Gingipain RgpB Is Excreted as a Proenzyme in the vimA -Defective Mutant Porphyromonas gingivalis FLL92

Abstract

We have previously shown that the unique vimA (virulence-modulating) gene could modulate proteolytic activity in Porphyromomas gingivalis. Although a reduction in cysteine protease activity was observed in the vimA-defective mutant, P. gingivalis FLL92, compared to that of the wild-type strain, no changes were seen in the expression of the gingipain genes. This result might suggest posttranscriptional regulation of protease expression. To determine whether there was a defect in the translation, transport, or maturation of the gingipains, P. gingivalis FLL92 was further characterized. In contrast to the wild-type strain, a 90% reduction was seen in both Rgp and Kgp protease activities in strain FLL92 during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. Throughout all the growth phases, Rgp and Kgp activities were mostly soluble, in contrast to those of the wild-type strain. Western blot analyses identified unique Rgp-and Kgp-immunoreactive bands in extracellular protein fractions from FLL92 grown to late exponential phase. Also, the RgpB proenzyme was identified in this fraction by mass spectrometry. In addition, in vitro protease activity could be induced by a urea denaturationrenaturation cycle in this fraction. These results indicate that protease activity in P. gingivalis may be growth phase regulated, possibly by multiple mechanisms. Furthermore, the gingipain RgpB is excreted in an inactive form in the vimA mutant. In addition, these results provide the first evidence of posttranslational regulation of protease activity in P. gingivalis and may suggest an important role for the vimA gene in protease activation in this organism.Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic adult periodontitis (reviewed in references 23, 25, and 43) and is associated with other systemic diseases, including atherosclerosis (reviewed in reference 11). While several virulence factors have been implicated in the pathogenicity of P. gingivalis, the high-level proteolytic abilities of this organism have been the focus of much attention and appear to play an important role in its virulence. The major proteases, called gingipains, are both extracellular and cell associated. They consist of arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]) (35). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp (28,33). In addition to being essential for growth, proteases have other functions. Several reports (reviewed in reference 22) have documented the multiple effects of proteases, including degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increases in vascular permeability and blood clotting prevention. Furthermore, these gingipains, which have been suggested...

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.…”

“…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).…”

“…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.…”

HtrA in Porphyromonas gingivalis can regulate growth and gingipain activity under stressful environmental conditions

“…One-litre cultures of P. gingivalis strains FLL203 and W83 were grown to stationary phase (OD 600 1?5) from actively growing cells. Preparations of whole-cell culture, cell-free medium, cell suspension, vesicles and vesicle-free medium were made as previously reported (Olango et al, 2003). The whole-cell culture (WC) fraction is a sample of the culture after the bacterium has been grown to a specific growth phase.…”

“…SDS-PAGE and transfer to nitrocellulose membranes were as described by Vanterpool et al (2006). The blots were probed with antibodies against specific protease domains (Olango et al, 2003) or HagA (generously donated by Ann Progulske-Fox, University of Florida). The secondary antibody was immunoglobulin G (heavy plus light chains)-horseradish peroxidase conjugate (Zymed Laboratories).…”