Porphyromonas gingivalis Gingipains Induce Cyclooxygenase-2 Expression and Prostaglandin E2 Production via ERK1/2-Activated AP-1 (c-Jun/c-Fos) and IKK/NF-κB p65 Cascades.

Porphyromonas gingivalis is commonly known as one of the major pathogens contributing to periodontitis, and its persistent infection may increase the risk for the disease. The proinflammatory mediators, including IL-6, TNF-α, and cyclooxygenase-2 (COX-2)/PGE2, are closely associated with progression of periodontitis. In this study, we focused on the cysteine protease "gingipains," lysine-specific gingipain, arginine-specific gingipain (Rgp) A, and RgpB, produced by P. gingivalis, and used the wild-type strain and several gene-deletion mutants (rgpA, rgpB, kgp, and fimA) to elucidate the involvement of gingipains in COX-2 expression and PGE2 production. We infected human monocytes, which are THP-1 cells and primary monocytes, with these bacterial strains and found that gingipains were involved in induction of COX-2 expression and PGE2 production. We have shown that the protease activity of gingipains was crucial for these events by using gingipain inhibitors. Furthermore, activation of ERK1/2 and IκB kinase was required for gingipain-induced COX-2 expression/PGE2 production, and these kinases activated two transcription factors, c-Jun/c-Fos (AP-1) and NF-κB p65, respectively. In particular, these data suggest that gingipain-induced c-Fos expression via ERK is essential for AP-1 formation with c-Jun, and activation of AP-1 and NF-κB p65 plays a central role in COX-2 expression/PGE2 production. Thus, we show the (to our knowledge) novel finding that gingipains with the protease activity from P. gingivalis induce COX-2 expression and PGE2 production via activation of MEK/ERK/AP-1 and IκB kinase/NF-κB p65 in human monocytes. Hence it is likely that gingipains closely contribute to the inflammation of periodontal tissues.

Porphyromonas gingivalis enhances pneumococcal adhesion to human alveolar epithelial cells by increasing expression of host platelet-activating factor receptor.

Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.

Molecular Strategies Underlying Porphyromonas gingivalis Virulence.

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is considered the keystone of periodontitis diseases, a set of inflammatory conditions that affects the tissues surrounding the teeth. In the recent years, the major virulence factors exploited by P. gingivalis have been identified and characterized, including a cocktail of toxins, mainly proteases called gingipains, which promote gingival tissue invasion. These effectors use the Sec pathway to cross the inner membrane and are then recruited and transported across the outer membrane by the type IX secretion system (T9SS). In P. gingivalis, most secreted effectors are attached to anionic lipopolysaccharides (A-LPS), and hence form a virulence coat at the cell surface. P. gingivalis produces additional virulence factors to evade host immune responses, such as capsular polysaccharide, fimbriae and outer membrane vesicles. In addition to periodontitis, it is proposed that this broad repertoire of virulence factors enable P. gingivalis to be involved in diverse human diseases such as rheumatoid arthritis, and neurodegenerative, Alzheimer, and cardiovascular disorders. Here, we review the major virulence determinants of P. gingivalis and discuss future directions to better understand their mechanisms of action.

A novel gingipain regulatory gene in Porphyromonas gingivalis mediates host cell detachment and inhibition of wound closure.

The black pigmentation-related genes in Porphyromonas gingivalis are primarily involved in regulating gingipain functions. In this study, we identified a pigmentation-related gene, designated as pgn_0361. To characterize the role of pgn_0361 in regulating P. gingivalis-mediated epithelial cell detachment and inhibition of wound closure, PgΔ0361, an isogenic pgn_0361-defective mutant strain, and PgΔ0361C, a complementation strain, were constructed using P. gingivalis ATCC 33277. The gingipain and hemagglutination activities, as well as biofilm formation, were examined in all three strains. The effect of P. gingivalis strains on epithelial cell detachment was investigated using the HO-1-N-1 and Ca9-22 epithelial cell lines. The inhibition of wound closure by heat-killed P. gingivalis cells and culture supernatant was analyzed using an in vitro wound closure assay. Compared to the wild-type strain, the PgΔ0361 strain did not exhibit gingipain or hemagglutination activity but exhibited enhanced biofilm formation. Additionally, the PgΔ0361 strain exhibited attenuated ability to detach the epithelial cells and to inhibit wound closure in vitro. Contrastingly, the culture supernatant of PgΔ0361 exhibited high gingipain activity and strong inhibition of wound closure. The characteristics of PgΔ0361C and wild-type strains were comparable. In conclusion, the pgn_0361 gene is involved in regulating gingipains. The PGN_0361-defective strain exhibited reduced virulence in terms of epithelial cell detachment and inhibition of wound closure. The culture supernatant of the mutant strain highly inhibited wound closure, which may be due to high gingipain activity.

Secreted gingipains from Porphyromonas gingivalis induce microglia migration through endosomal signaling by protease-activated receptor 2.

Much attention has been paid to the connection between periodontitis and Alzheimer's disease (AD). We previously showed that infection of P. gingivalis, one of major periodontal pathogen causing periodontitis, induced migration and inflammatory responses in murine microglia through gingipain-induced activation of protease-activated receptor 2 (PAR2). In this study, we have attempted to clarify effects of secreted gingipains on cell migration of human microglial cell line, cleavage sites of PAR2 by gingipains and subsequent signaling pathways. P. gingivalis culture supernatant induced migration and membrane ruffling, which is necessary for microglia migration, in human microglial cell line HMC3 cells through PAR2. These effects were mainly mediated by gingipains, because cell migration and membrane ruffling were dramatically inhibited by treatment with gingipain inhibitors. Furthermore, pharmacological and genetic inhibition of Src kinase and ß-arrestin, which are important for the internalization of G protein-coupled receptors, significantly inhibited P. gingivavlis culture supernatant-induced membrane ruffling in HMC3 cells. After treatment with P. gingivalis culture supernatant in Flag-PAR2-HA transfected HEK293T cells, Flag was removed from the cell surface, and HA was detected in the cytosol, indicating the internalization of PAR2. Furthermore, the phosphorylation level of ERK1/2 increased in PAR2-transfected HEK293T cells after treatment with P. gingivalis culture supernatant. The gingipain inhibitors, Src kinase inhibitor and ß-arrestin knockdown suppressed PAR2 internalization and ERK1/2 phosphorylation. These observations suggest that secreted gingipains from P. gingivalis induce Src- and ß-arrestin-dependent internalization of PAR2 and further activate the ERK1/2 pathway to promote migration of microglia. PAR2 are activated by the tethered ligands exposed by cleavage of extracellular N-terminal of PAR2. We also estimated potential gingipain cleavage sites in PAR2 and exposed tethered ligands, which are required for PAR2 internalization and membrane ruffling. The identified mechanism in this study might contribute to the retrogression of sporadic AD in patients after infection with P. gingivalis.

Identification and Modification of Porphyromonas gingivalis Cysteine Protease, Gingipain, Ideal for Screening Periodontitis.

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis-specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis. Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis.

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver.

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 ß (GSK-3ß) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.