Citrullination and Proteolytic Processing of Chemokines by Porphyromonas gingivalis

The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tissue destruction, loss of alveolar bone, periodontal pocket formation, and eventually, tooth loss. Potential virulence factors of P. gingivalis are peptidylarginine deiminase (PPAD), an enzyme modifying free or peptide-bound arginine to citrulline, and the bacterial proteases referred to as gingipains (Rgp and Kgp). Chemokines attract leukocytes during inflammation. However, posttranslational modification (PTM) of chemokines by proteases or human peptidylarginine deiminases may alter their biological activities. Since chemokine processing may be important in microbial defense mechanisms, we investigated whether PTM of chemokines by P. gingivalis enzymes occurs. Upon incubation of interleukin-8 (IL-8; CXCL8) with PPAD, only minor enzymatic citrullination was detected. In contrast, Rgp rapidly cleaved CXCL8 in vitro. Subsequently, different P. gingivalis strains were incubated with the chemokine CXCL8 or CXCL10 and their PTMs were investigated. No significant CXCL8 citrullination was detected for the tested strains. Interestingly, although considerable differences in the efficiency of CXCL8 degradation were observed with full cultures of various strains, similar rates of chemokine proteolysis were exerted by cell-free culture supernatants. Sequencing of CXCL8 incubated with supernatant or bacteria showed that CXCL8 is processed into its more potent forms consisting of amino acids 6 to 77 and amino acids 9 to 77 (the 6-77 and 9-77 forms, respectively). In contrast, CXCL10 was entirely and rapidly degraded by P. gingivalis, with no transient chemokine forms being observed. In conclusion, this study demonstrates PTM of CXCL8 and CXCL10 by gingipains of P. gingivalis and that strain differences may particularly affect the activity of these bacterial membrane-associated proteases.     

Role of Porphyromonas gingivalis Phosphoserine Phosphatase Enzyme SerB in Inflammation,Immune Response,and Induction of Alveolar Bone Resorption in Rats

Porphyromonas gingivalis secretes a serine phosphatase enzyme, SerB, upon contact with gingival epithelial cells in vitro. The SerB protein plays a critical role in internalization and survival of the organism in epithelial cells. SerB is also responsible for the inhibition of interleukin-8 (IL-8) secretion from gingival epithelial cells infected with P. gingivalis. This study examined the ability of a P. gingivalis SerB mutant to colonize the oral cavity and induce gingival inflammation, immune responses, and alveolar bone resorption in a rat model of periodontal disease. Both P. gingivalis ATCC 33277 and an isogenic ΔSerB mutant colonized the oral cavities of rats during the 12-week experimental period. Both of the strains induced significant (P < 0.05) systemic levels of immunoglobulin G (IgG) and isotypes IgG1, IgG2a, and IgG2b, indicating the involvement of both T helper type 1 (Th1) and Th2 responses to infection. Both strains induced significantly (P < 0.05) higher levels of alveolar bone resorption in infected rats than in sham-infected control rats. However, horizontal and interproximal alveolar bone resorption induced by the SerB mutant was significantly (P < 0.05) lower than that induced by the parental strain. Rats infected with the ΔSerB mutant exhibited significantly higher levels of apical migration of the junctional epithelium (P < 0.01) and polymorphonuclear neutrophil (PMN) recruitment (P < 0.001) into the gingival tissues than rats infected with the wild type. In conclusion, in a rat model of periodontal disease, the SerB phosphatase of P. gingivalis is required for maximal alveolar bone resorption, and in the absence of SerB, more PMNs are recruited into the gingival tissues.One of the predominant polymicrobial infections of humans is expressed clinically as periodontal disease. A bacterial etiology for periodontal diseases is well established, and a group of Gram-negative, mostly anaerobic bacteria is associated with the initiation and progression of disease. Porphyromonas gingivalis is considered one of the more pathogenic members of this group, and elevated levels of this organism are associated with an increased risk of periodontal breakdown (4, 54). P. gingivalis produces a variety of virulence factors that enable colonization of the periodontal pocket and destruction of the structural components of the periodontium. These virulence factors include proteolytic enzymes that can damage host cells, tissues, and immune response mediators; toxic metabolites; surface components with immune-modulating activity; and adherence factors that promote colonization and persistence (7, 8, 15, 18, 21, 22, 35, 38). Moreover, the role of some of these virulence factors, such as fimbriae and proteases, has been verified in animal models of periodontal disease (36, 44, 46). P. gingivalis is also an intracellular organism that can invade gingival epithelial cells in culture (37, 39, 58). In addition, P. gingivalis has been observed within epithelial cells in ex vivo samples such as gingival biopsy samples (45, 49), and high numbers of P. gingivalis have been observed within gingival and buccal epithelial cells obtained from healthy and disease subjects (5, 50, 51). Mechanistically, P. gingivalis invasion is mediated through fimbria-mediated attachment to gingival epithelial cell integrin receptors and cytoskeletal rearrangements that allow the bacteria to enter the cell (64, 65). A key effector of the invasive process is SerB, a haloacid dehydrogenase family phosphoserine phosphatase enzyme. SerB is present in the outer membrane of P. gingivalis, and contact with gingival epithelial cells induces secretion into the extracellular milieu (67). Cell-associated and extracellular SerB impacts host cell signal transduction pathways that control the cytoskeletal architecture, and treatment of epithelial cells with SerB induces actin microfilament and tubulin microtubule rearrangements (19, 59). Furthermore, SerB is required for intracellular persistence, as a mutant lacking SerB is compromised in intracellular survival (59). An additional role of SerB concerns involvement in the stealth-like properties of P. gingivalis (18). P. gingivalis is capable of both inhibiting secretion of interleukin-8 (IL-8) from gingival epithelial cells and antagonizing IL-8 production stimulated by other organisms (9, 23, 26, 60). SerB activity is required for this innate immune suppression, known as localized chemokine paralysis (9, 19).While SerB is important for invasion, intracellular survival, and immune suppression in vitro, its contribution to in vivo pathogenicity has not been addressed. Study of the in vivo role of P. gingivalis virulence factors has employed a variety of animal models (16). Among these, the oral infection model in rodents has been used to study colonization, periodontal inflammation, immune responses, and induction of alveolar bone resorption by P. gingivalis (3, 29, 32). The pathological processes induced by oral infection with periodontal pathogens in rodent models, including gingival and periodontal ligament destruction and resorption of the alveolar bone, resemble those that occur in humans (34). In this study, we examine the in vivo role of SerB in colonization, inflammation, immune response, alveolar bone resorption, and induction of periodontal disease in rats.     

Negative Correlation of Distributions of Streptococcus cristatus and Porphyromonas gingivalis in Subgingival Plaque

Porphyromonas gingivalis is one of the major causative agents of adult periodontitis. One of the features of this periodontal pathogen is its ability to attach to a variety of oral bacterial surfaces and to colonize subgingival dental plaque. We have shown that Streptococcus cristatus CC5A inhibits expression of fimA, a gene encoding the major protein subunit of long fimbriae in P. gingivalis; as a result, S. cristatus interrupts formation of P. gingivalis biofilms. Here we further demonstrate that the inhibitory activity of S. cristatus affects multiple strains of P. gingivalis and that optimal inhibitory activity correlates with levels of arginine deiminase expression in S. cristatus. More strikingly, the impact of S. cristatus on P. gingivalis colonization was revealed by comparing levels of P. gingivalis and S. cristatus in subgingival dental plaque. Spearman correlation analysis indicated a negative correlation between the distributions of S. cristatus and P. gingivalis (r = −0.57; P < 0.05). These data suggest that some early colonizers of dental plaque, such as S. cristatus, may be beneficial to the host by antagonizing the colonization and accumulation of periodontal pathogens such as P. gingivalis.Periodontitis is a bacterial biofilm (dental plaque)-related infectious disease. Although over 750 oral bacterial taxa can be detected in the oral cavity, three gram-negative species, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola—known as the red complex—have strong associations with chronic periodontal disease (9, 28). It has been suggested that these gram-negative bacteria are later colonizers of dental plaques and are recruited to microbial communities by earlier colonizers, such as oral streptococci and Actinomyces species, via specific interactions of surface molecules (10). While there is no direct evidence that the earlier colonizers are associated with periodontitis, some of these organisms can provide a favorable environment for periodontal pathogens such as P. gingivalis. A well-studied interaction of earlier colonizers and later colonizers of dental plaque is coadhesion of Streptococcus gordonii and P. gingivalis. Specific adherence of P. gingivalis to S. gordonii strains was demonstrated in in vitro experiments (12). Studies by independent laboratories showed involvement of multiple sets of adhesins in the two bacteria. The first set of adhesins is the P. gingivalis long fimbriae (FimA) and glyceraldehyde-3-phosphate dehydrogenase present on the surfaces of streptococci (17). The second set involves the P. gingivalis short fimbriae (Mfa1) and the streptococcal SspA/B (antigen I/II) adhesins (4, 23). It is likely that these specific protein-protein interactions promote P. gingivalis colonization on existing biofilms consisting of S. gordonii and related oral streptococci (4, 27).We previously reported an antagonistic relationship between P. gingivalis and Streptococcus cristatus CC5A. P. gingivalis was unable to form microcolonies with S. cristatus, due to repression of fimA expression in the presence of S. cristatus (32). Moreover, the long fimbriae are important for aspects of P. gingivalis colonization. Previous studies have shown that a P. gingivalis strain with a fimA deficiency has a diminished capacity to adhere to human gingival fibroblasts and epithelial cells (6) and is deficient in invasion of epithelial cells (35). The fimA mutant also is less able to induce periodontal bone loss in a gnotobiotic rat model (18). Our recent study identified an S. cristatus surface protein, arginine deiminase (ArcA), responsible for eliciting repression of fimA expression in P. gingivalis 33277 (34). The arcA gene is found in a number of oral bacteria, mainly in streptococci (3). However, arcA is differentially expressed among oral streptococcal strains (16). Higher-level expression of arcA was observed in S. cristatus than in S. gordonii, which may contribute to the ability of the organism to prevent P. gingivalis colonization in the oral cavity. In this study, we tested an antagonistic role of S. cristatus in P. gingivalis colonization. We postulate that inhibition of FimA production in P. gingivalis by S. cristatus requires higher expression of ArcA and that colonization of S. cristatus strains expressing an elevated level of ArcA plays an important role in antagonizing P. gingivalis colonization.     

Effect of Lactobacillus acidophilus and Porphyromonas gingivalis on proliferation and apoptosis of gingival epithelial cells

PurposeThis study aimed to evaluate the possible antagonistic effects of Lactobacillus acidophilus on Porphyromonas gingivalis, and detect inhibition of Lactobacillus acidophilus on Porphyromonas gingivalis when they are co-cultured with human gingival epithelial cells.Materials and methodsHuman gingival epithelial cells were co-cultured with Lactobacillus acidophilus and Porphyromonas gingivalis alone or together. The amount of Porphyromonas gingivalis adhering to or invading the epithelial cells were determined by bacterial counts. The cellular proliferation was assayed by the MTT method. Apoptosis was detected by flow cytometry with apoptosis detection kit.ResultsOn one hand, Lactobacillus acidophilus reduced the inhibitory effect of Porphyromonas gingivalis on the human gingival epithelial cells proliferation in a dose dependent manner. On the other hand, Porphyromonas gingivalis induced significant apoptosis on human gingival epithelial cells, and Lactobacillus acidophilus inhibited this apoptosis-inducing effect of Porphyromonas gingivalis in a dose dependent manner.ConclusionsPorphyromonas gingivalis inhibits the proliferation and induces the apoptosis of human gingival epithelial cells. Lactobacillus acidophilus could attenuate this effect in a dose-dependent manner, and it thus reduces the destruction from pathogens. Lactobacillus acidophilus could be an effective candidate for probiotic therapy in periodontal diseases.     

Contact-Dependent Protein Secretion in Porphyromonas gingivalis

Porphyromonas gingivalis can induce its uptake by host epithelial cells; however, the nature and role of the P. gingivalis molecules involved in this invasion process have yet to be determined. In this study, modulation of secreted P. gingivalis proteins following association with gingival epithelial cells was investigated. Western immunoblot analysis showed that contact with epithelial cells or epithelial cell growth media induces P. gingivalis 33277 to secrete several proteins with molecular masses between 35 and 95 kDa. Secretion of the Arg-gingipain and Lys-gingipain proteases was repressed under these conditions. The contact-induced secreted protein profile was altered in Arg-gingipain-deficient and Lys-gingipain-deficient mutants, indicating a possible role for these proteases in the secretion pathway. The P. gingivalis contact-dependent protein secretion pathway differs to some extent from type III protein secretion pathways in enteric pathogens, as a gene homologous to the invA family genes was not detected in P. gingivalis. The secreted proteins of P. gingivalis may play a role in the interactions of the organism with host cells.     

Differential ability of periodontopathic bacteria to modulate invasion of human gingival epithelial cells by Porphyromonas gingivalis

Periodontitis is a polymicrobial infection caused by selected gram-negative bacteria including Porphyromonas gingivalis. Host cell invasion by P. gingivalis has been proposed as a possible mechanism of pathogenesis in periodontitis. The aim of the present study was to assess the influence of periodontopathogens on P. gingivalis invasion of gingival epithelial cells in polymicrobial infection. P. gingivalis was tested for its ability to invade a human gingival epithelial cell line Ca9-22 in co-infection with periodontopathogens, using an antibiotic protection assay. Among the pathogens tested, only Fusobacterium nucleatum demonstrated the ability to significantly promote P. gingivalis invasion (P < 0.01). This increased invasion was confirmed by confocal scanning laser microscopy utilizing a dual labeling technique. In contrast, co-infection with Aggregatibacter actinomycetemcomitans or Tannerella forsythia attenuated P. gingivalis invasion. The fusobacterial enhancement of host cell invasion was not observed in co-incubation with other periodontopathogens tested. These results suggested that complex synergistic or antagonistic physiologic mechanisms are intimately involved in host cell invasion by P. gingivalis in polymicrobial infection.     

Differential ability of periodontopathic bacteria to modulate invasion of human gingival epithelial cells by Porphyromonas gingivalis

Periodontitis is a polymicrobial infection caused by selected gram-negative bacteria including Porphyromonas gingivalis. Host cell invasion by P. gingivalis has been proposed as a possible mechanism of pathogenesis in periodontitis. The aim of the present study was to assess the influence of periodontopathogens on P. gingivalis invasion of gingival epithelial cells in polymicrobial infection. P. gingivalis was tested for its ability to invade a human gingival epithelial cell line Ca9-22 in co-infection with periodontopathogens, using an antibiotic protection assay. Among the pathogens tested, only Fusobacterium nucleatum demonstrated the ability to significantly promote P. gingivalis invasion (P < 0.01). This increased invasion was confirmed by confocal scanning laser microscopy utilizing a dual labeling technique. In contrast, co-infection with Aggregatibacter actinomycetemcomitans or Tannerella forsythia attenuated P. gingivalis invasion. The fusobacterial enhancement of host cell invasion was not observed in co-incubation with other periodontopathogens tested. These results suggested that complex synergistic or antagonistic physiologic mechanisms are intimately involved in host cell invasion by P. gingivalis in polymicrobial infection.     

Porphyromonas gingivalis RgpA-Kgp Proteinase-Adhesin Complexes Penetrate Gingival Tissue and Induce Proinflammatory Cytokines or Apoptosis in a Concentration-Dependent Manner

The RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis were observed, using immunostaining, in human gingival tissue associated with periodontitis but not in healthy tissue. The staining pattern suggested a concentration gradient from the subgingival plaque into the subjacent gingival connective tissue. Intense immunostaining was observed in areas displaying gross disturbance of tissue architecture. P. gingivalis cells and the RgpA-Kgp complexes at low concentrations were shown to stimulate secretory intercellular adhesion molecule 1, interleukin-8 (IL-8), IL-6, and macrophage chemoattractant protein secretion from cultured human epithelial (KB) and fibroblast (MRC-5) cells. However, at high concentrations a reduction in the level of these mediators was observed. In contrast, macrophage inflammatory protein 1α and IL-1α were stimulated only at high P. gingivalis cell concentrations. P. gingivalis cells and the RgpA-Kgp complexes were shown to induce apoptosis in KB and MRC-5 cells in a time- and dose-dependent manner. These data suggest that the RgpA-Kgp complexes penetrate the gingival connective tissue; at low concentrations distal from the plaque the complexes stimulate the secretion of proinflammatory mediators, while at high concentrations proximal to the plaque they induce apoptosis and attenuate the secretion of proinflammatory mediators.Chronic periodontitis is an inflammatory disease associated with specific bacteria in subgingival plaque that results in the destruction of the tooth''s supporting tissues. The presence of the three bacterial species Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as a consortium in subgingival plaque has been associated with chronic periodontitis, and of these bacteria, P. gingivalis is reported to be most closely associated with the severity of disease (51, 79). P. gingivalis produces extracellular complexes of proteinases and adhesins, designated the RgpA-Kgp complexes (or high-molecular-weight gingipains). Isogenic mutants of P. gingivalis lacking the RgpA-Kgp complexes are avirulent in an animal periodontitis model, and therefore the complexes have been proposed to be a major virulence factor for this bacterium (51, 56). The chronic interaction of the host immune system with subgingival plaque containing P. gingivalis and the RgpA-Kgp complexes in the subjacent tissue is believed to be a major factor in tissue destruction in chronic periodontitis (6, 21, 51, 83). Compared with healthy subjects, gingival tissues and gingival crevicular fluid of patients with chronic periodontitis are reported to have significantly increased amounts of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-α); chemokines such as IL-8, macrophage chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α); and adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1) (12, 25, 27, 28, 32, 33, 37, 44, 46, 62, 84, 87). These cytokines and chemokines play a significant role in mediating the recruitment of a dense mononuclear infiltrate, consisting mainly of T cells and macrophages (21, 22, 39, 55, 81). Furthermore, a concentration gradient of secretory ICAM-1 (sICAM-1) across the junctional epithelium is reported to be an important mechanism leading to leukocyte recruitment into the gingival sulcus (85). Assuma et al. (2) have reported that blocking IL-1 and TNF-α activity in a nonhuman primate model significantly reduced tissue destruction and alveolar bone loss. These studies suggest that the chronic presence of specific pathogens such as P. gingivalis in subgingival plaque results in the secretion of inflammatory mediators which, in turn, may cause inappropriate accumulation and activation of circulating and resident leukocytes at the site of infection, producing chronic inflammation and tissue destruction. Another contributing factor for periodontal tissue destruction is reported to be the induction of host cell apoptosis by specific subgingival plaque pathogens (5). In gingival biopsies from patients with chronic periodontitis, apoptotic cells have been reported to constitute about 10% of the total cell population and included epithelial and fibroblast cells (31, 36). Tonetti et al. (86) have reported that exposure of clinically healthy gingival tissues to plaque bacteria induces apoptosis-associated DNA damage and the expression of proapoptotic p53 protein. Furthermore, in gingival biopsies from patients with periodontitis, epithelial cell apoptosis was reported to be more prevalent in the most apical part of the sulcus closest to the subgingival plaque (31, 86). These observations suggest that interactions with certain bacterial products may play an important role in inducing apoptosis in gingival tissue cells. Furthermore, a high prevalence of apoptotic cells expressing the p53 protein was detected in the subgingival inflammatory infiltrate, suggesting that apoptotic cell death may be important in the regulation of the inflammatory response to chronic bacterial challenge (86).A number of prior studies have investigated the ability of P. gingivalis to induce secretion of proinflammatory mediators from oral epithelial and fibroblast cells. It has been reported that P. gingivalis induces expression of proinflammatory mediators such as IL-1β, IL-8, IL-6, and sICAM-1 from gingival epithelial or fibroblast cells (1, 70, 80, 93). However, in contrast, P. gingivalis cells have also been reported to degrade existing inflammatory cytokines and antagonize IL-1β, IL-8, IL-6, and sICAM-1 production by gingival epithelial or fibroblast cells (13, 16-18, 43). These apparent contradictions may be partially explained by the assay conditions related to factors such as bacterial strain variability and inclusion of human serum (38, 74, 80). However, we have postulated that this paradoxical situation where P. gingivalis both suppresses and induces the host immune response is a concentration-dependent phenomenon that is related to the level of P. gingivalis cells and the P. gingivalis major virulence factor, the RgpA-Kgp proteinase-adhesin complexes (51).In the present study, we demonstrate the presence of RgpA-Kgp in gingival tissue associated with periodontitis, and we investigate the hypothesis that P. gingivalis W50 whole cells and RgpA-Kgp complexes differentially regulate the expression of proinflammatory mediators and the induction of apoptosis in human epithelial and fibroblast cells in a concentration-dependent fashion.     

Hemin-binding protein 35 (HBP35) plays an important role in bacteria–mammalian cells interactions in Porphyromonas gingivalis

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.     

Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis

Although Candida albicans has been isolated from periodontal pockets, its relationship to periodontitis is unclear. In this study, we investigated the effect of C. albicans on the adhesion and invasion of Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts by Porphyromonas gingivalis. Heat-killed C. albicans and water-soluble mannoprotein-β-glucan complex from C. albicans (CAWS) did not enhance P. gingivalis adhesion or upregulate the expression of β1 integrin and ICAM-1, which are required for P. gingivalis invasion; both the epithelial cells and fibroblasts expressed dectin-1, which recognizes components of the C. albicans cell wall. However, pretreatment of Ca9-22 cells and human gingival fibroblasts with heat-killed C. albicans or CAWS significantly enhanced P. gingivalis invasion. These results suggest that C. albicans may exacerbate infectious disease by enhancing the invasion of host cells by anaerobic bacteria.     

P2X7 receptor-mediated leukocyte recruitment and Porphyromonas gingivalis clearance requires IL-1β production and autocrine IL-1 receptor activation

The Gram-negative bacterium Porphyromonas gingivalis is strongly associated with periodontitis. We previously demonstrated that P2X7 receptor activation by extracellular ATP (eATP) triggers elimination of intracellular pathogens, such as Leishmania amazonensis, Toxoplasma gondii and Chlamydia trachomatis. We also showed that eATP-induced IL-1β secretion via the P2X7 receptor is impaired by P. gingivalis fimbriae. Furthermore, enhanced P2X7 receptor expression was detected in the maxilla of P. gingivalis-orally infected mice as well as in human periodontitis patients. Here, we examined the effect of P2X7-, caspase-1/11- and IL-1 receptor-mediated responses during P. gingivalis infection. P2X7 receptor played a large role in controlling P. gingivalis infection and P. gingivalis-induced recruitment of inflammatory cells, especially neutrophils. In addition, IL-1β secretion was detected at different time points only when P2X7 receptor was expressed and in the presence of eATP treatment ex vivo. Activation of P2X7 receptor and IL-1 receptor by eATP and IL-1β, respectively, promoted P. gingivalis elimination in macrophages. Interestingly, eATP-induced P. gingivalis killing was inhibited by the IL-1 receptor antagonist (IL-1RA), consistent with autocrine activation of the IL-1 receptor for P. gingivalis elimination. In vivo, caspase-1/11 and IL-1 receptor were also required for bacterial clearance, leukocyte recruitment and IL-1β production after P. gingivalis infection. Our data demonstrate that the P2X7-IL-1 receptor axis activation is required for effective innate immune responses against P. gingivalis infection.     

Diverse effects of Porphyromonas gingivalis on human osteoclast formation

Porphyromonas gingivalis is associated with periodontitis, a chronic inflammatory disease of the tooth-supporting tissues. A major clinical symptom is alveolar bone loss due to excessive resorption by osteoclasts. P. gingivalis may influence osteoclast formation in diverse ways; by interacting directly with osteoclast precursors that likely originate from peripheral blood, or indirectly by activating gingival fibroblasts, cells that can support osteoclast formation. In the present study we investigated these possibilities.Conditioned medium from viable or dead P. gingivalis, or from gingival fibroblasts challenged with viable or dead P. gingivalis were added to human mononuclear osteoclast precursors. After 21 days of culture the number of multinucleated (≥3 nuclei) tartrate resistant acid phosphatase (TRACP)-positive cells was determined as a measure for osteoclast formation.Conditioned medium from viable P. gingivalis, and from fibroblasts with viable P. gingivalis stimulated osteoclast formation (1.6-fold increase p < 0.05). Conditioned medium from dead bacteria had no effect on osteoclast formation, whereas conditioned medium from fibroblasts with dead bacteria stimulated formation (1.4-fold increase, p < 0.05). Inhibition of P. gingivalis LPS activity by Polymyxin B reduced the stimulatory effect of conditioned medium. Interestingly, when RANKL and M-CSF were added to cultures, conditioned media inhibited osteoclast formation (0.6-0.7-fold decrease, p < 0.05).Our results indicate that P. gingivalis influences osteoclast formation in vitro in different ways. Directly, by bacterial factors, likely LPS, or indirectly, by cytokines produced by gingival fibroblasts in response to P. gingivalis. Depending on the presence of RANKL and M-CSF, the effect of P. gingivalis is either stimulatory or inhibitory.     

Noncanonical Activation of β-Catenin by Porphyromonas gingivalis

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.     

Beyond Toll-Like Receptors: Porphyromonas gingivalis Induces IL-6, IL-8, and VCAM-1 Expression Through NOD-Mediated NF-κB and ERK Signaling Pathways in Periodontal Fibroblasts

To investigate whether oligomerization domains (NODs) are involved in Porphyromonas gingivalis-induced interleukin (IL)-6, IL-8, and vascular cell adhesion molecule (VCAM)-1 expression beyond Toll-like receptors (TLRs), we investigated the role of NOD1/2 in P. gingivalis-induced IL-6, IL-8, and VCAM-1 expression in human gingival fibroblasts (hGFs) and periodontal ligament cells (hPDLCs). The mechanism was explored by activation and silence of NODs, electrophoretic mobility shift assay (EMSA), and pathway blockade assays. Results showed that P. gingivalis could induce NOD1, NOD2, IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLs at mRNA and protein levels. Activation of NOD1/2 by agonists could clearly upregulate the expression of these genes, while silence of NOD1/2 could remarkably attenuate them. EMSA and blockade of NF-κB and extracellular-signal-regulated kinase (ERK)1/2 pathway assays also verified that the two pathways were involved in NOD1/2-mediated IL-6, IL-8, and VCAM-1 expression. In conclusion, our findings demonstrated that P. gingivalis induced IL-6, IL-8, and VCAM-1 expression in hGFs and hPDLCs through NOD1/2-mediated NF-κB and ERK1/2 signaling pathways beyond TLRs.     

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.     

Proteome Analysis of Coinfection of Epithelial Cells with Filifactor alocis and Porphyromonas gingivalis Shows Modulation of Pathogen and Host Regulatory Pathways

Changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. The relative abundances of several newly recognized microbial species, including Filifactor alocis, as-yet-unculturable organisms, and other fastidious organisms have raised questions on their impact on disease development. We have previously reported that the virulence attributes of F. alocis are enhanced in coculture with Porphyromonas gingivalis. We have evaluated the proteome of host cells and F. alocis during a polymicrobial infection. Coinfection of epithelial cells with F. alocis and P. gingivalis strains showed approximately 20% to 30% more proteins than a monoinfection. Unlike F. alocis ATCC 35896, the D-62D strain expressed more proteins during coculture with P. gingivalis W83 than with P. gingivalis 33277. Proteins designated microbial surface component-recognizing adhesion matrix molecules (MSCRAMMs) and cell wall anchor proteins were highly upregulated during the polymicrobial infection. Ultrastructural analysis of the epithelial cells showed formation of membrane microdomains only during coinfection. The proteome profile of epithelial cells showed proteins related to cytoskeletal organization and gene expression and epigenetic modification to be in high abundance. Modulation of proteins involved in apoptotic and cell signaling pathways was noted during coinfection. The enhanced virulence potential of F. alocis may be related to the differential expression levels of several putative virulence factors and their effects on specific host cell pathways.