ABSTRACT The oral microbiome comprises several hundreds of bacterial species that contribute to periodontitis, the most complex polymicrobial inflammatory disorder. Porphyromonas gingivalis is a prominent periodontitis pathogen that produces gingipains as a major virulent factor. Gingipain facilitates P. gingivalis survival, pathogenicity, and growth. Several genes were identified to have a role in the regulating of P. gingivalis pathogenesis. Studies suggest that gingipains inhibition is key for the successful treatment of periodontitis. As of now, several gingipain inhibitors have been developed, some exhibit high inhibition activity against gingipains. However, most inhibitors offer unknown toxicity and undesirable side effects. Hence, the development of highly potent and safe gingipain inhibitor is a major concern for periodontitis treatment. The present review highlights the connectivity between P. gingivalis, virulent factors, and its gene, periodontitis, and gingipain inhibitors. Development of gingipains inhibitors would not only treat periodontitis but would also assist in the treatment of other associated systemic diseases, for example: rheumatoid arthritis, cardiovascular diseases, diabetes, and Alzheimer's disease.
Porphyromonas gingivalis--pathogenicity ; Gingipain Cysteine Endopeptidases

Fimbriae Proteins ; genetics ; Genotype ; Humans ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Virulence

OBJECTIVETo identify the differential genes in Porphyromonas gingivalis (P.gingivalis) highly toxic strain W83 and minimally toxic strain ATCC 33277.

METHODSUsing suppression subtractive hybridization (SSH) to compare P.gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 (driver). The chromosomal DNAs were purified from P.gingivalis W83 and P.gingivalis ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T vector and transformed to competent cells E.coli JM109. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically.

RESULTSSubtractive library which had high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88 bp to 372 bp were enriched in P.gingivalis highly toxic strain W83 sequences which were absent from P.gingivalis ATCC 33277. Through dot blot analysis confirmed that all these fragments were present in P.gingivalis W83 but absent from ATCC 33277. The GenBank homology search indicated that among them, several genes were associated with two paralogous regions of the chromosome; Some genes are associated with evasion of P.gingivalis W83; Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides.

CONCLUSIONSComparative whole-genome analysis of highly toxic and minimally toxic strains of P.gingivalis has identified the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.

Genes, Bacterial ; Genome, Bacterial ; Nucleic Acid Hybridization ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Sequence Analysis, DNA ; Virulence ; genetics

Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.
Animals ; Colitis, Ulcerative/microbiology* ; Mice ; Mice, Inbred C57BL ; Porphyromonas gingivalis/pathogenicity* ; Protein-Arginine Deiminases ; Virulence Factors

OBJECTIVETo investigate the adhesive and invasive ability of four common periodontal pathogens, Pg33277, Pi25611, Aa29522 and Fn10953 in human umbilical vein endothelial cells (HUVEC).

METHODSThe model of infection of HUVEC by periodontal pathogens was established in vitro. The invasive ability of four periodontal pathogens in HUVEC was tested by scanning electron microscope (SEM) and antibiotic protection assays-colony-forming units (CFU).

RESULTSAll of the four periodontal pathogens were found to adhere to HUVEC by SEM and invaded HUVEC at invasion numbers of (0.8 +/- 0.1) x 10(8), (4.1 +/- 0.5) x 10(6), (1.6 +/- 0.3) x 10(6) and (5.0 +/- 0.4) x 10(6) CFU/L respectively by antibiotic protection assays-CFU. The invasion efficiencies were (0.400 +/- 0.050)%, (0.021 +/- 0.003)%, (0.008 +/- 0.002)% and (0.025 +/- 0.002)%, respectively. The invasive ability of Pg33277 was significantly greater than those of the other three periodontal pathogens (P < 0.001). There was no difference in invasive abilities among Pi25611, Aa29522 and Fn10953 (P > 0.05).

CONCLUSIONSAll of the four common periodontal pathogens, Pg33277, Pi25611, Aa29522 and Fn10953 could adhere to and invaded HUVEC, with Pg33277 being the strongest.

Aggregatibacter actinomycetemcomitans ; pathogenicity ; ultrastructure ; Bacterial Adhesion ; Cells, Cultured ; Fusobacterium nucleatum ; pathogenicity ; ultrastructure ; Human Umbilical Vein Endothelial Cells ; cytology ; microbiology ; Humans ; Microscopy, Electron, Scanning ; Porphyromonas gingivalis ; pathogenicity ; ultrastructure ; Prevotella intermedia ; pathogenicity ; ultrastructure

BACKGROUNDThe occurrence and development of aortic aneurysm (AA) are associated with infection. Some researchers have detected the DNA of periodontal pathogens in AA samples in certain populations. However, it has not been done in Chinese population. The objective of this study was to evaluate the prevalence of periodontal pathogens in oral tissue samples and aneurysm samples of AA patients.

METHODSEighty-nine subjects with AA and 59 subjects without AA were examined. Periodontal clinical parameters were evaluated. Unstimulated saliva and subgingival plaque samples were collected from all subjects. Twenty-six dissected AA samples were obtained. Evidence of eight periodontal pathogens including Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Tannerella forsythensis (Tf), Treponema denticola (Td), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), and Prevotella nigrescens (Pn) was ascertained in all samples by 16S rRNA-based polymerase chain reaction (PCR) assay.

RESULTSThe periodontal indexes including plaque index (PLI), probing depth (PD), bleeding index (BI), and clinical attachment loss (CAL), of the six Ramfjord index teeth were significantly higher in the AA group than those in the control group (P < 0.01). Eight periodontal pathogens in subgingival plaque samples were more frequently detected in the AA group than in control group. The difference in prevalence between the groups was significant for six (out of eight) periodontal pathogens assayed (Pg, Pi, Fn, Pn, Tf, and Td, P < 0.01). Additionally, all eight periodontal pathogens were more frequently detected in saliva samples of the AA group than in those of the control group, again with six (out of eight) (Pg, Pi, Fn, Cr, Tf, and Td) displaying significant differences in prevalence between the two groups (P < 0.01). Out of 26 aneurysm samples examined, Pg, Pi, Fn, Cr and Tf were detected in 6 (23.1%), 2 (7.7%), 3 (11.5%), 1 (3.8%), 2 (7.7%), respectively, and Aa, Pn, and Td were not detected in dissected aneurysm samples.

CONCLUSIONResults of this study suggested that periodontal infection is associated with the occurrence of AA.

Aged ; Aggregatibacter actinomycetemcomitans ; genetics ; pathogenicity ; Aortic Aneurysm ; microbiology ; Female ; Humans ; Male ; Middle Aged ; Periodontitis ; microbiology ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Prevotella intermedia ; genetics ; pathogenicity ; RNA, Ribosomal, 16S ; genetics ; Treponema denticola ; genetics ; pathogenicity

OBJECTIVETo evaluate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) expression in human periodontal ligament cells (HPDLC) and the effect of Pg on bone resorption in periodontitis.

METHODSHPDLC were exposed to 25, 50 mg/L sonicated extracts of Pg for 6 h, HPDLC without treatment served as control. The expression of RANKL-OPG mRNA and protein were examined by real time polymerase chain reaction and Western blotting. OPG protein in the supernatant was examined by enzyme linked immunosorbent assay (ELISA). The data were statistically analyzed by SPSS 13.0 and one-way analysis of variance (ANOVA).

RESULTSWhen HPDLC were exposed to sonicated extracts of Pg, the expression of RANKL mRNA and protein in 25 mg/L and 50 mg/L groups were higher than that of control group (P < 0.05), the expression of OPG mRNA in 50 mg/L group (0.087 ± 0.021) was lower than that of control group (0.240 ± 0.019) (P < 0.05), and OPG protein in 25 mg/L and 50 mg/L groups (0.813 ± 0.007, 0.398 ± 0.009) was lower than that of control group (1.131 ± 0.005) (P < 0.01). OPG protein expression in the supernatant was not significantly different between experimental group and control group.

CONCLUSIONSSonicated extracts of Pg exposed to HPDLC can up-regulate RANKL expression, down-regulate OPG expression and influence bone metabolism.

Adult ; Cells, Cultured ; Humans ; Osteoprotegerin ; genetics ; metabolism ; Periodontal Ligament ; cytology ; metabolism ; Porphyromonas gingivalis ; pathogenicity ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Sonication ; Young Adult

OBJECTIVETo analyse the genetic polymorphism of Arg-gingipainB (rgpB), a virulent factors of Porphyromonas gingivalis (P.gingivalis) and discuss the role of the different genotypes in the genesis and progress of chronic periodontitis.

METHODSA total of 104 subgingival plaque samples were included in this study. The extracted DNA was amplified with the primers designed to obtain the gene encoding the catalytic domain of rgpB (rgpB-cd), P.gingivalis was typed into four genotypes by restriction fragment length polymorphism (RFLP).

RESULTSIn lesion site, the detection rate of type IV was the highest (52.78%), which was higher than those of type I and III (P < 0.05 or P < 0.01). While in non-lesion site, The detection rate of type II was the highest (75.86%), which was higher than those of other types (P < 0.01).

CONCLUSIONSThe polymorphism of rgpB-cd gene may influence the virulence of P.gingivalis. P.gingivalis type IV may be well related to periodontitis, while type II may be a indigenous flora.

Adhesins, Bacterial ; genetics ; Adult ; Aged ; Chronic Periodontitis ; microbiology ; Cysteine Endopeptidases ; genetics ; Dental Plaque ; microbiology ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Porphyromonas gingivalis ; genetics ; pathogenicity

OBJECTIVETo study the role of fibrinogen molecule in the pathogenesis of periodontal diseases.

METHODSAn in vitro cell culture model was used. Methyl-(3)H Thymidine radiolabeled Porphyromonas gingivalis (Pg) ATCC 33277 were examined for their ability to adhere to and invade the confluent monolayers of human oral epithelial KB cells with or without exogenous human fibrinogens by scintillation spectrometry.

RESULTSThe addition of exogenous fibrinogens made more amount of and higher ratios of adhesive and invasive Pg, in contrast to the group without exogenous fibrinogen (P < 0.001). At different concentrations of exogenous fibrinogen, the amount and ratios of adhesive and invasive Pg varied significantly (P < or = 0.007). The higher concentrations of exogenous fibrinogen was added, the greater amount and ratios of adhesive and invasive Pg were found.

CONCLUSIONSFibrinogen promotes the adherence of Pg to human oral epithelial cells and may play an important role in the pathogenesis of periodontal diseases.

Bacterial Adhesion ; drug effects ; Fibrinogen ; administration & dosage ; pharmacology ; Humans ; KB Cells ; Mouth Mucosa ; drug effects ; microbiology ; Periodontitis ; etiology ; Porphyromonas gingivalis ; pathogenicity

OBJECTIVEThis study measures the glutaredoxin (Grx) gene and protein expression in umbilical vein endothelial cells upon exposure to Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The involvement of the Akt-signaling pathway is also determined.

METHODSEA-hy926 cells were pretreated with 1,000 ng · mL⁻¹ P. gingivalis LPS for 4, 12, 18, and 24 h, and then real-time reverse transcription polymerase chain reaction was employed to detect Grx1 expression. The effect of Grx on Akt activity was investigated using Western blot for the control, LPS (1,000 ng · mL⁻¹ LPS), and carmus- tine (BCNU) groups (1,000 ng · mL⁻¹ LPS, and the EA-hy926 cells were pretreated with 25 μmol · ml⁻¹ BCNU for 30 min).

RESULTSGene expression of Grx1 significantly increased in LPS group compared with that in the control group. The Grx1 expression reached the peak level in 12 h, and the variation between the expression in 4 and 12 h was significant (P < 0.05). After 12 h, the protein levels of Grx and phosphorylated-Akt (p-Akt) significantly increased in the LPS group (P < 0.05), whereas the BCNU group showed a considerable decrease in both Grx and p-Akt expression levels (P < 0.05). Moreover, a slight difference was observed in the total Akt protein levels in the three groups (P > 0.05).

CONCLUSIONGrx expression increased upon exposure of EA-hy926 cells to the LPS. Akt activity could be inhibited by BCNU (a Grx inhibitor), which indicated that Akt might act as a downstream regulator of Grx.

Endothelial Cells ; Glutaredoxins ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Oxidative Stress ; drug effects ; Phosphorylation ; Porphyromonas gingivalis ; pathogenicity ; Proto-Oncogene Proteins c-akt ; drug effects ; Signal Transduction ; drug effects ; Umbilical Veins