Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

Selective growth inhibition of Porphyromonas gingivalis by bestatin

Abstract Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis . The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis . Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 μg ml⁻¹ in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 μg ml⁻¹. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asacharolytic bacteria, was not affected even at bestatin concentrations up to 50 μg ml⁻¹. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestasin is a highly effective inhibitor of cell growth of P. gingivalis . Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis , a suspected pathogen in adult chronic periodontitis.     

Development of a simple chemically defined medium for Porphyromonas gingivalis: requirement for α-ketoglutarate

Abstract The aim of this study was the development of a simple, defined medium for the growth of laboratory and clinical isolates of Porphyromonas gingivalis . A medium was designed in which the carbon and nitrogen requirements were provided by a single protein source — bovine serum albumin. High cell yields were achieved in this medium but growth was accompanied by a heavy blackening of the cells due to the deposition of metal sulfide(s), most probably iron(II) sulfide, at the cell surface. Good growth in the absence of blackening was achieved when the iron salt in the medium was substituted with α-ketoglutarate. The resultant α-ketoglutarate/BSA medium was able to support the growth of all laboratory and clinical P. gingivalis strains examined and should prove useful in the investigation of the physiology and nutritional regulation of virulence of this organism.     

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis

Abstract 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.     

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1–2 ppm) and under anaerobic conditions (≤0.1 ppm O₂). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth — biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O₂-grown biofilm.     

Immunological characterization and localization of a Porphyromonas gingivalis BApNA-hydrolyzing protease possessing hemagglutinating activity

Abstract A monoclonal antibody (mAb-PC) was produced against a BA p NA-hydrolyzing protease possessing hemagglutinating activity (Pase-C) from Porphyromonas gingivalis . Other P. gingivalis BA p NA-hydrolyzing enzymes (Pase-B and Pase-S) did not react with this antibody. By ELISA or SDS-PAGE and Western immunoblotting analysis, mAb-PC recognized all P. gingivalis and P. endodontalis strains tested but did not recognize other members of the Porphyromonas genus nor other putative periodontopathogenic organisms. Pase-C, extracellular vesicles (ECV) and human strains of P. gingivalis showed two major immunoreactive bands (44 kDa and 40 kDa), whereas a different pattern was obtained with animal strains of P. gingivalis . Biotinylarginyl chloromethane, an irreversible inhibitor of trypsin-like proteases, did not affect the reactivity of Pase-C with mAb-PC on immunoblot. By reversed-phase electronmicroscopy following immunogold labeling, the antibody was shown to bind to the cell surface of P. gingivalis . mAb-PC inhibited the hemagglutinating activity of both P. gingivalis cells and ECV whereas a monoclonal antibody against LPS of P. gingivalis did not. These results suggest that Pase-C is located on the cell surface of P. gingivalis and may participate in erythrocyte binding.     

牙龈卟啉单胞菌感染通过Wnt通路调节牙周膜干细胞的成骨分化

目的 观察牙龈卟啉单胞菌(P.gingivalis)感染通过Wnt通路调节牙周膜干细胞(PDLSCs)成骨分化的作用。 方法 培养原代PDLSCs,分为常规处理的对照组、P.gingivalis感染的P.gingivalis组和P.gingivalis感染并用Wnt3a处理的P.gingivalis+Wnt3a组,成骨诱导后茜素红染色并检测A405值,Western blot检测Wnt通路分子的蛋白表达量,碱性磷酸酶(ALP)试剂盒检测ALP活力,PCR检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素(OCN)的mRNA表达量。 结果 与对照组比较,P.gingivalis组Wnt3a、βcatenin、pGSK3β的蛋白表达水平(0.33±0.07)、(0.27±0.08)、(0.44±0.09)以及成骨诱导后A405值(0.55±0.08)、ALP活力(20.14±6.54)U/mL和Runx2、OCN的mRNA表达量(0.45±0.09)、(0.51±0.07)均明显减少;与P.gingivalis组比较,P.gingivalis+Wnt3a组成骨诱导后A405值(0.89±0.15)、ALP活力(29.44±5.26)U/mL及Runx2、OCN的mRNA表达量(0.89±0.17)、(0.81±0.18)均明显增加。 结论 P.gingivalis感染能够抑制PDLSCs的成骨分化,抑制Wnt通路是可能的分子机制。     

Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: Molecular cloning and sequence analysis of its gene and purification of the recombinant protein

Abstract Porphyromonas gingivalis is associated with human periodontal disease. We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P. gingivalis FDC381. The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein. The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans. Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti- Mycobacterium leprae HSP60 monoclonal antibody. We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography. This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease. This immunological reactivity and the existence of molecular mimicry between the P. gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.     

Porphyromonas gingivalis fimbriae and their synthetic peptides induce proinflammatory cytokines in human peripheral blood monocyte cultures

Abstract Porphyromonas gingivalis fimbriae as well as synthetic peptides that mimic the fimbrial subunit protein, which includes the amino acid sequence XLTXXLTXXNXX, induced high production of proinflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-α in human peripheral blood monocyte/macrophage cultures. Responses induced by some peptide segments were comparable to those induced by Escherichia coli lipopolysaccharides. A chemically modified peptide analogous to an active peptide segment was found to be antagonistic with regard to interleukin-6 production induced by the native fimbriae. It may be suggested that P. gingivalis fimbriae and their degraded peptides function as proinflammatory agents in vivo, while certain analog peptides inhibited the process.     

Porphyromonas gingivalis infection and cell death in human aortic endothelial cells

Porphyromonas gingivalis is a periodontal pathogen that promotes a proatherogenic response in endothelial cells. Cell death responses of human aortic endothelial cells to P. gingivalis at various multiplicities of infection (MOI) were investigated by assessment of cell detachment, histone-associated DNA fragmentation, lactate dehydrogenase release and ADP:ATP ratio. Porphyromonas gingivalis at MOI 1:10-1:100 did not have a cytotoxic effect, but induced apoptotic cell death at MOI 1:500 and 1:1000. Monocyte chemoattractant protein-1 production was significantly enhanced by P. gingivalis at MOI 1:100. At higher MOI, at least in vitro, P. gingivalis mediates endothelial apoptosis, thereby potentially amplifying proatherogenic mechanisms in the perturbed vasculature.     

牙龈卟啉单胞菌感染通过激活NLRP3小体诱导牙周膜细胞炎症反应及凋亡效应

目的 观察牙龈卟啉单胞菌感染通过激活含NLR家族PYRIN域蛋白3(NLRP3)小体诱导人牙周膜细胞(hPDLCs)炎症反应及凋亡的效应。 方法 取健康前磨牙样本并分离培养hPDLCs,分为牙龈卟啉单胞菌感染的感染组和常规处理的对照组,检测细胞中NLRP3小体[NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-1]、凋亡基因[自杀相关因子(Fas)、Fas配体(FasL)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Caspase-3]的表达量及培养基中炎症细胞因子[白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)]的含量。 结果 感染组hPDLCs中NLRP3、ASC、Caspase-1、Fas、FasL、Bax、Caspase-3的表达量及培养基中IL-1β、IL-18、TNF-α的含量明显高于对照组,细胞中Bcl-2的表达量明显低于对照组。 结论 牙龈卟啉单胞菌感染能够诱导hPDLCs的炎症反应及凋亡且该作用与NLRP3小体的激活有关。     

Stimulation with Porphyromonas gingivalis enhances malignancy and initiates anoikis resistance in immortalized oral keratinocytes

The aim of this study was to get new insights into molecular processes involved in tumor propagation of immortalized oral keratinocytes induced by the keystone pathogen Porphyromonas gingivalis. Cell culture experiments with immortalized OKF6 cells were performed to analyze cellular effects caused by bacterial stimulation focusing on altered gene expression, signaling pathways, proliferation rate, cell viability, migration and invasion behavior, and on the development of antiapoptotic pathways. Gene and protein expression were analyzed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, western blot, and protein arrays. Trypan blue staining was used to analyze proliferation and viability, transwell assays for cellular migration, Matrigel assays for invasion, and anoikis-assays for evaluating anoikis resistance. Stimulation of OKF6 cells with Porphyromonas gingivalis led to an alteration in the molecular repertoire of proteins which are involved in cell proliferation, epithelial–mesenchymal transition, stem cell formation, migration, invasion, and anoikis resistance. Higher proliferation rates were detected in conjunction with an activation of PI3K/Akt signaling and the mTOR-pathway. Additionally, inhibition of glycogen-synthase-kinase3-β led to stabilization of β-catenin and Snail, which resulted in a switch from predominant E-cadherin to N-cadherin expression and increased expression of the stem cell markers Oct3/4, Sox2, and Nanog. Enhanced biosynthesis and enzyme activity of matrix metalloproteinase-9 was accompanied by elevated invasion behavior. Finally, anoikis resistance was detected in stimulated keratinocytes by decreased apoptosis of nonadherent cells and elevated expression of epidermal growth factor receptor and c-Met. Hence, Porphyromonas gingivalis is able to induce a more aggressive tumor-like phenotype in immortalized oral keratinocytes, thus contributing to enhanced tumor features.     

COPD患者口腔和呼吸道内牙龈卟啉单胞菌、齿垢密螺旋体和福赛坦菌的同源性分析

目的分析慢性阻塞性肺疾病(COPD)患者口腔和呼吸道内牙龈卟啉单胞菌(Pg)、齿垢密螺旋体(Td)和福赛坦菌(Tf)的同源性,探讨牙周炎与COPD之间的关系。方法收集53例急性加重期COPD患者,采集龈下菌斑及呼吸道分泌物,同时进行牙周指标的检查。通过16S r DNA序列分析,构建患者口腔及呼吸道内Pg、Td和Tf的系统进化树,进行同源性分析。结果 53例患者中,5例患者口腔和呼吸道内Pg具有同源性,6例患者Td具有同源性,3例患者Tf具有同源性。结论 COPD患者口腔和呼吸道内Pg、Td和Tf具有同源性,COPD患者呼吸道内的这3种病原体均来自于口腔。     

Genetic variation of a fimbrial protein from Porphyromonas gingivalis and its distribution in patients with periodontal diseases

Pg-II fim from various strains of Porphyromonas gingivalis was classified on the basis of each nucleotide sequence, while the distribution of Pg-II fim types in 141 subgingival plaque samples was analyzed using PCR assays. Pg-II fim was divided into two types as follows: strains OMZ409, HG405, 381, ATCC 33277 and BH18/10 (type 1) and strains OMZ314 and HW24D-1 (type 2). The presence of P. gingivalis was demonstrated in 2.8% of healthy subjects and 56.1% of patients with periodontal diseases, and Pg-II fim was detected in 91.8% of the P. gingivalis-positive subjects. We also analyzed the distribution of the Pg-II fim types among Pg-II fim-positive patients, with the following results: type 1 (38.2%), type 2 (56.4%) and types 1 and 2 (5.4%). These findings strongly suggest that P. gingivalis organisms possessing Pg-II fim type 2 was principally detected in patients with periodontal diseases.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

Candidate salivary biomarkers associated with alveolar bone loss: cross-sectional and in vitro studies

This cross-sectional study evaluated the association between radiographic evidence of alveolar bone loss and the concentration of host-derived bone resorptive factors (interleukin-1 beta, tumor necrosis factor-alpha, interleukin-6, prostaglandin-E2), and markers of bone turnover [pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), osteocalcin, osteonectin] in stimulated human whole saliva collected from 110 untreated dental patients. Alveolar bone loss scores for each patient were derived from radiographic examination. Variables positively associated with increased bone loss score were: age, current smoking, use of bisphosphonate drugs, and salivary interleukin-1beta levels above the median. Salivary osteonectin levels above the median were associated with a decreased bone loss score. Additional in vitro studies were carried out to determine the fate of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha added to whole and parotid saliva. All cytokines added to saliva were detected in significantly lower concentrations than when added to buffer alone. Protease inhibitors added to saliva did not prevent the reduction in detection of biomarkers. Variation in time of incubation, repeated cycles of freezing and thawing, or exposure to dimethylsulfoxide did not appreciably affect the measurement of cytokines in saliva. These results suggest that detection of biomarkers by conventional immunoassays may underestimate the actual quantity of molecules in saliva.     

Unchanged survival rates of Shadoo knockout mice after infection with mouse-adapted scrapie

Previous studies have demonstrated that Shadoo (Sho), a GPI-linked glycoprotein encoded by the Sprn gene with a membrane localization similar to PrP^C, is reduced in the brains of rodents with terminal prion disease. To determine the functional significance of Sho in prion disease pathogenesis, Sho-deficient mice were generated by gene targeting. Sho knockout and control wild-type (WT) mice were infected with themouse-adapted scrapie strains 22L or RML. No significant differences in survival, the incubation period of prion disease or other disease features were observed between Sho mutant and WT mice. In this model of prion disease, Sho removal had no effect on disease pathogenesis.