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Background: Periodontitis is a common chronic inflammatory disorder that affects supporting tissues of the teeth. An increased risk of pregnancy complications has been reported in patients with periodontitis; however, its pathophysiology remains uncharacterized to date. In addition, Porphyromonas gingivalis (Pg) is detectable in a few placentae derived from diseased pregnancies. Thus, the aim of this study is to examine the roles of soluble factors produced by Pg on trophoblast invasion in vitro to clarify the remote effects of periodontitis on pregnancy outcomes., Methods: The immortalized trophoblast cell line HTR-8/SVneo was cultured on plates or via hanging drop to evaluate viability, apoptosis, and morphology in the presence of the culture supernatant of Pg (PG-sup). Cells were plated on solubilized extracellular matrix rich membrane preparation plates to evaluate cell invasion. Morphologic changes were evaluated via stereomicroscopy, optical microscopy, and transmission electron microscopy., Results: After 24 hours of culture, cell invasion was inhibited by PG-sup in a dose-dependent manner. Although cell viability and apoptotic counts were not affected by PG-sup, spheroid formation in hanging drop culture was inhibited. Spheroids became fragile and irregular in the presence of PG-sup. Transmission electron microscopy revealed shortened microvilli and increased intracellular spaces., Conclusions: PG-sup inhibits trophoblast invasion and affects trophoblast morphology without direct cytotoxicity. These results indicate that Pg produces soluble factor(s) that suppress trophoblast invasion and subsequent vascular remodeling, which affect placental growth and fetal well-being. It is expected that the current findings will explain the increased prevalence of pregnancy complications in patients with periodontitis.

Porphyromonas gingivalis requires heme to grow, however, heme availability and concentration in the periodontal pockets vary. Fluctuations in heme concentration may affect each P. gingivalis strain differently, however, this was never fully demonstrated. Here, we elucidated the effects of varying hemin concentrations in representative P. gingivalis strains. Throughout this study, representative P. gingivalis strains [FDC381 (type I), MPWIb-01 (type Ib), TDC60 (type II), ATCC49417 (type III), W83 (type IV), and HNA99 (type V)] were used and grown for 24 h in growth media under varying hemin concentrations (5 × , 1 × , 0.5 × , 0.1 × ). Samples were lysed and protein standardized. Arg-gingipain (Rgp), H2O2, and superoxide dismutase (SOD) levels were subsequently measured. We focused our study on 24 h-grown strains which excluded MPWIb-01 and HNA99. Rgp activity among the 4 remaining strains varied with Rgp peaking at: 1 × for FDC381, 5 × for TDC60, 0.5 × for ATCC49417, 5 × and 0.5 × for W83. With regards to H2O2 and SOD amounts: FDC381 had similar H2O2 amounts in all hemin concentrations while SOD levels varied; TDC60 had the lowest H2O2 amount at 1 × while SOD levels became higher in relation to hemin concentration; ATCC49417 also had similar H2O2 amounts in all hemin concentrations while SOD levels were higher at 1 × and 0.5 × ; and W83 had statistically similar H2O2 and SOD amounts regardless of hemin concentration. Our results show that variations in hemin concentration affect each P. gingivalis strain differently., (Published by Elsevier Ltd.)

Objective: A number of studies have recently suggested Epstein-Barr virus (EBV) involvement in the pathogenesis of periodontitis. In this study, we investigated the association between major periodontopathic bacteria Porphyromonas gingivalis (P. gingivalis) and EBV in Japanese chronic periodontitis (CP) patients., Materials and Methods: A group of 25 patients with CP participated in the study along with 13 individuals without periodontitis. Subgingival samples were obtained with paper points. Quantitative real-time polymerase chain reaction (PCR) was used to detect EBV DNA and P. gingivalis., Results: In the CP patients, EBV DNA and P. gingivalis were detected in both 80 % of sites with probing pocket depths (PPD) of ≥5 mm and in 40 and 36 % of sites with PPD ≤3 mm, respectively. EBV DNA and P. gingivalis were detected in 50 and 27 % of the sites in periodontally healthy individuals. Coexistence of EBV DNA and P. gingivalis was significantly higher in the deeper PPD sites of CP patients (68 %) than in the PPD sites of the healthy controls (15 %) and shallow PPD sites of CP patients (12 %). PCR-positive deeper PPD sites of CP patients for EBV DNA and P. gingivalis range between 3.74 × 10(3)∼2.83 × 10(9) and 2.73 × 10(5)∼6.65 × 10(9) (copies/ml), respectively., Conclusion: These results suggest an association between EBV DNA, P. gingivalis, and CP in Japanese individuals. Further studies are required to clarify this association; however, we believe that our enhanced understanding of the pathogenesis of periodontal diseases involving viral infections will lead to new treatments.

Porphyromonas gingivalis FimA is a major aetiological agent in periodontal disease development, however, its structure has never been determined. Here, we established the mature P. gingivalis FimA ab initio model of all six FimA variants. We determined the conserved amino acid sequences of each FimA variant and generated mature FimA models. Subsequently, we validated their quality, protein empirical distribution, and radius of gyration. Similarly, structural comparison and topological orientation were elucidated, and the probable protein-protein docking was investigated. We found that the putative mature FimA model is β-sheet-rich and, likewise, we observed that each mature FimA model has varying levels of structural differences which can be topologically subdivided into the upper, middle, and lower FimA sections. Moreover, we found that the FimA epithelial cell-binding domain (EBD) is structurally conserved within the middle FimA section of all variants and FimA-FimA docking suggests that the FimA EBDs are oriented in opposite and alternating directions of each other., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis sialidase activity is associated with virulence and initiated by sialic acid (SA) binding to the β-propeller domain (BPD). Sialidase BPD is structurally conserved in various bacterial species and the protein binding interfaces have the tendency to form salt bridges, whereas uncommitted charged residues may affect binding and protein structure. However, it is not clear whether the sialidase BPD of varying strains of the same bacterial species differ, particularly with regards to salt bridge formation. Here, we determined the P. gingivalis ATCC 33277 and W50 sialidase homology models and sialidase activities, while the putative salt bridge residues found in the sialidase BPDs were compared. We established that both ATCC 33277 and W50 have different sialidase homology models and activities, whereas, the BPD (β1-6) is structurally conserved with most salt bridge-forming residues following a common orientation. Moreover, β2D444-β6K338 distance measurement in ATCC 33277 (5.99 Å) and W50 (3.09 Å) differ, while β1K396A substitution alters the β2D444-β6K338 distance measurements in ATCC 33277 (3.09 Å) and W50 (3.01 Å) consequentially affecting each model. P. gingivalis plays a major role in periodontitis induction and its virulence is greatly influenced by the sialidase enzyme wherein the sialidase BPD is highly conserved. Our results suggest that alterations in the salt bridge formation within the BPD interface may affect the P. gingivalis sialidase structure. This would imply that disrupting the salt bridge formation within the P. gingivalis sialidase BPD could serve as a potential therapeutic strategy for the treatment of P. gingivalis-related periodontitis.

Porphyromonas gingivalis requires optimal hemin to grow while non-optimal hemin hampers growth. Hemin induces H2O2 production while H2O2 has a dual function. In P. gingivalis ATCC 33277, we found similar physiological effects under hemin-excess and hemin-limited concentrations which we propose is related to two different functions of the H2O2 molecule., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis., (© 2013.)

A wide variety of infections, including bacteria, viruses, fungi and protozoa occur in the immunocompromised condition associated with human immunodeficiency virus type 1 (HIV-1) infection and acquired immunodeficiency syndrome (AIDS). Although these opportunistic infections are believed to arise as an effect of the immunodeficiency, these microbes sometimes promote the disease progression of HIV-1 infection by enhancing viral replication or modulating host immune responses. Here we review the experimental and clinical evidence supporting such causal relationships associated with periodontogenic bacteria. Periodontal disease, caused by subgingival infection with oral anaerobic bacteria, typically Porphyromonas gingivalis (P. gingivalis) belonging to the phylum Bacteroidetes, is found worldwide and is one of the most prevalent microbial diseases of mankind. Emerging evidence implicates the involvement of P. gingivalis infection in the progression of HIV-1 infection. We demonstrate that P. gingivalis can induce HIV-1 reactivation via chromatin modification, and that the bacterial metabolite butyric acid produced in anaerobic conditions is responsible for this effect. These findings suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to AIDS progression. Furthermore, it would imply that prevention and early treatment of periodontitis involving P. gingivalis infection could effectively block further clinical progression of AIDS.

Helicobacter pylori can be found in the oral cavity and is mostly detected by the use of PCR techniques. Growth of H. pylori is influenced by various factors in the mouth, such as the oral microflora, saliva and other antimicrobial substances, all of which make colonization of the oral cavity by H. pylori difficult. In the present study, we analysed the effect of the cell supernatant of a representative periodontal bacterium Porphyromonas gingivalis on H. pylori and found that the cell supernatant destroyed the H. pylori cell envelope. As P. gingivalis produces butyric acid, we focused our research on the effects of butyrate and found that it significantly inhibited the growth of H. pylori. H. pylori cytoplasmic proteins and DNA were detected in the extracellular environment after treatment with butyrate, suggesting that the integrity of the cell envelope was compromised and indicating that butyrate has a bactericidal effect on H. pylori. In addition, levels of extracellular H. pylori DNA increased following treatment with the cell supernatant of butyric acid-producing bacteria, indicating that the cell supernatant also has a bactericidal effect and that this may be due to its butyric acid content. In conclusion, butyric acid-producing bacteria may play a role in affecting H. pylori colonization of the oral cavity.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that usually results in latent infection of B cells. The EBV BZLF1 gene product ZEBRA is a master regulator of the transition from latency to the lytic replication cycle. In the latent state, hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases (HDACs) is primarily involved in maintaining EBV latency. Although the mechanism that regulates the switch between latency and lytic replication has been a central research focus in EBV infection, the causal link between HDAC inhibition and the disruption of viral latency is not well understood. Periodontal disease is a complex chronic inflammatory disease caused by subgingival infection with oral anaerobic bacteria, typically Porphyromonas gingivalis. Periodontal disease occurs worldwide and is among the most prevalent microbial diseases in humans. In this study, we examined the biological effect of P. gingivalis infection on EBV reactivation and found that P. gingivalis induced expression of ZEBRA. This activity was associated with supernatant from bacterial culture, but not with other bacterial components such as lipopolysaccharide or fimbriae. We demonstrated that culture supernatant from P. gingivalis, which contained high concentrations of butyric acid, inhibited HDACs, thus increasing histone acetylation and the transcriptional activity of the BZLF1 gene. Chromatin immunoprecipitation assays revealed that HDACs were present in the BZLF1 promoter during latent state and that they were dissociated from the promoter concomitantly with the association of acetylated histone H3, upon stimulation by culture supernatant from P. gingivalis. Thus, P. gingivalis induced EBV reactivation via chromatin modification, and butyric acid-a bacterial metabolite-was responsible for this effect. These findings suggest that periodontal disease is a risk factor for EBV reactivation in infected individuals and might therefore contribute to progression of EBV-related diseases., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Although approximately 200 distinct cell types - including fibroblasts, neurons, and hematopoietic cells - possess the same DNA sequence, they have diverse functions in humans and exhibit considerably different gene expression patterns. It has become increasingly clear that epigenetic regulation plays an important role in gene expression. There are two major forms of epigenetic regulation: posttranslational modification of DNA-associated histone proteins in chromatin and methylation of DNA. These forms are regulated by distinct but coupled pathways. Notably, histone Lys acetylation by histone acetyltransferase and deacetylation by histone deacetylases play a crucial role in on-off regulation of gene expression. It is now understood that epigenetics plays an important role not only in the regulation of gene expression but also in the pathogenesis of a broad range of diseases such as cancer and microbial infections. We have determined that epigenetic regulation is involved in the establishment and maintenance of HIV-1 latency and in the reactivation of HIV-1 by periodontopathic bacteria. In this review, we focus on the effect of histone modification on transcriptional regulation and the contribution thereof to the regulation of HIV-1 gene expression during the lytic and latent stages of HIV-1 infection. Likewise, we discuss the mechanisms by which periodontal diseases may accelerate AIDS progression in infected individuals as a new systemic disease caused by periodontitis and describe potential therapeutic interventions based on epigenetic mechanisms.

We previously reported that mutation of galE in Porphyromonas gingivalis has pleiotropic effects, including a truncated lipopolysaccharide (LPS) O-antigen and deglycosylation of the outer membrane protein OMP85 homolog. In the present study, further analysis of the galE mutant revealed that it produced little or no outer membrane vesicles (OMVs). Using three mouse antisera raised against whole cells of the P. gingivalis wild type strain, we performed ELISAs to examine the reactivity of these antisera with whole cells of the wild type or the galE mutant. All three antisera had significantly lower reactivity against the galE mutant compared to wild type. OMVs, but not LPS, retained the immunodominant determinant of P. gingivalis, as determined by ELISAs (with wild type LPS or OMVs as antigen) and absorption assays. In addition, we assessed the capacity of OMVs as a vaccine antigen by intranasal immunization to BALB/c mice. Synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [Poly (I∶C)], an agonist of Toll-like receptor 3 (TLR3), was used as the mucosal adjuvant. Vaccination with OMV elicited dramatically high levels of P. gingivalis-specific IgA in nasal washes and saliva, as well as serum IgG and IgA. In conclusion, the OMVs of P. gingivalis have an important role in mucosal immunogenicity as well as in antigenicity. We propose that P. gingivalis OMV is an intriguing immunogen for development of a periodontal disease vaccine.

Latently infected cells harbor the HIV-1 proviral DNA genome primarily integrated into heterochromatin, allowing the persistence of transcriptionally silent proviruses. Hypoacetylation of histone proteins by histone deacetylases (HDAC) is involved in the maintenance of HIV-1 latency by repressing viral transcription. In addition, periodontal diseases, caused by polymicrobial subgingival bacteria including Porphyromonas gingivalis, are among the most prevalent infections of mankind. Here we demonstrate the effects of P. gingivalis on HIV-1 replication. This activity could be ascribable to the bacterial culture supernatant but not to other bacterial components such as fimbriae or LPS. We found that this HIV-1-inducing activity was recovered in the lower molecular mass (<3 kDa) fraction of the culture supernatant. We also demonstrated that P. gingivalis produces high concentrations of butyric acid, acting as a potent inhibitor of HDACs and causing histone acetylation. Chromatin immunoprecipitation assays revealed that the corepressor complex containing HDAC1 and AP-4 was dissociated from the HIV-1 long terminal repeat promoter upon stimulation with bacterial culture supernatant concomitantly with the association of acetylated histone and RNA polymerase II. We thus found that P. gingivalis could induce HIV-1 reactivation via chromatin modification and that butyric acid, one of the bacterial metabolites, is responsible for this effect. These results suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to the systemic dissemination of the virus.

Background/aim: We investigated the mechanisms of adherence of salivary and serum proteins, which mimic gingival crevicular fluid (GCF), to Porphyromonas gingivalis, and the effects of these adhered proteins on coaggregation and hemagglutination properties., Methods: The amounts of salivary and serum proteins adhering to P. gingivalis were determined using (3)H-labeled and non-labeled proteins. The coaggregation between P. gingivalis and Streptococcus oralis or Streptococcus gordonii was observed. Hemagglutination was evaluated using sheep erythrocytes. Proteins that interacted with zinc or copper in saliva and serum and on P. gingivalis were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis., Results: The amount of salivary or serum proteins that adhered to the surface of P. gingivalis strains was increased by cations, especially zinc and copper ions. The pretreatment of bacterial cells with salivary or serum proteins before the assay inhibited coaggregation with gram-positive bacteria and hemagglutination. These phenomena were enhanced by the presence of zinc or copper ions during the pretreatment of P. gingivalis with proteins. We detected protein bands that were related to these cations in saliva and serum and on P. gingivalis., Conclusions: Our findings suggest that zinc and copper ions markedly enhanced the adhesion and accumulation of salivary and serum proteins on cells of P. gingivalis and inhibited the coaggregation and hemagglutination of P. gingivalis. These cations might be useful for limiting the settlement of P. gingivalis in the gingival sulcus with the goal of preventing periodontal disease.

OMP85 is a highly conserved outer membrane protein in all Gram-negative bacteria. We studied an uncharacterized OMP85 homolog of Porphyromonas gingivalis, a primary periodontal pathogen forming subgingival plaque biofilms. Using an outer-loop peptide antibody specific for the OMP85 of P. gingivalis, loop-3 Ab, we found a difference in the mobility of OMP85 on SDS-PAGE gel between the P. gingivalis wild-type and the isogenic galE mutant, a deglycosylated strain, suggesting that OMP85 naturally exists in a glycosylated form. This was also supported by a shift in OMP85 PAGE mobility after chemical deglycosylation treatment. Further, loop-3 Ab cross-reacted with the galE mutant stronger than the wild-type strain; and could inhibit biofilm formation in the galE mutant more than in the wild-type strain. In conclusion, this is the first report providing the evidence of OMP85 glycosylation and the involvement of OMP85 in biofilm formation.

Purpose: The present study was undertaken to determine the presence of 3 periodontopathic bacteria in the supragingival plaques of 3- to 16-year-old children with different oral health conditions., Methods: DMFT and dft, PMA index (P=papillary gingivitis, M=marginal gingivitis, and A=attached gingivitis), OHI (oral hygiene index), and oral malodor of each subject were determined prior to the collection of supragingival plaques. Periodontopathic bacteria (P. gingivalis, P. intermedia, and A. actinomycetemcomitans) in supragingival plaques were detected using an immunoslot blot assay with monoclonal 3 periodontopathic bacteria in the 2 subject groups (children with and without caries). P. gingivalis-positive subjects, but not their P. intermedia or A. actinomycetemcomitans counterparts, were correlated to oral malodor. Oral malodor was also correlated to debris index, a component of OHI., Results: The group with the higher OHI showed a higher prevalence of periodontopathic bacteria. For the 3 periodontopathic bacteria in the subjects tested, P. gingivalis-, P. intermedia-, and A. actinomycetemcomitans-positive plaques were not age related., Conclusions: The supragingival plaques in children can harbor periodontopathic bacteria such as P. gingivalis, P. intermedia, and A. actinomycetemcomitans.

A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity.

Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.