Filifactor alocis, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis. In contrast to P. gingivalis, F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesiclemediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.

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...

Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A 1.2-kb open reading frame, a putative glycosyltransferase, downstream of vimE, was cloned, insertionally inactivated using the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, this mutant, designated P. gingivalis FLL95, was nonpigmented and nonhemolytic when plated on Brucella blood agar. Arginine-and lysine-specific gingipain activities were reduced by approximately 97% and 96%, respectively, relative to that of the parent strain. These activities were unaffected by the growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of the rgpA, rgpB, and kgp gingipain genes was unaffected in P. gingivalis FLL95 in comparison to the wild-type strain. In nonactive gingipain extracellular protein fractions, multiple high-molecular-weight proteins immunoreacted with gingipain-specific antibodies. The specific gingipain-associated sugar moiety recognized by monoclonal antibody 1B5 was absent in FLL95. Taken together, these results suggest that the vimE downstream gene, designated vimF (virulence modulating gene F), which is a putative glycosyltransferase group 1, is involved in the regulation of the major virulence factors of P. gingivalis.As a common theme, the expression of extracellular proteolytic activities is highly regulated in both prokaryotic and eukaryotic systems (reviewed in references 11 and 53). This regulation can occur at multiple levels including expression of the protease genes, secretion, processing of an inactive secreted precursor to its active form, and/or the posttranslational glycosylation of the proteins (16, 49). The multiple layers of regulation are vital to ensure that expression is tightly controlled in the appropriate temporal and spatial patterns.Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe has been implicated as an important etiological agent in adult periodontitis (reviewed in references 26, 32, and 48). While several virulence factors have been implicated in the pathogenicity of P. gingivalis, the high proteolytic abilities of this organism are the focus of much attention as they are considered to play the most significant role in virulence (34). The major proteases, called gingipains, are both extracellular and cell associated. They consist of arginine-specific proteases (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]) (34). Although glycosylation appears to be important for their activation (reviewed in references 16 and 41), there remains a gap in our knowledge about the regulation/ activation of the P. gingivalis gingipains.Previously, we have reported that the recA locus can affect the phenotypic expression and distribution of the gingipains in P. gingivalis (1, 2, 37). Using the cloned vimA gene, which is downstream of the recA gene and part of the bcp-recA-vimA transcriptional unit, a defective mutant was constructed by allel...

The Porphyromonas gingivalis recombinant VimA can interact with the gingipains and several other proteins, including a sialidase. Sialylation can be involved in protein maturation; however, its role in virulence regulation in P. gingivalis is unknown. The three sialidase-related proteins in P. gingivalis showed the characteristic sialidase Asp signature motif (SXDXGXTW) and other unique domains. To evaluate the roles of the associated genes, randomly chosen P. gingivalis isogenic mutants created by allelic exchange and designated FLL401 (PG0778::ermF), FLL402 (PG1724::ermF), and FLL403 (PG0352::ermF-ermAM) were characterized. Similar to the wild-type strain, FLL402 and FLL403 displayed a black-pigmented phenotype in contrast to FLL401, which was not black pigmented. Sialidase activity in P. gingivalis FLL401 was reduced by approximately 70% in comparison to those in FLL402 and FLL403, which were reduced by approximately 42% and 5%, respectively. Although there were no changes in the expression of the gingipain genes, their activities were reduced by 60 to 90% in all the isogenic mutants compared to that for the wild type. Immunoreactive bands representing the catalytic domains for RgpA, RgpB, and Kgp were present in FLL402 and FLL403 but were missing in FLL401. While adhesion was decreased, the capacity for invasion of epithelial cells by the isogenic mutants was increased by 11 to 16% over that of the wild-type strain. Isogenic mutants defective in PG0778 and PG0352 were more sensitive to hydrogen peroxide than the wild type. Taken together, these results suggest that the P. gingivalis sialidase activity may be involved in regulating gingipain activity and other virulence factors and may be important in the pathogenesis of this organism.

The authors have shown previously that the vimA gene, which is part of the bcp-recA-vimA operon, plays an important role in protease activation in Porphyromonas gingivalis. The gingipain RgpB proenzyme is secreted in the vimA-defective mutant P. gingivalis FLL92. An important question that is raised is whether the vimA gene product could directly interact with the proteases for their activation or regulate a pathway responsible for protease activation. To further study the mechanism(s) of VimA-dependent protease activation, the vimA gene product was further characterized. A 39 kDa protein consistent with the size of the predicted VimA protein was purified. In protein–protein interaction studies, the VimA protein was shown to interact with gingipains RgpA, RgpB and Kgp. Immune sera from mice immunized with P. gingivalis immunoreacted with the purified VimA protein. Taken together, these data suggest an interaction of VimA with the gingipains and further confirm the role of this protein in their regulation or maturation.