A 0.9-kb open reading frame encoding a unique 32-kDa protein was identified downstream of the recA gene of Porphyromonas gingivalis. Reverse transcription-PCR and Northern blot analysis showed that both the recA gene and this open reading frame are part of the same transcriptional unit. This cloned fragment was insertionally inactivated using the ermF-ermAM antibiotic resistance cassette to create a defective mutant by allelic exchange. When plated on Brucella blood agar, the mutant strain, designated P. gingivalis FLL92, was non-black pigmented and showed significant reduction in beta-hemolysis compared with the parent strain, P. gingivalis W83. Arginine-and lysine-specific cysteine protease activities, which were mostly soluble, were approximately 90% lower than that of the parent strain. Expression of the rgpA, rgpB, and kgp protease genes was the same in P. gingivalis FLL92 as in the wild-type strain. In contrast to the parent strain, P. gingivalis FLL92 showed increased autoaggregration in addition to a significant reduction in hemagglutinating and hemolysin activities. In in vivo experiments using a mouse model, P. gingivalis FLL92 was dramatically less virulent than the parent strain. A molecular survey of this mutant and the parent strain using all known P. gingivalis insertion sequence elements as probes suggested that no intragenomic changes due to the movement of these elements have occurred in P. gingivalis FLL92. Taken together, these results suggest that the recA downstream gene, designated vimA (virulence-modulating gene), plays an important role in virulence modulation in P. gingivalis W83, possibly representing a novel posttranscriptional or translational regulation of virulence factors in P. gingivalis.

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...

Porphyromonas gingivalis FLL32, a recA mutant, was isolated during construction of a recA defective mutant of P. gingivalis W83 by allelic exchange mutagenesis. In contrast to W83 and FLL33, the typical recA- mutant previously reported, FLL32 was non-pigmented, lacked beta-hemolytic activity on blood agar and produced significantly less proteolytic activity. The proteolytic activity in FLL32 was mostly soluble. Expression of the rgpA, rgpB and kgp protease genes was unaltered in FLL32 when compared to FLL33 and the wild-type strain. FLL32 exhibited reduced virulence in a murine model and partially protected the animals immunized with that strain against a subsequent lethal challenge by the wild-type strain. These results indicate that the reduced level of proteolytic activity in FLL32 may be due to a defect in the processing of the proteases. Further, immunization with a non-virulent recA defective mutant of P. gingivalis can partially protect against a lethal wild-type challenge. The results from this study suggest that the recA locus may be involved in expression and regulation of proteolytic activity in P. gingivalis.