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Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection., Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis. No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1β (IL-1β) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1β production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis. Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to P. gingivalis might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

Angiopoietin 1 (Angpt1) and angiopoietin 2 (Angpt2) are the ligands of tyrosine kinase (Tie) receptors, and they play important roles in vessel formation and the development of inflammatory diseases, such as atherosclerosis. Porphyromonas gingivalis is a Gram-negative periodontal bacterium that is thought to contribute to the progression of cardiovascular disease. The aim of this study was to investigate the role of P. gingivalis infection in the modulation of Angpt1 and Angpt2 in human aortic smooth muscle cells (AoSMCs). We exposed AoSMCs to wild-type (W50 and 381), gingipain mutant (E8 and K1A), and fimbrial mutant (DPG-3 and KRX-178) P. gingivalis strains and to different concentrations of tumor necrosis factor (TNF). The atherosclerosis risk factor TNF was used as a positive control in this study. We found that P. gingivalis (wild type, K1A, DPG3, and KRX178) and TNF upregulated the expression of Angpt2 and its transcription factor ETS1, respectively, in AoSMCs. In contrast, Angpt1 was inhibited by P. gingivalis and TNF. However, the RgpAB mutant E8 had no effect on the expression of Angpt1, Angpt2, or ETS1 in AoSMCs. The results also showed that ETS1 is critical for P. gingivalis induction of Angpt2. Exposure to Angpt2 protein enhanced the migration of AoSMCs but had no effect on proliferation. This study demonstrates that gingipains are crucial to the ability of P. gingivalis to markedly increase the expressed Angpt2/Angpt1 ratio in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. These findings further support the association between periodontitis and cardiovascular disease.

Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on β-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of β-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for β-catenin processing. The β-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3β were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that β-catenin fragments were translocated to the nucleus. The accumulation of β-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the β-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis -induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of β-catenin and disassociation of the β-catenin destruction complex by gingipain-dependent proteolytic processing. β-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype.

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 Gram-negative anaerobic bacteriumPorphyromonas gingivalisis a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. ThegtfBmutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation ofP. gingivalis.

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-872aa]) and nonimmune (IL-877aa) cells. IL-877aahas low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-877aasignificantly enhance burst activation by fMLP and chemotactic activity (P< 0.05) but decrease burst activation and chemotactic activity of IL-872aatoward neutrophil-like HL60 cells and primary neutrophils (P< 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-877aaand the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.

Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogenPorphyromonas gingivalisand are encoded byrgpAandrgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpAcat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-catalytic chain (mt-RgPAcat), are derived fromrgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight.rgpBencodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated thatrgpBis involved in the glycosylation process, since inactivation ofrgpBresults in the loss of not only RgpB and mt-RgpB but also mt-RgpAcat. This work aims to confirm the role of RgpB in the posttranslational modification of RgpAcatand the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivatedrgpBstrain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream ofrgpBhad no effect onrgpA-derived enzymes, suggesting that the phenotype of thergpBmutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpAcatfrom W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpAcatfrom D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived fromrgpAand that this process is required for enzyme stabilization.

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 ( vi rulence m odulating gene F ), which is a putative glycosyltransferase group 1, is involved in the regulation of the major virulence factors of P. gingivalis .

The presence ofPorphyromonas gingivalisin the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells and human microvascular endothelial cells to gingipain-active extracellular protein preparations and/or purified gingipains fromP. gingivalis. Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin β1 was observed in immunoblots of cell lysates. There was a direct correlation between the kinetics of cleavage of N- and VE-cadherin and loss of cell adhesion properties. Loss of cell adhesion, as well as N- and VE-cadherin and integrin β1 cleavage, could be inhibited or significantly delayed by preincubation ofP. gingivalisW83 gingipain-active extracellular extracts with the cysteine protease inhibitorNα-p-tosyl-l-lysine chloromethylketone. Furthermore, purified gingipains also induced endothelial cell detachment and apoptosis. Apoptosis-associated events, including annexin V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase and topoisomerase I (Topo I), were observed in endothelial cells after detachment. All of the effects observed were correlated with the different levels of cysteine-dependent proteolytic activity of the extracts tested. Taken together, these results indicate that gingipains fromP. gingivaliscan alter cell adhesion molecules and induce endothelial cell death, which could have implications for the pathogenicity of this organism.

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) arePorphyromonas gingivaliscysteine proteinases implicated as major virulence factors in pathologies of periodontitis. We purified a 660-kDa cell-associated gingipain complex existing as a homodimer of two catalytically active monomers which comprises their catalytic and adhesin domains. Electron microscopy revealed that the complex was composed of a globular particle with a 10-nm external diameter possessing one or two electron-dense hole-like structures. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide (LPS) with the catalytic domains and a hemagglutinin domain, Hgp44, of Rgp and Kgp in the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endotherial cells with an efficiency which was higher than that of the monomeric gingipains. The native complex produced only a small amount of nitrogen dioxide, tumor necrosis factor alpha, and interleukin-6 by macrophages, whereas the heat-denatured complex resulted in increased production. Inhibition of the proteolytic activities of the gingipain complex did not up-regulate the cytokine production, indicating that the functional domains in LPS are structurally masked by the complex proteins. These results indicate the importance of the complex in evasion of host defense mechanisms as well as in host tissue breakdown.

Porphyromonas gingivalis , a gram-negative anaerobic bacterium associated with active lesions of chronic periodontitis, produces several proteinases which are presumably involved in host colonization, perturbation of the immune system, and tissue destruction. The aims of this study were to investigate the degradation of human transferrin by gingipain cysteine proteinases of P. gingivalis and to demonstrate the production of toxic hydroxyl radicals (HO · ) catalyzed by the iron-containing transferrin fragments generated or by release of iron itself. Analysis by polyacrylamide gel electrophoresis and Western immunoblotting showed that preparations of Arg- and Lys-gingipains of P. gingivalis cleave transferrin (iron-free and iron-saturated forms) into fragments of various sizes. Interestingly, gingival crevicular fluid samples from diseased periodontal sites but not samples from healthy periodontal sites contained fragments of transferrin. By using 55 Fe-transferrin, it was found that degradation by P. gingivalis gingipains resulted in the production of free iron, as well as iron bound to lower-molecular-mass fragments. Subsequent to the degradation of transferrin, bacterial cells assimilated intracellularly the radiolabeled iron. Growth of P. gingivalis ATCC 33277, but not growth of an Arg-gingipain- and Lys-gingipain-deficient mutant, was possible in a chemically defined medium containing 30% iron-saturated transferrin as the only source of iron and peptides, suggesting that gingipains play a critical role in the acquisition of essential growth nutrients. Finally, the transferrin degradation products generated by Arg-gingipains A and B were capable of catalyzing the formation of HO · , as determined by a hypoxanthine/xanthine oxidase system and spin trapping-electron paramagnetic resonance spectrometry. Our study indicates that P. gingivalis gingipains degrade human transferrin, providing sources of iron and peptides. The iron-containing transferrin fragments or the release of iron itself may contribute to tissue destruction by catalyzing the formation of toxic HO · .

The gingipains have been implicated in the pathogenicity of Porphyromonas gingivalis , a major etiologic agent of chronic periodontitis. Mature gingipains often present as a membrane-bound glycosylated proteinase-adhesin complex comprising multiple adhesin domains (HA1 to -4) and a catalytic domain. Using recombinant adhesin domains, we were able to show that patients with chronic periodontitis produce significantly more immunoglobulin G reactive with gingipain domains than a corresponding group with healthy periodontium. Titers were predominantly directed toward the carbohydrate epitopes shared between the gingipains and the lipopolysaccharide of P. gingivalis with little recognition of the peptide backbone of the catalytic domains. Distribution of titers to peptide epitopes of the adhesin domains was as follows: HA4 ≈ HA1 > HA3 ≫ HA2. No correlation was observed between markers of disease severity and titers to individual adhesins within the disease group. Posttreatment titers showed no change or a decrease in titers for the majority of patients except for titers to the HA2 domain which showed marked increases in a few responding patients. Since the HA2 domain is important in hemoglobin binding and acquisition of essential porphyrin, boosting titers of antibodies to this domain may have the potential to control the growth of this organism.

Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis . Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis , inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis , including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.

Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-γ) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-γ have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogenPorphyromonas gingivalisstimulated monocytes primed with IFN-γ to release IL-12, thereby enhancing IFN-γ accumulation in T-cell populations. P. gingivalisLPS was shown to enhance IL-12 induction of IFN-γ in T cells in a manner independent from TNF-α contribution. The levels of T-cell IL-12 receptors were not affected byP. gingivalisLPS and played only a minor role in the magnitude of the IFN-γ response. These data suggest that LPS fromP. gingivalisestablishes an activation loop with IL-12 and IFN-γ with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-γ accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-γ levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) ofP. gingivaliscould lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.

Cysteine proteases, including Arg-gingipain of Porphyromonas gingivalis, have been implicated as important virulence factors in periodontal diseases. These enzymes are also involved in the hemagglutinating activity of the organisms. In order to determine the role of proteases in the colonization of the gingival margin, we have compared the attachment properties of P. gingivalis 381 with those of its Arg-gingipain-defective mutant, G-102. Interactions with gram-positive bacteria, human oral epithelial cells, extracellular matrix proteins, and type I collagen were evaluated. In all cases, mutant G-102 was deficient in attachment relative to the parental strain. The mutant's defects could be explained, in part, by the weak autoaggregation displayed by the mutant, which appeared to result from altered fimbrial expression. Both Western blot (immunoblot) and Northern (RNA) blot analyses indicated reduced expression of the major 43-kDa fimbrillin subunit in the mutant. These results suggest that Arg-gingipain may play both direct and indirect roles in the colonization of the gingival margin. In addition, fimbriae may play a direct role in interacting with some host surfaces.

The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA -defective mutant in a different P. gingivalis genetic background were evaluated. The vimA -defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA -defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA -defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting.

Porphyromonas gingivalis is a major periodontal pathogen that has the pathogenic proteinases Arg-specific gingipain and Lys-specific gingipain. We previously found that a cell surface component on P. gingivalis is able to induce Toll-like receptor 2 (TLR2)- and TLR4-independent signaling in 7.19 cells and that this component can be degraded by gingipains. In this study, we purified this component from the P. gingivalis gingipain-null mutant KDP136 and obtained two candidate proteins. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis showed that the proteins, with molecular masses of 123 and 43 kDa, were encoded by PGN_0748 and PGN_0728 (pgm6), respectively, in the P. gingivalis ATCC 33277 genome sequence. The PGN_0748-encoded protein, which we refer to as gingipain-sensitive ligand A (GslA), reacted with antiserum that could effectively inhibit the activity of KDP136 to induce NF-kappaB activation in 7.19 cells, but Pgm6 did not. To further determine what protein is responsible for the NF-kappaB activation, we constructed gslA, pgm6, and pgm6 pgm7 deletion mutants from KDP136. When 7.19 cells were exposed to those mutants, the gslA deletion mutant did not induce NF-kappaB activation, whereas the pgm6 and pgm6 pgm7 deletion mutants did. Furthermore, NF-kappaB activation in 7.19 cells induced by KDP136 was partially inhibited by antiserum against a recombinant protein expressed from the 5'-terminal third of gslA. These results indicate that GslA is one of the factors that induce NF-kappaB activation in 7.19 cells. Interestingly, the gslA gene was present in four of seven P. gingivalis strains tested. This restricted distribution might be associated with the virulence potential of each strain., Infection and immunity, 77(10), pp.4414-4420; 2009

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 membrane-associated 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.

The arginine gingipains RgpA and RgpB ofPorphyromonas gingivalisare well-documented virulence factors of this organism. Structurally, RgpA and RgpB have nearly identical catalytic domains, while RgpA possesses an additional hemagglutinin domain. In this study, we examined the abilities of these proteins to elicit protection againstP. gingivalis-mediated oral bone loss in a murine oral challenge model. Mice immunized subcutaneously with heat-killedP. gingivalisor purified RgpA or RgpB possessed elevated levels ofP. gingivalis-specific immunoglobulin G; however, only the animals immunized withP. gingivaliswhole cells or RgpA were protected from maxillary bone loss. These data suggest that immunization with RgpA stimulates the production of hemagglutinin domain-specific antibodies, which contribute to the prevention ofP. gingivalis-mediated periodontal disease.

Porphyromonas gingivalis is one of the principal organisms associated with adult periodontitis. Bacterial surface proteins such as fimbriae and gingipain hemagglutinin domains have been implicated as adhesins that actuate colonization of epithelium lining the gingival sulcus. We investigated the genetics of P. gingivalis adhesion to monolayers of epithelial cells using wild-type and gingipain mutant strains. These experiments suggested that arginine-specific gingipain (Rgp) catalytic activity modulated adhesion. From the data obtained with rgp mutants, we constructed a working hypothesis predicting that attachment and detachment of P. gingivalis to epithelial cells were mediated by gingipain adhesin and Rgp catalytic domains, respectively. A membrane-based epithelial cell binding assay, used to locate adhesins in extracellular fractions of wild-type and mutant strains, recognized gingipain peptides as adhesins rather than fimbriae. We developed a capture assay that demonstrated the binding of gingipain adhesin peptides to oral epithelial cells. The adherence of fimbrillin to epithelial cells was detected after heat denaturation of cell fractions. The prediction that Rgp catalytic activities mediated detachment was substantiated when the high level of attachment of an rgp mutant was reduced in the presence of wild-type cell fractions that contained gingipain catalytic activities.

Suffusion of gingipain RgpA (GRgpA) elicited a significant concentration-dependent increase in the clearance of macromolecules from in situ hamster cheek pouch which was attenuated by NPC 17647, a selective bradykinin B2receptor antagonist. Leupeptin and a mixture of proteinase inhibitors also attenuated GRgpA-induced responses. These data indicate that GRgpA elicits plasma exudation from in situ oral mucosa in a catalytic site-dependent fashion by elaborating bradykinin.

We have previously reported on a Tn 4351 -generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459–2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS 1126 is capable of transposition within P. gingivalis . We have also determined that IS 1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K ( kgp ) and the arginine-specific proteinase, gingipain R2 ( rgpB ). Sequence analysis of P. gingivalis MSM-3 revealed that Tn 4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS 1126 . Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS 1126 compared to the parental strain A7436. Examination of the first additional IS 1126 element, IS 1126 1 , indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS 1126 element, IS 1126 2 , was found to have inserted upstream of a newly identified gene, hmuR , which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS 1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn 4351 into the P. gingivalis chromosome has resulted in two previously undocumented phenomena in P. gingivalis : (i) the transposition of the endogenous insertion sequence element IS 1126 and (ii) the modulation of gingipain transcription and translation as a result of IS 1126 transposition.

The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils. Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsullonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at both potential Lys-gingipain sites (i.e., between residues 17 and 18 [K-D] and 28 and 29 [K-T]) and at two chymotrypsin sites (between residues 14 and 15 [Y-D] and 20 and 21 [L-D]), respectively. These studies suggest that P. gingivalis contains at least two enzymes capable of cleaving the C5aR, Lys-gingipain and a second nontryptic serine proteinase that is distinct from either Arg- or Lys-gingipain.