Haemin inhibits the trypsin-like enzyme activity of Porphyromonas gingivalis W83

The effect of haemin and related porphyrins on the activity of the trypsin-like enzyme activity in cell sonicates of Porphyromonas gingivalis was examined using a spectrophotometric assay and by following the degradation of human IgG. Haemin was inhibitory in both assay systems and the effect was shown to be reversible. The high concentration of haemin accumulated by P. gingivalis may protect the organism against autodegradative effects of the trypsin-like protease.     

Lysis of erythrocytes by the secreted cysteine proteinase of Porphyromonas gingivalis W83

The cysteine proteinase produced in the culture supernatant of Porphyromonas gingivalis was extensively purified. Haemagglutination type assays in which the enzyme was titrated against a fixed concentration of erythrocytes, showed that low levels of enzyme directly caused lysis of the red blood cells. However, using the same assay, the presence of stoichiometric amounts of the thiol blocking agent, 2,2'-dipyridyl disulphide (2-PDS) specifically inhibited the action of the enzyme or its haemagglutination with W83 cells or vesicles. In all cases, electron micrographs revealed that in the presence of 2-PDS the erythrocytes remained intact. Thiol activator free enzyme or aerated, inactivated enzyme had no effect on the red blood cells. These results show conclusively that the secreted cysteine proteinase of P. gingivalis causes lysis of erythrocytes and must now be regarded as a potent virulence determinant of P. gingivalis.     

Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III

Abstract We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme ( M _r 47 000) is inhibited by ATIII with an association rate constant ( k _ass) of 5.65 × 10⁴ M⁻¹ s⁻¹ but does not form SDS-stable complexes. Heparin enhances the k _ass and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R³⁹³-S³⁹⁴. In the absence of heparin this is accompanied by N-terminal cleavage between K³⁹-I⁴⁰.     

Hemoglobinase Activity of the Lysine Gingipain Protease (Kgp) of Porphyromonas gingivalis W83

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.     

8-oxo-7,8-dihydroguanine is removed by a nucleotide excision repair-like mechanism in Porphyromonas gingivalis W83

A consequence of oxidative stress is DNA damage. The survival of Porphyromonas gingivalis in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in P. gingivalis under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in P. gingivalis FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-oxoG repair activity was also increased in cell extracts from P. gingivalis FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in Escherichia coli. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to P. gingivalis. Taken together, the increased 8-oxoG levels in P. gingivalis FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in P. gingivalis.     

The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O₂ consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance.     

Generation and characterization of a monoclonal antibody specific for the major thiol-activated cysteine proteinase of Porphyromonas gingivalis W83

An IgM monoclonal antibody specified for the thiol-activated proteinase of the oral pathogen Porphyromonas gingivalis W83 was generated. The antibody reacted with a single protein of approximate molecular mass 43 kDa in outer membrane preparations of P. gingivalis. Immuno-electron microscopy using the monoclonal antibody and immunogold labelling confirmed the cell surface location of the thiol-activated proteinase. The monoclonal antibody failed to detect any proteins in Western blot analysis of other closely related oral bacteria. The specificity of this monoclonal antibody to the thiol-activated proteinase of P. gingivalis should allow its use as a diagnostic tool for the rapid enumeration of P. gingivalis in clinical samples.     

Determination of the Genome Sequence of Porphyromonas gingivalis Strain ATCC 33277 and Genomic Comparison with Strain W83 Revealed Extensive Genome Rearrangements in P. gingivalis

The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).Key words: Porphyromonas gingivalis, whole genome sequence, genome rearrangement, conjugative transposon, MITE     

Identification of a second lipopolysaccharide in Porphyromonas gingivalis W50

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.     

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ～70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.     

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements

Abstract The nucleotide sequence of IS 1126 , the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis , has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transpsosase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposashad homology to putative transposases of IS 1106 and IS 1186 both of which belong to the IS 5 group within the IS₄ super-family of insertion elements. On the basis of this homology we propose that IS 1126 should also be included in the IS 5 group. Southern-blot analysis of a number of P. gingivalis strains using IS 1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS 1126 from other closely related Porphyromonas species. This should allow IS 1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis .     

Cytoglobin Up-regulated by Hydrogen Peroxide Plays a Protective Role in Oxidative Stress

Cytoglobin (Cygb) is a recently discovered intracellular respiratory globin, which exists in all types of cells. It has been suggested that Cygb has a role in protecting cells against oxidative stress. In the present study we have tested this hypothesis. The N2a neuroblastoma cells were exposed to various kinds of insults, including hydrogen peroxide (H₂O₂), hypoxia, kainic acid, high extracellular CaCl₂, high osmolarity, UV irradiation and heat shock. Among them, only H₂O₂-treatment induced a significant up-regulation of cytoglobin mRNA level. We stably transfected N2a cells with Cygb-siRNA vectors and successfully knocked down Cygb. The Cygb-siRNA could exacerbate cell death upon H₂O₂-treatment, as demonstrated by MTT cell viability assay. Thus, Cygb in neuronal cells might be specifically induced under oxidative stress to protect them from death.     

Complete genome sequence of the oral pathogenic Bacterium porphyromonas gingivalis strain W83

The complete 2,343,479-bp genome sequence of the gram-negative, pathogenic oral bacterium Porphyromonas gingivalis strain W83, a major contributor to periodontal disease, was determined. Whole-genome comparative analysis with other available complete genome sequences confirms the close relationship between the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum and the green-sulfur bacteria. Within the CFB phyla, the genomes most similar to that of P. gingivalis are those of Bacteroides thetaiotaomicron and B. fragilis. Outside of the CFB phyla the most similar genome to P. gingivalis is that of Chlorobium tepidum, supporting the previous phylogenetic studies that indicated that the Chlorobia and CFB phyla are related, albeit distantly. Genome analysis of strain W83 reveals a range of pathways and virulence determinants that relate to the novel biology of this oral pathogen. Among these determinants are at least six putative hemagglutinin-like genes and 36 previously unidentified peptidases. Genome analysis also reveals that P. gingivalis can metabolize a range of amino acids and generate a number of metabolic end products that are toxic to the human host or human gingival tissue and contribute to the development of periodontal disease.