Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis.

The cloning and sequencing of the gene encoding porphypain, a cysteine proteinase previously isolated from detergent extracts of the Porphyromonas gingivalis W12 cell surface, are described. The prtP gene encoded a unique protein of 1,732 amino acids, including a putative signal sequence for protein secretion. The predicted molecular mass for the mature protein was 186 kDa, which was close to the observed molecular mass of 180 kDa. There was one copy of prtP in the genomes of seven P. gingivalis strains examined. The gene was located 5' to a region with a high degree of homology to the insertion element IS1126 in P. gingivalis W12. The PrtP protein had regions of high homology to HagA, a hemagglutinin of P. gingivalis, and to several purported proteinases of P. gingivalis that have Arg-X specificity. A detailed comparison of genes encoding the latter and cpgR suggested that rgp-1, prpR1, prtR, agp, cpgR, and possibly prtH were derived from identical genetic loci. Although an rgp-1-like locus was detected in seven P. gingivalis strains by Southern blot analyses, agp and cpgR were not detected, not even in the strains from which they were originally isolated. In addition, at least 20 copies of a repeat region common to PrtP, the Rgp-1-like proteins, and HagA were observed in each of the seven genomes examined. The repeat region hybridization patterns for strains W83 and W50 were very similar, and they were identical for strains 381 and ATCC 33277, providing further evidence that these strains are closely related genetically.     

Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis

Porphyromonas gingivalis, a putative pathogen in human periodontal disease, possesses a 60-kDa heat shock protein (hsp60, GroEL). The GroEL homologs are known to be key molecules in auto-immune reactions because of the sequence similarity with human hsp60. In this study, B-cell epitopes on P. gingivalis GroEL (PgGroEL) were analyzed by both Western immunoblotting with truncated PgGroEL and by the multi-pin synthetic peptide approach. To examine auto-antibody production in periodontitis patients, Western immunoblotting with human gingival fibroblasts was performed. Deletion mutants were constructed from the cloned PgGroEL gene (P. gingivalis groEL), and four C-terminal truncated PgGroEL and one N-terminal truncated PgGroEL were prepared from the deletants. Sera from periodontitis patients reacted with all truncated PgGroEL used in this study. The results suggest that the B-cell epitopes were overlaid throughout PgGroEL. To determine the detailed locations of the B-cell epitope, 84 decapeptides covering the entire PgGroEL were synthesized and the serum IgG response to the peptides was examined. Epitope mapping using the synthetic peptides confirmed that the B-cell epitopes were overlaid throughout the length of PgGroEL and revealed that highly conserved peptides between PgGroEL and human hsp60 were recognized by the serum antibodies. Immuno-reactivity against human gingival fibroblasts was examined with sera from 30 periodontitis patients and 10 periodontally healthy subjects. IgG antibody against the 65-kDa antigen in human gingival fibroblasts (same molecular mass as human hsp60) was detected in two patients. Although IgG production against human hsp60 may be rare case in periodontitis patients, the results of epitope mapping demonstrated the potential of PgGroEL to cause the cross-reactions with human hsp60.     

Tobacco-induced alterations to Porphyromonas gingivalis–host interactions

Smokers are more susceptible than non-smokers to persistent infection by Porphyromonas gingivalis , a causative agent of periodontitis. Patients who smoke exhibit increased susceptibility to periodontitis and are more likely to display severe disease and be refractory to treatment. Paradoxically, smokers demonstrate reduced clinical inflammation. We show that P. gingivalis cells exposed to cigarette smoke extract (CSE) induce a lower proinflammatory response (tumour necrosis factor-α, interleukin-6, interleukin-12 p40) from monocytes and peripheral blood mononuclear cells than do unexposed bacteria. This effect is reversed when CSE-exposed bacteria are subcultured in fresh medium without CSE. Using microarrays representative of the P. gingivalis genome, CSE-exposure resulted in differential regulation of 6.8% of P. gingivalis genes, including detoxification and oxidative stress-related genes; DNA repair genes; and multiple genes related to P. gingivalis virulence, including genes in the major fimbrial and capsular operons. Exposure to CSE also altered the expression of outer membrane proteins, most notably by inducing the virulence factors RagA and RagB, and a putative lipoprotein cotranscribed with the minor fimbrial antigen. Therefore, CSE represents an environmental stress to which P. gingivalis adapts by altering gene expression and outer membrane proteins. These changes may explain, in part, the altered virulence and host–pathogen interactions that have been documented in vivo in smokers with periodontal disease.     

Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical,microbiological and serological study

Introduction

The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.

Methods

In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.

Results

A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.

Conclusions

Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.     

牙龈卟啉单胞菌、嵴链球菌、口腔链球菌属在口腔内不同解剖部位的分布

目的探讨牙龈卟啉单胞菌(Porphyromonas gingivalis)、嵴链球菌(Streptococcus cristatus)、口腔链球菌属(Streptococci oralis)在慢性牙周炎患者及牙周健康者不同口腔解剖部位生物膜的分布情况。方法选取慢性牙周炎患者25例,牙周健康者24例,分别作为慢性牙周炎组及健康对照组。测量临床指标(探诊深度、附着丧失和探诊出血),取受试者龈下菌斑、舌背、颊黏膜和唾液样品。Real-time PCR分析受试者不同受检部位S.cristatus、P.gingivalis、Streptococci oralis相对数量。结果慢性牙周炎组四个受检部位中P.gingivalis数量均大于健康对照组;慢性牙周炎组龈下菌斑中P.gingivalis数量大于其余受检部位;而慢性牙周炎组龈下菌斑、舌背、颊黏膜三个受检部位S.cristatus、Streptococci oralis数量小于健康对照者。结论与牙周健康者比较,慢性牙周炎患者口腔内不同解剖位置P.gingivalis数量增多,S.cristatus、Streptococci oralis数量减少;P.gingivalis检出数量增加提示牙周炎患病风险增加,而S.cristatus、Streptococci oralis检出数量降低提示牙周炎患病风险降低。     

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis.     

PCR直接检测龈下菌斑主要可疑牙周致病菌

目的：应用PCR方法直接检测龈下菌斑主要可疑牙周致病菌与牙周病活动部位的关系,探讨其方法的可行性并探讨其主要可疑牙周致病菌的分布规律。方法：应用聚合酶链反应(polymerase chain reaction,PCR)直接检测龈下菌斑主要可疑致病菌16s RNA保守区域片段。40名受试者包括牙周病患者20人,每人同口取一个牙周病活动部位,一个相对健康或牙周病静止对照部位;成人健康者20人,每人各取一个标本。结果：龈下菌斑5种可疑牙周致病菌在牙周病活动部位的检出率牙龈卟啉菌为86％,福赛类杆菌为95％,螺旋体为86％,中间普氏菌和黑色普氏菌分别为95％和33％,均显著高于同口部位对照组和健康对照组。结论：PCR直接检测菌斑牙龈卟啉单胞菌、中间普氏菌、福赛类杆菌、齿密螺旋体及黑色普氏菌匀与牙周炎活动部位相关。