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.     

Expression of the short fimbriae of Porphyromonas gingivalis is regulated in oral bacterial consortia

The Mfa1 protein of Porphyromonas gingivalis is the structural subunit of the short fimbriae and mediates coadhesion between P. gingivalis and Streptococcus gordonii. We utilized a promoter-lacZ reporter construct to examine the regulation of mfa1 expression in consortia with common oral plaque bacteria. Promoter activity of mfa1 was inhibited by S. gordonii, Streptococcus sanguinis and Streptococcus mitis. In contrast, Streptococcus mutans, Streptococcus cristatus, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum did not affect mfa1 expression. Expression of SspA/B, the streptococcal receptor for Mfa1, was not required for regulation of mfa1 promoter activity. Proteinaceous molecule(s) in oral streptococci may be responsible for regulation of Mfa1 expression. Porphyromonas gingivalis is capable of detecting heterologous organisms, and responds to selected organisms by specific gene regulation.     

不同fimA基因型牙龈卟啉单胞菌超微结构观察

目的 探查具有不同粘附、侵入能力的各fimA基因型P.gingivalis菌体表面结构特点.方法 选取临床培养的经PCR鉴定、筛选的fimA基因Ⅰ、Ⅱ和Ⅳ型P.gingivalis野生菌株,制备超薄切片,在透射电镜下进行菌体表面结构观察.结果 fimA基因Ⅰ、Ⅱ和Ⅳ型P.gingivalis的菌毛存在差别：Ⅰ型和Ⅱ型菌体表面有放射状菌毛,Ⅱ型略显致密,Ⅳ型表面未见明显菌毛.同时也观察到各型荚膜存在差别：Ⅰ型荚膜最厚,Ⅳ型荚膜较薄,Ⅱ型荚膜最薄.结论 fimA基因Ⅰ、Ⅱ和Ⅳ型P.gingivalis的菌毛和荚膜存在较大差别,推测其粘附、侵入或其它致病能力的差异可能不仅与菌毛有关,还与荚膜或其它因素有关.     

Maturation of fimbria precursor protein by exogenous gingipains in Porphyromonas gingivalis gingipain-null mutant

Porphyromonas gingivalis expresses several virulence factors such as fimbriae and proteases, termed gingipains, which are enzymes that process precursor fimbriae proteins. Thus, gingipain-null mutants lack mature fimbriae. Membrane vesicle-depleted supernatants (VDS) containing soluble gingipains were prepared as an exogenous gingipain fraction. Precursor proteins were treated with VDS and a fimbriated gingipain-null mutant was successfully generated. Experiments showed that the wild strain adhered to and invaded epithelial cells at a greater level than the fimbriated gingipain-null mutant, while adhesion/invasion was prevented in the presence of fetal calf serum, which inhibits gingipain activity. The findings of this study suggest that gingipains expose cellular cryptic ligands in a proteolytic manner and promote fimbriae binding to epithelial cells.     

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.     

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.     

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.     

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。     

Inconsistency between the fimbrilin gene and the antigenicity of lipopolysaccharides in selected strains of Porphyromonas gingivalis

Abstract Immunochemical specificity of lipopolysaccharide an the molecular property of the gene encoding the fimbrilin ( fimA ) of Porphyromonas gingivalis strains were examined using 'fimbriated' strains 381 and HG564 and 'non-fimbriated' strains 381FL and W50. Lipopolysaccharide from strains 381, 381FL and HG564 reacted with monoclonal antibody raised to lipopolysaccharide from strain 381 to give a fused precipitin band by the immunodiffusion test. However, silver staining and Western blotting of lipopolysaccharide clearly revealed a difference in profile of bands between strains 381 and 381FL. On the other hand, lipopolysaccharide from W50 formed another precipitin band and reacted with the antibody, but only at higher concentrations of lipopolysaccharide. The fimA genes in these strains were amplified by polymerase chain reaction and cloned. Sequencing of the fimA gene revealed thatthe fimA _(W50) was almost identical to fimA _(HG564), but a notable difference was observed at the start codon of the open reading frame, while the fimA _(381FL) was considerably different from fimA of other strains and its open reading frame was found to be missing. These results indicate that the molecular structure of the fimA genes of these strains is not homologous, indicating that moe molecular modifications in the fimA gene should occur during in vitro passages and maintenance of strains of P. gingivalis in laboratories.     

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.     

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.     

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.     

Purification and characterization of hemolysin from Porphyromonas gingivalis A7436

Porphyromonas gingivalis, a periodontal pathogen, has the ability to lyse erythrocytes. The hemolytic activity of P. gingivalis A7436 was purified as a 45-kDa protein from the culture supernatant of a 3-days old culture using nickel-nitrilotriacetic acid chromatography. Erythrocytes treated with purified P. gingivalis hemolysin showed the presence of pores and extracellular debris by scanning electron microscopy. Active immunization of mice with 15 micrograms hemolysin induced neutralizing antibodies to hemolysin. Heating at 60 degrees C and treatment with trypsin and dithiothreitol abolished hemolytic activity, while incubation with the protease inhibitor Na-p-tosyl-L-lysine chloromethyl ketone caused no effect. We report here for the first time purification of a hemolysin from P. gingivalis A7436. The amino acid sequence of an internal peptide of hemolysin showed sequence similarity with fimbrillin from P. gingivalis HG564. However, the amino acid composition of purified hemolysin was different from that of P. gingivalis fimbrillin. Also, the ability to lyse but not agglutinate erythrocytes and to bind to nickel-nitrilotriacetic acid differentiates P. gingivalis hemolysin from fimbrillin.     

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.     

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.     

Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components

The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis.