Inactivation ofvimF, a Putative Glycosyltransferase Gene Downstream ofvimE, Alters Glycosylation and Activation of the Gingipains inPorphyromonas gingivalisW83

Abstract

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...

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

“…Curtis and colleagues were the first to demonstrate that the gingipains are posttranslationally modified with carbohydrate additions that are cross-reactive with monoclonal antibodies to P. gingivalis lipopolysaccharide (LPS) (41,60). Our recent publications confirm and extend these results (213,214,215). Most of the LPS-like glycan moieties appear to occur at the C-terminus of the polypeptide chain and seem to serve to anchor the gingipain molecule into the outer membrane (146,178,188).…”

“…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).…”

“…There is emerging evidence that this process may also be important in gingipain biogenesis in P. gingivalis (60,213,214,215). For glycosylation, different glycosyl transferases catalyze the transfer of different carbohydrate moieties from active donors to specific acceptors (including lipids, proteins and nucleic acids) (26).…”

“…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). Taken together, these observations suggest a role for the Vim proteins in the regulation of gingipain activation; however, their mechanisms are unclear and are the subject of ongoing investigation (see further discussion below in section 3.3.…”

Por Secretion System of Porphyromonas gingivalis

“…These gingipain adhesin complexes are anchored by cell surface polysaccharides. The porR related to the biosynthesis of APS 12 , vimA 13 , vimE, vimF putative glycosyltransferase 14 , rfa related to the biosynthesis of lipopolysaccharides LPS and APS 15 , gtfB glycosyltransferase 16 , and PG1051 putative O antigen ligase 17 genes were identified as being associated with the biosynthesis of LPS and or cell surface polysaccharides that can function as anchorage points for gingipain adhesin complexes. Gingipain activity was detected in the supernatant but not in intact cells.…”