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.     

Haemin-restriction influences haemin-binding haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown in a chemostat either under haemin limitation or haemin excess at pH 7.3. Cells and the extracellular vesicle (ECV) and extracellular protein (EP) fractions were separated, quantified, and assayed for haemagglutination, protease activity and haemin binding. Under haemin-limitation, despite a reduction in cell yield, there was a 2.5-fold increase in the gravimetric yield of extracellular vesicles. Cells and vesicles from haemin-limited cultures, haemagglutinated sheep red blood cells to higher titres than their haemin-excess counterparts. Growth in haemin-excess conditions resulted in increased haemin-binding capacities of ECV, cells and EDTA-extracted outer membrane. Cells grown under haemin-excess showed a 2-fold elevation in specific activity towards the substrate N-alpha-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) compared to haemin-limited cells. The specific activities against L-BAPNA for haemin-limited ECV were 3-fold greater than their haemin-excess counterparts. These vesicle activities represented 25% and 3% of the total culture protease activity under haemin limited and haemin excess conditions respectively.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

Roles of porphyrins and host iron transport proteins in regulation of growth of Porphyromonas gingivalis W50.

Porphyromonas gingivalis (Bacteroides gingivalis) requires iron in the form of hemin for growth and virulence in vitro, but the contributions of the porphyrin ring structure, porphyrin-associated iron, host hemin-sequestering molecules, and host iron-withholding proteins to its survival are unknown. Therefore, the effects of various porphyrins, host iron transport proteins, and inorganic iron sources on the growth of P. gingivalis W50 were examined to delineate the various types of iron molecules used for cellular metabolism. Cell envelope-associated hemin and iron stores contributed to the growth of P. gingivalis in hemin-free culture, and depletion of these endogenous reserves required eight serial transfers into hemin-free medium for total suppression of growth. Comparable growth of P. gingivalis was observed with 7.7 microM equivalents of hemin as hemoglobin (HGB), methemoglobin, myoglobin, hemin-saturated serum albumin, lactoperoxidase, cytochrome c, and catalase. Unrestricted growth was recorded in the presence of haptoglobin-HGB and hemopexin-hemin complexes, indicating that these host defense proteins do not sequester HGB and hemin from P. gingivalis. The iron chelator 2,2'-bipyridyl functionally chelated hemin-associated iron, resulting in dose-dependent inhibition of growth in hemin-restricted cultures at 1 to 25 microM 2,2'-bipyridyl concentrations. In the absence of an exogenous iron source, protoporphyrin IX did not support P. gingivalis growth. These findings suggest that the iron atom in the hemin molecule is the critical constituent for growth and that the tetrapyrrole porphyrin ring structure may represent an important vehicle for delivery of iron into the P. gingivalis cell. P. gingivalis does not have a strict requirement for porphyrins, since growth occurred with nonhemin iron sources, including high concentrations (200 muM) of ferric, ferrous, and nitrogenous inorganic iron, and P. gingivalis exhibited unrestricted growth in the presence of host transferrin, lactoferrin, and serum albumin. The diversity of iron substrates utilized by P. gingivalis and the observation that growth was not affected by the bacteriostatic effects of host iron-withholding proteins, which it may encounter in the periodontal pocket, may explain why P. gingivalis is such a formidable pathogen in the periodontal disease process.     

Structural analysis of a novel anionic polysaccharide from Porphyromonas gingivalis strain W50 related to Arg-gingipain glycans

The Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cell-surface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of alpha-1,6-linked mannose residues and the side-chains contain alpha-1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Manalpha1-2Manalpha1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O-phosphorylation of APS abolished cross-reactivity suggesting that Manalpha1-2Manalpha1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.     

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.     

NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells

Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption. Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue. However, whether the cells contribute to bone metabolism in periodontitis remains unknown. In this study, we investigated the effect of P. gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs). We showed that P. gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC. P. gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB. In addition, incubation of OPG with P. gingivalis supernatant resulted in loss of the protein. These results indicate that P. gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria. Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.     

The prpR1 and prR2 arginine-specific protease genes of Porphyromonas gingivalis W50 produce five biochemically distinct enzymes

The arginine-specific protease activity of Porphyromonas gingivalis is considered to be an important factor in the pathogenic potential of this organism in destructive periodontal disease. Multiple forms of closely related Arg-x proteases are present in the culture supernatants of P. gingivalis W50. RI is a heterodimer (α/β) in which the catalytic α chain is associated with a second β chain which functions as a haemagglutinin. RIA is a single-chain enzyme (α) and RIB is a highly post-translationally lipid-modified enzyme (LPS-α) with reduced solubility compared to the other two forms. The N-terminal sequence of the α chain of all three forms is identical, suggesting that all these enzymes may arise by differential processing of the prpR1 (protease polyprotein for RI). In the present study we constructed a prpR1^? strain of P. gingivalis W50 by insertional gene inactivation and characterized the residual extracellular Arg-x protease activity of the resulting mutant. Loss of prpR1 expression led to the abolition of RI, RIA and RIB but the total Arg-x activity in the supernatant of this strain was reduced by only c. 66%. The remaining activity was composed of two novel forms of Arg-x protease (RIIA and RIIB) which appeared to be structurally and kinetically almost identical to RIA and RIB, respectively, except for two amino acid differences in the N-terminus at position 8 (Q→E) and position 17 (A→P) and with respect to their stability to high pH. Confirmation that RIIA and RIIB are the products of a homologous locus (prR2) was obtained by cloning and sequencing the prR2 which showed the predicted substitutions in the deduced translation. These data indicate that RI, RIA and RIB are produced by prpR1 expression and a maturation pathway which can give rise to a dimer and an unmodifed- or LPS-modified catalytic monomer. Furthermore, RIIA and RIIB, the products of prR2, are exported into the culture supernatant in the absence of prpR1 expression and these forms may also contribute to the pathogenic potential of this organism in destructive disease.     

Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia.

A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia. The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components. Under these conditions, heme-starved P. intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M. Heme-starved P. gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M. Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P. intermedia. Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red. Hemopexin also inhibited bacterial hemin binding. These findings indicate that both P. gingivalis and P. intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin. P. gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P. intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition.     

Hemolytic toxin produced by Porphyromonas gingivalis

Abstract Porphyromonas gingivalis no. 381 and ATCC 33277 produced an extracellular hemolytic toxin which was heat-labile, trypsin-sensitive, and lytic to human, horse, sheep and rabbit erythrocytes. The hemolytic toxin is a 'hot-cold', thiol-independent toxin. The production of the hemolytic toxin was greatly enhanced by addition of hemoglobin to the culture medium.     

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.     

Glycylprolyl dipeptidase activity of Bacteroides gingivalis W50 and the avirulent variant W50/BEI

Glycylprolyl dipeptidase activity was measured in cells, extracellular vesicles (ECV) and the soluble extracellular protein fraction (EP) of batch cultures of strains W50 and W50/BEI. Total culture enzyme activity of W50 dropped with age whilst that of W50/BEI remained constant. Activity was highest in the cellular fraction, greater for W50/BEI than W50 and rose with culture age. Both strains showed similar ECV activities but these declined with culture age. The EP glycylprolyl dipeptidase activity of W50/BEI in older cultures rose to a level 13-fold greater than W50. The majority of extracellular activity was represented by the ECV for strain W50 but by EP for W50/BEI. Variable but incomplete attenuation of activity was achieved by dithiothreitol. ECV and EP activities were associated with a high molecular mass fraction, but a smaller fraction (molecular mass 30,000) was detected in W50/BEI EP.