OBJECTIVETo study the effect of para-aminobenzoic acid (PABA) on the growth and metabolism of Porphyromonas gingivalis (P.g).

METHODSAfter adding different concentrations of PABA into the medium, anaerobic technique was applied to culture P.g. The products' A value and action of TLP was assayed, and P.g grew in the medium was observed by a scanning electron microscope.

RESULTSPABA promoted the growth of P.g and action of TLP, which would reach the highest level when PABA was 1 mg/L, and would decrease with the increasing of concentration of PABA. When the concentration arrived at 100 mg/L, PABA had no effect on them. In the mean time, PABA had effect on the form and adherence of P.g. When the concentration was 1 mg/L and 100 mg/L, this effect was strong, but as the concentration was 10 mg/L, the effect disappeared.

CONCLUSIONSPABA influences the growth and metabolism of P.g, which indicate that Streptococcus sanguis has regulative effect on the microecology of subgingival plaque.

4-Aminobenzoic Acid ; Dental Plaque ; Porphyromonas gingivalis ; metabolism ; Streptococcus sanguis

OBJECTIVETo test whether Porphyromonas gingivalis (P. gingivalis) could produce bacterial signal molecule, bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and lay the foundation for explorations of its roles in life metabolism and periodontitis immunity of P. gingivalis.

METHODSP. gingivalis standard strain ATCC33277 was used as the experimental strain to extract nucleic acids from the bacteria. Then, c-di-AMP was detected using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Subsequently, HPLC was used to validate the sample further.

RESULTSBased on the signal/noise (S/N) for 3 : 1, the limit of determination of HPLC-MS/MS for peak time of c-di-AMP standard substances was 7.49 min and nucleic acid extractions from P. gingivalis was 8.82 min (S/N > 3). Further confirmation of HPLC showed that nucleic acid extractions from both P. gingivalis and c-di-AMP standard substances pre- sented goal absorbent peaks at 15.7 min, with the same ultraviolet absorbent spectrum.

CONCLUSIONThe nucleic acid extrac- tions from P. gingivalis contained c-di-AMP, which shows that P. gingivalis could produce c-di-AMP.

Chromatography, High Pressure Liquid ; Cyclic AMP ; chemistry ; metabolism ; Periodontitis ; Porphyromonas gingivalis ; metabolism ; Tandem Mass Spectrometry

OBJECTIVE: To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting. RESULTS: In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm²), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05). CONCLUSION P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.
Humans ; Cadherins/metabolism* ; Escherichia coli/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Occludin ; Porphyromonas gingivalis/metabolism* ; Umbilical Veins/metabolism*

Porphyromonas gingivalis is among the major etiological pathogens of chronic periodontitis. The virulence mechanisms of P. gingivalis is yet to be identified as its activity is largely unknown in actual disease process. The purpose of this study is to identify antigens of P. gingivalis expressed only in patients with chronic periodontitis using a unique immunoscreening technique. Change Mediated Antigen Technology (CMAT), an antibody-based screening technique, was used to identify virulence-associated proteins of P. gingivalis that are expressed only during infection stage in patients having chronic periodontitis. Out of 13,000 recombinant clones screened, 22 tested positive for reproducible reactivity with rabbit hyperimmune anti-sera prepared against dental plaque samples acquired from periodontitis patients. The DNA sequences of these 18 genes were determined. CMAT-identified protein antigens of P. gingivalis included proteins involved in energy metabolism and biosynthesis, heme and iron binding, drug resistance, specific enzyme activities, and unknown functions. Further analysis of these genes could result in a novel insight into the virulence mechanisms of P. gingivalis.
Base Sequence ; Chronic Periodontitis ; Clone Cells ; Dental Plaque ; Drug Resistance ; Energy Metabolism ; Heme ; Humans ; Iron ; Mass Screening ; Periodontitis ; Porphyromonas gingivalis ; Porphyromonas ; Virulence ; Virulence Factors

Objectives: To study the effects of Porphyromonas gingivalis (Pg) injected through tail vein on the molecular expression levels of biomarkers of neural stem cells (NSC) and neurons in the hippocampus of wild-type adult rats, and the effects on hippocampal neurogenesis. Methods: Eighteen male Sprague-Dawley (SD) rats were randomly divided into 3 groups based on the table of random numbers (n=6 in each group). In low-intensity group and high-intensity group, rats were injected intravenously through tail vein with 200 μl Pg ATCC33277 [1.0×10³ and 1.0×10⁸ colony forming unit (CFU), respectively] 3 times per week for 8 weeks. In the sham group, 200 μl of phosphate buffer saline (PBS) was given instead. Behavioral tests: the navigation and the exploration tests using Morris water maze (MWM) were applied to evaluate learning and memory ability of rats. Immunohistochemistry was performed to detect cells positively expressing nestin, doublecortin (DCX) and neuronal nuclei (NeuN) in the subgranular zone (SGZ) of rats in each group. Western blotting was used to evaluate the expression levels of nestin, DCX and NeuN in rat hippocampus. Results: Learning and memory abilities: on day 5 of navigation test, the lagency time was 22.83 (16.00, 38.34) s in the high-intensity group, significantly longer than the sham group [5.59 (5.41, 6.17) s] (t=-11.17, P<0.001). There were no significant differences between the low-intensity group [9.85 (8.75, 21.01) s] and the sham group (t=-6.83, P=0.080). Results in the exploration test showed that, in the high-intensity group, the number of fime crossing over the previous platform area within 60 s was 1.50 (1.00, 2.00), significantly less than the sham group [4.00 (2.75, 4.00)] (t=9.75, P=0.003); no significant differences between the low-intensity group [2.50 (2.00, 3.00)] and the sham one (t=4.50, P=0.382). Immunohistochemistry showed that the nestin⁺ cell density in the low-intensity group [(35.36±4.32) cell/mm²] and high-intensity group [(26.51±5.89) cell/mm²] were significantly lower than the sham group [(59.58±14.15) cell/mm²] (t=24.21, P=0.018; t=33.07, P=0.005); as for the mean absorbance of DCX⁺ cells, the low-intensity group (0.007±0.002) and the high-intensity group (0.006±0.002) were significantly lower than the sham group (0.011±0.001) (t=0.004, P=0.018; t=0.006, P=0.005); compared with the sham group [(1.13±0.14)×10³ cell/mm²], the density of NeuN⁺ neurons in the high-intensity group [(0.75±0.08)×10³ cell/mm²] was significantly reduced (t=0.38, P=0.017), and was not significantly changed in the low-intensity group [(0.88±0.19)×10³ cell/mm²] (t=0.25, P=0.075). Western blotting results showed that, compared with the sham group, the expression levels of nestin, DCX, and NeuN were significantly reduced in the high-intensity group (t=0.74, P<0.001; t=0.18, P=0.014; t=0.35, P=0.008), but were not statistically changed in the low-intensity group (t=0.18, P=0.108; t=0.08, P=0.172; t=0.19, P=0.077). Conclusions: Pg injected through tail vein may reduce learning and memory abilities of wild-type rats, and may reduce the number of nestin, DCX, and NeuN-positive cells, and the protein expression levels of the above molecules in the hippocampus.
Animals ; Biomarkers/metabolism* ; Hippocampus/metabolism* ; Male ; Nestin/metabolism* ; Neural Stem Cells/metabolism* ; Neurons/metabolism* ; Porphyromonas gingivalis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Tail/metabolism*

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

OBJECTIVE: Soluble triggering receptors expressed by myeloid cells-1 (sTREM-1) and inflammatory cytokine tumor necrosis factor-α (TNF-α) in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) to investigate the expression of triggering receptors expressed by myeloid cells-1 (TREM-1) and further explore the correlation between TREM-1 and the pathogenesis of periodontitis. METHODS: THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0 (blank control), 0.5, or 1.0 μg·mL⁻¹ Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated (P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL⁻¹ Pg-LPS group than in the 0.5 μg·mL⁻¹ group; this expression was statistically significant since the 6, 4, and 4 h time point (P<0.05). Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS (1.0 μg·mL⁻¹) for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages (P<0.05). The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TNF-α was significantly higher in 1.0 μg·mL⁻¹ Pg-LPS stimulated groups than in 0.5 μg·mL⁻¹ Pg-LPS-stimulated groups since the 6 h time point (P<0.05). The expressions of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in 0.5 μg·mL⁻¹ Pg-LPS-stimulated macrophages were positively correlated with one another (r=1, P<0.05), but no statistically significant correlation was found in the expression of TNF-α. The positive correlation between sTREM-1 and TNF-α expressions was detected when macrophages were stimulated by 1.0 μg·mL⁻¹ Pg-LPS (r=1, P<0.05). CONCLUSIONS The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the culture supernatant in Pg-LPS-stimulated macrophages was significantly upregulated on the basis of the concentration of Pg-LPS; moreover, their upregulation was positively correlated with one another. The expression of TNF-α in the supernatant of cell culture was also upregulated and was positively correlated with the expression of sTREM-1 at the group of high Pg-LPS concentration (1.0 μg·mL⁻¹). Results reveal that TREM-1, which has been realized as a proinflammatory receptor protein, can promote the development of periodontitis by regulating the expression of TNF-α in macrophages.
Adult ; Humans ; Lipopolysaccharides ; Macrophages ; metabolism ; Myeloid Cells ; Periodontitis ; metabolism ; microbiology ; Porphyromonas gingivalis ; pathogenicity ; Triggering Receptor Expressed on Myeloid Cells-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer's disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.
Animals ; Rats ; Bacteremia/metabolism* ; Blood-Brain Barrier/microbiology* ; Caveolin 1/metabolism* ; Gingipain Cysteine Endopeptidases/metabolism* ; Permeability ; Porphyromonas gingivalis/pathogenicity* ; Transcytosis ; Virulence Factors/metabolism*

OBJECTIVETo detect and compare the activity and intensity of gingipain K (Kgp)-caspase like subdomain in culture medium and cell extract of Porphyromonas gingivalis (Pg) isolates in puberty gingivitis and to reveal the possible association of Kgp with puberty gingivitis.

METHODSThirty-six children of 14 to 17 years old were enrolled in this study. Clinical parameters including gingival index (GI), sulcus bleeding index (SBI) and probing depth (PD) were evaluated. Subgingival plaque samples were collected and Pg isolates were obtained. 16S rRNA PCR was used to confirm Pg clinical isolates. Bacteria were grown in batches of BHI base and harvested at the end of log-phase growth. Culture fractions (culture medium and cell extract) of 10 Pg isolates were performed with SDS-PAGE and Western blot technique using primary antibody against specific Kgp-caspase like subdomain. Activity of Kgp in both samples was detected as well. The data were statistically analyzed using SPSS 11.5 software. The relationship between the Kgp intensity/activity of Kgp and the clinical parameters was statistically analyzed using Spearman correlation coefficient.

RESULTSThere was positive correlation between the intensity/activity of Kgp and the clinical parameters (P < 0.05).

CONCLUSIONSThe Kgp in clinical isolates of Pg from puberty gingivitis is in complicated forms. Caspase-like molecules with low molecular weight may exist as intracellular functional protein molecules which can affect the interaction between Pg and host. Kgp was contributes in certain degree to the pathogenesis of puberty gingivitis.

Adhesins, Bacterial ; genetics ; metabolism ; Adolescent ; Cysteine Endopeptidases ; genetics ; metabolism ; Dental Plaque ; microbiology ; Female ; Gingivitis ; enzymology ; microbiology ; Humans ; Male ; Porphyromonas gingivalis ; genetics ; isolation & purification ; metabolism

OBJECTIVETo clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis.

METHODSUsing PCR, the prtH was amplified and cloned into pGEM-T vector. To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe.

RESULTSRecombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay. Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns. Strain 47A-1 seemed even not to contain prtH gene.

CONCLUSIONSDifferent prtH gene sequences exist in different P.g strains. This polymorphism may indicate various potential virulent effects during the infection and pathogenesis. Established PCR protocol is sensitive for identification of prtH gene.

Bacterial Proteins ; Blotting, Southern ; Cloning, Molecular ; Cysteine Endopeptidases ; genetics ; DNA, Bacterial ; genetics ; metabolism ; Deoxyribonuclease BamHI ; metabolism ; Deoxyribonuclease HindIII ; metabolism ; Polymorphism, Genetic ; Porphyromonas gingivalis ; genetics ; Species Specificity