Purification and partial characterization of a lysine-specific protease of Porphyromonas gingivalis

Abstract A lysine-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion-exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and the p I value was 7.3. The enzyme hydrolysed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins.     

Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gingivalis strain 381

We previously reported the existence of two different kinds of fimbriae expressed by Porphyromonas gingivalis ATCC 33277. In this study, we isolated and characterized a secondary fimbrial protein from strain FPG41, a fimA-inactivated mutant of P. gingivalis 381. FPG41 was constructed by a homologous recombination technique using a mobilizable suicide vector, and failed to express the long fimbriae (41-kDa fimbriae) that were produced on the cell surface of P. gingivalis 381. However, short fimbrial structures were observed on the cell surface of FPG41 by electron microscopy. The fimbrial protein was purified from FPG41 by DEAE-Sepharose CL-6B column chromatography. The secondary fimbrial protein was eluted at 0.15 M NaCl, and the molecular mass of this protein was approximately 53 kDa as estimated by SDS-PAGE. An antibody against the 53-kDa fimbrial protein reacted with the short fimbriae of the FPG41 and the wild-type strain. However, the 41-kDa long fimbriae of the wild-type strain and the 67-kDa fimbriae of ATCC 33277 did not react with the same antibody. Moreover, the N-terminal amino acid sequence of the 53-kDa fimbrial protein showed only 2 of 15 residues that were identical to those of the 41-kDa fimbrial protein. These results show that the properties of the 53-kDa fimbriae are different from those of the 67-kDa fimbriae of ATCC 33277 as well as those of the 41-kDa fimbriae.     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用

目的探讨牙龈卟啉单胞菌血凝素2（Porphyromonas gingivalis hemagglutinin-2,PgHA-2）的氯化血红素结合位点多肽对牙龈卟啉单胞菌（Porphyromonasgingivalis,Pg）摄取氯化血红素生长的影响。方法合成多肽DHYAVMISK（肽1）,DEYAVMISK（肽2,肽1中第2位氨基酸突变为谷氨酸）,ALHPDHYLI（肽3,HA-2结合位点不相关多肽）。将肽l、肽2、肽3分别与氯化血红素琼脂糖珠预孵育,加入Pg重组血凝素2（Porphyromonas gingivalis recombinant HA-2,PgrHA-2）,收集与氯化血红素结合的PgrHA-2,SDS—PAGE电泳,分析多肽对PgrHA-2与氯化血红素结合的抑制作用。肽1、肽2、肽3与氯化血红素预孵育后,加入到CDC液体培养基中培养Pg,测定菌液A600值,分析多肽对Pg生长的抑制作用。结果肽1浓度依赖性抑制PgrHA-2与氯化血红素结合,而肽2和肽3对PgrHA-2与氯化血红素的结合无抑制作用。在24、48和72h时间点,肽1组的A600值较肽2、肽3和PBS组明显降低（P〈0．05）。结论本研究表明PgHA-2氯化血红素结合位点多肽DHYAVMISK与Pg竞争结合氯化血红素,抑制Pg的生长,为开发新的牙周病防治方法奠定基础。     

Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product

A clone expressing a Porphyromonas gingivalis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between BamHI and HindIII sites. When a 3.2-kb HindIII fragment of pYS307 was used as a probe in Southern hybridization, HindIII-digested chromosomal DNA of P. gingivalis W83, as well as those of W50 and W12, showed a single 3.2-kb hybridizing band, while that of P. gingivalis 33277 showed a 5.0-kb band. Colonies of E. coli containing pYS307 showed pronounced proteolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coli containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-dependent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coli JM83 containing pYS307 was prepared for electrophoresis in the absence of beta-mercaptoethanol. When samples were solubilized in the presence of beta-mercaptoethanol prior to electrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The activity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCl2.     

Isolation and preliminary characterization of the Porphyromonas gingivalis prtC gene expressing collagenase activity

A gene, prtC, has been isolated from Porphyromonas gingivalis ATCC 53977 in Escherichia coli utilizing the plasmid vector pPL-lambda. The resultant protease positive clone NHS1, harboring plasmid pS1 with a 5.9-kilobase P. gingivalis insert, expressed an enzyme capable of hydrolyzing the synthetic collagenase substrate PZ-PLGPA as well as solubilized type I collagen. Subcloning and deletion analysis located the prtC gene at one end of the P. gingivalis DNA insert on plasmid pS1.     

Intra- and inter-individual comparison of Porphyromonas gingivalis genotypes

Abstract Genetic analysis of 31 clinical strains of Porphyromonas gingivalis isolated from nine subjects, 2–6 strains per subject, was performed by Southern hybridization. Chromosomal DNA was extracted by the method of Moncla et al. [1] and digested to completion with restriction endonucleases Pst I, Cla I and Bgl I. The DNA fragments were separated electrophoretically on agarose gels, transferred to nylon membranes and hybridized to the non-radioactively labelled plasmid pKK 3535 which contains the rrn B ribosomal RNA operon of the Escherichia coli chromosome. Of the three enzymes, Bgl I was the most suitable for the genetic analysis of P. gingivalis . With this enzyme, the intra-individual strains were shown to be identical in eight of the nine subjects, whereas inter-individual strains were different.     

Biological activities of surface-associated material from Porphyromonas gingivalis

Abstract Surface-associated material (SAM) from Porphyromonas gingivalis was tested for in vitro biological activities that may be relevant to the pathogenesis of chronic periodontitis. SAM was found to stimulate bone resorption at a concentration of 1.0 μg/ml and this was inhibited by indomethacin, interleukin-1 receptor antagonist protein and anti-tumour necrosis factor antibody. At a concentration of 10 ng/ml, the SAM inhibited DNA and collagen synthesis in osteoblasts and murine calvaria and DNA synthesis in fibroblasts, monocytes and epidermal cells. Therefore, easily solubilised surface components from P. gingivalis could play a role in the pathogenesis of chronic periodontitis if these activities operate in vivo.     

Quantitative proteomics of intracellular Porphyromonas gingivalis

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.     

Separation of novel phosphoproteins of Porphyromonas gingivalis using phosphate‐affinity chromatography

Phosphorylation of serine, threonine and tyrosine is a central mechanism for regulating the structure and function of proteins in both eukaryotes and prokaryotes. However, the action of phosphorylated proteins present in Porphyromonas gingivalis, a major periodontopathogen, is not fully understood. Here, six novel phosphoproteins that possess metabolic activities were identified, namely PGN_0004, PGN_0375, PGN_0500, PGN_0724, PGN_0733 and PGN_0880, having been separated by phosphate‐affinity chromatography. The identified proteins were detectable by immunoblotting specific to phosphorylated Ser (P‐Ser), P‐Thr, and/or P‐Tyr. These results imply that novel phosphorylated proteins might play an important role for regulation of metabolism in P. gingivalis.     

Quantitative detection of Porphyromonas gingivalis fimA genotypes in dental plaque

We developed quantitative fimA genotype assays and applied them in a pilot study investigating the fimbrial genotype distribution of Porphyromonas gingivalis in European subjects with or without chronic periodontitis. P. gingivalis was found in 71% and 9% of the samples from patients and healthy subjects, respectively. Enumeration of total P. gingivalis cell numbers by polymerase chain reaction and immunofluorescence showed excellent correspondence (r = 0.964). 73% of positive samples contained multiple fimA genotypes, but generally one genotype predominated by one to three orders of magnitude. Genotype II predominated in 60% of the samples. Genotype IV occurred with similar prevalence (73%) as genotype II but predominated in only 20% of the samples. Genotypes I, III and V were of much lower prevalence and cell densities of the latter two remained sparse. Our results suggest marked differences among the fimA genotypes' ability to colonize host sites with high cell numbers.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Abstract Porphyromonas gingivalis culture supernate was found to induce hemotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of aglutination induced by the secretagogues PMA and FMLP. Lipopolysaccaharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors of PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity fot P. gingivalis cells but when PMN were treated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutinatin or enhanced adherence could also lead to decreased phagocytic capacity of the adherant or agglutinated cells.     

A natural anti-periodontitis agent,epimedokoreanin B,inhibits virulence activities of gingipains from Porphyromonas gingivalis

Gingipains are potent virulence cysteine proteases secreted by Porphyromonas gingivalis, a major pathogen of periodontitis. We previously reported that epimedokoreanin B inhibits the activities of gingipains. In this report, we show that epimedokoreanin B inhibits the virulence of gingipains-containing P. gingivalis culture supernatants, indicating the potential use of this prenylated flavonoid as a new agent to combat against periodontal pathogens.     

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.     

Cloning and characterization of heavy and light chain genes encoding the FimA-specific monoclonal antibodies that inhibit Porphyromonas gingivalis adhesion

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis.     

Some binding properties of the envelope of Porphyromonas gingivalis to hemoglobin

Abstract Porphyromonas gingivalis was found to bind to hemoproteins (hemoglobin, myoglobin, catalase, cytochrome c ) and the binding properties of the envelope of P. gingivalis to hemoglobin were investigated. Maximum amount of hemoglobin bound to 1 mg of the envelope was 58 μg. No significant binding was observed at 4°C and the binding was inhibited strongly by tosyl- l -lysine chloromethyl ketone, Leupeptin, EDTA and partially by meta-periodate. Heating of the envelope at 70°C for 15 min resulted in complete loss of the binding activity. The binding activity of the envelope was not influenced by the treatment with the endogenous proteases. The envelope saturated with hemoglobin could no longer bind to other hemoproteins tested, indicating that binding site for these hemoproteins are common.     

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.