,214,215). In both the vimE and vimF isogenic mutants, activation of the gingipain proenzyme species could not be achieved (213,214).…”