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.     

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.     

Fimbrial proteins of porphyromonas gingivalis mediate in vivo virulence and exploit TLR2 and complement receptor 3 to persist in macrophages

Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.     

Porphyromonas gingivalis fimbriae binds to neoglycoproteins: evidence for a lectin-like interaction

Porphyromonas gingivalis is a likely major pathogen in adult periodontitis. Fimbriae in particular have been suggested as playing an important role in facilitating the initial interaction between the bacteria and the host and triggers host responses. Murakami et al. [Biochem. Biophys. Res. Commun. 192 (1993) 826] have shown that fimbriae of P. gingivalis strongly induced TNF-alpha gene expression in macrophages and expression of TNF-alpha was inhibited by N-acetyl-D-galactosamine, but not inhibited by other sugars. Studies by Sojar et al. [FEBS Lett. 422 (1998) 205] suggested that the oligosaccharide moiety of lactoferrin is involved in the interaction of P. gingivalis fimbriae and human lactoferrin. In the present study, purified fimbriae from P. gingivalis and neoglycoproteins were used to assess lectin-like interaction of fimbriae. In dot blot and overlay assays, iodinated purified P. gingivalis fimbriae as well as biotinylated purified P. gingivalis fimbriae bound strongly to albumin-fucosylamide (albumin-1-amido-1-deoxy-L-fucose) and by lesser extent to albumin-N-acetyl-D-galactosamine (albumin-p-aminophenyl-N-acetyl-beta-D-galactosaminide). However, fimbriae failed to bind carbohydrate free bovine serum albumin, which was used in preparation of the neoglycoproteins. These results suggests that P. gingivalis fimbriae bind to glycoconjugates through lectin-like interaction with carbohydrate. This protein-carbohydrate interactions may be important for triggering events in these cells, which mediate the host response of this pathogen.     

Research on the roles of genes coding ATP-binding cassette transporters in Porphyromonas gingivalis pathogenicity

Porphyromonas gingivalis, as a major pathogen of periodontitis, could rapidly adhere to and invade host gingival epithelial cells (GECs) for the induction of infection. One ATP-binding cassette (ABC) transporter gene was found to be upregulated during this infection process, however, the molecular mechanisms remain unclear. In this study, we systemically investigated the messenger RNA level changes of all ABC transporter family genes in P. gingivalis while being internalized within GECs by real-time polymerase chain reaction. We identified that two ABC transporter genes, PG_RS04465 (PG1010) and PG_RS07320 (PG1665), were significantly increased in P. gingivalis after coculturing with GECs. Mutant strains with knockout (KO) of these two genes were generated by homogenous recombination. PG_RS04465 and PG_RS07320 KO mutants showed no change in the growth of bacteria per se. Knockdown of PG_RS07320, but not PG_RS04465, caused decreased endotoxin level in the bacteria. In contrast, both mutant strains showed decreased Arg- and Lys-gingipains activities, with significantly reduced adhesion and invasion capabilities. Secreted interleukin-1β (IL-1β) and IL-6 levels in GECs cocultured with PG_RS04465 or PG_RS07320 KO mutants were also decreased, whereas, only the cells cocultured with PG_RS07320 KO mutants showed significant decrease. In addition, virulence study using mouse revealed that both KO mutant strains infection caused less mouse death than wild-type strains, showing reduced virulence of two KO strains. These results indicated that ABC transporter genes PG_RS04465 and PG_RS07320 are positive regulators of the virulence of P. gingivalis.     

Expression of saliva-binding epitopes of the Porphyromonas gingivalis FimA protein on the surface of Streptococcus gordonii

Porphyromonas gingivalis, a gram-negative oral anaerobic bacterium, has been implicated in the onset and development of periodontitis. The P. gingivalis fimbriae which mediate bacterial adherence to host oral sites and induce host inflammatory responses have been suggested as a potential antigen candidate. for vaccine development. This study was undertaken to generate Streptococcus gordonii vectors expressing the major subunit protein (FimA) of P. gingivalis fimbriae for testing as a potential live vaccine against periodontitis. We report here the expression of the C-terminal saliva-binding epitopes of P. gingivalis FimA on the surface of S. gordonii and demonstrate that domains containing free cysteine residues are poorly expressed on the surface of S. gordonii.     

Porphyromonas gingivalis FimA Fimbriae: Fimbrial Assembly by fimA Alone in the fim Gene Cluster and Differential Antigenicity among fimA Genotypes

The periodontal pathogen Porphyromonas gingivalis colonizes largely through FimA fimbriae, composed of polymerized FimA encoded by fimA. fimA exists as a single copy within the fim gene cluster (fim cluster), which consists of seven genes: fimX, pgmA and fimA-E. Using an expression vector, fimA alone was inserted into a mutant from which the whole fim cluster was deleted, and the resultant complement exhibited a fimbrial structure. Thus, the genes of the fim cluster other than fimA were not essential for the assembly of FimA fimbriae, although they were reported to influence FimA protein expression. It is known that there are various genotypes for fimA, and it was indicated that the genotype was related to the morphological features of FimA fimbriae, especially the length, and to the pathogenicity of the bacterium. We next complemented the fim cluster-deletion mutant with fimA genes cloned from P. gingivalis strains including genotypes I to V. All genotypes showed a long fimbrial structure, indicating that FimA itself had nothing to do with regulation of the fimbrial length. In FimA fimbriae purified from the complemented strains, types I, II, and III showed slightly higher thermostability than types IV and V. Antisera of mice immunized with each purified fimbria principally recognized the polymeric, structural conformation of the fimbriae, and showed low cross-reactivity among genotypes, indicating that FimA fimbriae of each genotype were antigenically different. Additionally, the activity of a macrophage cell line stimulated with the purified fimbriae was much lower than that induced by Escherichia coli lipopolysaccharide.     

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.     

Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803

The adhesion properties of the recombinant fimbriae (r-fimbriae) recovered from a YH522 transformant of Porphyromonas gingivalis which harbors a chimeric plasmid, pYHF2, containing the fimA gene of strain 381 were compared with those of the endogenous fimA fimbriae of strain 33277. The adhesion level of the r-fimbriae to Actinomyces viscosus was clearly lower than that of the endogenous fimbriae. In addition, the r-fimbriae were shown to lack some minor components detectable in the endogenous fimbriae. The plasmid pYHF2 prepared from the YH522 transformant was then transformed into six different P. gingivalis strains and the resultant pYHF2-containing strains were examined for their fimbrial expression. In spite of the presence of a considerable diversity in the expression level of the r-fimbriae among these transformants, it was evident that the strains expressing higher levels of the r-fimbriae exhibited a greater decrease in adhesion activity to other bacteria and to oral epithelial cells, as well as in self-aggregation.     

Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine-specific protease

Porphyromonas gingivalis , a Gram-negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine-specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg-specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real-time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease-degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme-linked biotin–avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of l -Arg, or oligopeptides containing the Arg residue at the C-terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C-terminus. Taken together, these studies indicate that P. gingivalis arginine-specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C-terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae–Arg interaction (a novel host–parasite relationship).     

牙龈卟啉单胞菌影响牙龈上皮细胞miR1555p 通过JAK/STAR信号通路作用牙周炎的机制

目的 通过生物信息学对GEO数据库进行分析筛选miRNA后运用分子生物学手段验证并对机制进行深入探讨,为未来牙周炎治疗的生物标志物筛选及靶向治疗提供理论依据。 方法 通过生物信息学分析GEO数据库发现牙周炎患者中差异表达的miRNA。在DIANA生信预测网站中发现了与JAK/STAT信号传导方式有关的miRNA。随后,TargetScan被用于预测miRNA的靶mRNA,该mRNA不仅在牙周炎中差异表达,而且与JAK/STAT信号传导有关。利用基因集富集分析(GSEA)寻找与JAK/STAT信号转导途径紧密相关的基因集。通过实时定量PCR(qRTPCR)和免疫印迹法(Western blot)检测在牙周炎中差异表达并与JAK/STAT信号有关的miRNA和mRNA的表达。通过免疫组织化学(IHC)观察组织中IL6ST的表达。通过双重荧光素酶测定法证实了miRNA和mRNA之间的关系。此外,采用细胞内氧化活性氧红色荧光检测试剂盒检测活性氧(ROS)的变化。 结果 在牙周炎患者中,与正常组织比较,MiR1555p被下调,mRNA IL6ST被上调且差异均具有统计学意义(t=9.188 7、2.852 1,P=0.000 6、0.015 7)。与对照组比较,miR1555p在P.gingivalis处理组表达情况出现明显下降且IL6ST在处理后表达量出现明显升高,差异均有统计学意义(t=2.125 3、1.852 0,P=0.013 5、0.015 7)。miR1555p具有IL6ST 3′非翻译区的靶结合位点。通过过表达miR1555p能够明显抑制JAK/STAT信号通路pSTAT3、pJAK2、IL6ST蛋白的表达(t=1.924 8、2.530 8、3.107 5,P=0.023 1、0.011 6、0.010 0)。感染牙龈卟啉单胞菌后,细胞中的ROS产生增加(t=3.051 2、9.632 7,均P结论 牙龈卟啉单胞菌可抑制miR1555p表达,激活GECs中的JAK/STAR信号,促进牙周炎的发生和发展。     

Porphyromonas gingivalis fimbriae carbohydrate specificity assessment by glycomics

Porphyromonas gingivalis is a major pathogen in adult periodontitis. Fimbriae play an important role in the initial interaction between the bacteria and the host. Our earlier studies suggested that the oligosaccharide moiety of lactoferrin is involved in the interaction with fimbriae. Porphyromonas gingivalis fimbriae bound strongly to albumin-fucosylamide (albumin-1-amido-1-deoxy-l-fucose) and to a lesser extent to albumin-N-acetyl-d-galactosamine (albumin-p-aminophenyl-N-acetyl-β-d-galactosaminide, but failed to bind bovine serum albumin. In this study we explored the glycan array to determine the carbohydrate-binding specificity of P.?gingivalis fimbriae. Purified fimbriae bind to glycans which have a Lewis(x) , Galβ1-4(Fucα1-3) GlcNAcβ structure in common. Interestingly, all glycans have a terminal fucose moiety and if fucose is removed, the fimbriae fail to bind. This is the first study that suggests that fucose is important for P.?gingivalis fimbriae binding.     

Complement receptor 3 blockade promotes IL-12-mediated clearance of Porphyromonas gingivalis and negates its virulence in vivo

The ability of certain pathogens to exploit innate immune function allows them to undermine immune clearance and thereby increase their persistence and capacity to cause disease. Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with increased risk of systemic conditions. We have previously shown that the fimbriae of P. gingivalis interact with complement receptor 3 (CR3; CD11b/CD18) in monocytes/macrophages, resulting in inhibition of IL-12p70 production in vitro. The in vivo biological implications of this observation were investigated in this study using a CR3 antagonist (XVA143). XVA143 was shown to block CR3 binding of P. gingivalis fimbriae and reverse IL-12p70 inhibition; specifically, CR3 blockade resulted in inhibition of ERK1/2 phosphorylation and up-regulation of IL-12 p35 and p40 mRNA expression. Importantly, mice pretreated with XVA143 elicited higher IL-12p70 and IFN-gamma levels in response to P. gingivalis i.p. infection and displayed enhanced pathogen clearance, compared with similarly infected controls. The notion that CR3 is associated with reduced IL-12p70 induction and impaired P. gingivalis clearance was confirmed using i.p. infected wild-type and CR3-deficient mice. Moreover, XVA143 dramatically attenuated the persistence and virulence of P. gingivalis in experimental mouse periodontitis, as evidenced by reduced induction of periodontal bone loss. Therefore, CR3 blockade may represent a promising immunomodulatory approach for controlling human periodontitis and possibly associated systemic diseases.