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PubMed1986

Kinetic studies on the nitrite reductase of Wolinella succinogenes.

The six haem groups of the nitrite reductase enzyme isolated from Wolinella succinogenes are rapidly reduced by the addition of dithionite (S2O4(2-)). The reduction, however, is not homogeneous. Two of the haem groups, namely those that show spectral characteristics typical of five-co-ordinated haem groups, are reduced in a dithionite-concentration-dependent fashion with a rate limit of 1.5 S-1. The other four haem groups, which show spectral characteristics very similar to those of normal six-co-ordinate c-haem groups, reduce in a linear dithionite-concentration-dependent manner with a second-order rate constant of 150 M-1/2 X S-1. The ratio of the amplitudes of the two reduction phases observed in stopped-flow studies is found to be dependent on the concentration of dithionite used. A model is proposed to account for these observations, and computer simulations show that the model represents a good fit to the experimental data. The two haem groups with five-co-ordinate spectral characteristics bind CO. Flash photolysis of the CO complex exhibits one major recombination process with a linear dependence in rate on CO concentration with a second-order rate constant of 2 X 10(6) M-1 X S-1. By contrast, stopped-flow mixing of the reduced protein with CO shows a very complex pattern of combination, with most of the observed absorbance change associated with a concentration-independent step. These findings are rationalized in terms of structural changes in the protein consequent to ligand binding.

Bacteroidaceae↗
PubMed1990

The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes.

The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.

Bacteroidaceae↗
PubMed1990

Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.

Bacteroidaceae↗
PubMed1990

An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes.

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.

Anaerobiosis↗
PubMed2002

Invasion of vascular cells in vitro by Porphyromonas endodontalis.

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.

Bacteroidaceae Infections↗
PubMed2004

Chlamydia pneumoniae together with collagenase-2 (MMP-8) in periodontal lesions.

OBJECTIVE: Dental infections may be associated with subsequent atherosclerosis. In this regard we wanted to study if traces of Chlamydia pneumoniae can be found from deep periodontal pockets characterized with elevated collagenase-2 [matrix metalloproteinase (MMP)-8] levels in gingival crevicular fluid (GCF). Chlamydia pneumoniae has not previously been found to infect the oral cavity. SUBJECTS AND METHODS: Subgingival samples of dental plaque were collected from 31 teeth in 12 adult periodontitis patients by means of sterile curettes and examined for C. pneumoniae using a quantitative PCR technique. GCF samples were also collected and assayed by an immunofluorometric assay (IFMA) for MMP-8. RESULTS: Chlamydia pneumoniae RNA was demonstrated in a sample from one of the patients studied. Periodontal treatment eliminated the C. pneumoniae from the patient's subgingival dental plaque as well as reduced GCF MMP-8 level. CONCLUSIONS: Our findings suggest that C. pneumoniae, which is not normally thought to be involved in periodontitis, can be found in dental plaque.

Adult↗
PubMed1998

Characteristics of Prevotella intermedia-specific CD4+ T cell clones from peripheral blood of a chronic adult periodontitis patient.

Periodontitis is a chronic destructive inflammatory disease associated with periodontopathic bacteria. In addition, autoantigens such as collagen and heat shock proteins (hsp) have been suggested to play a role. Established periodontal lesions are characterized by dense infiltrations of immune cells such as cytokine-producing CD4+ and CD8+ T cells. CD4+ T cells specific for Prevotella intermedia can be isolated from lesional gingiva, suggesting an active role for CD4+ T cells in the response to this bacterium. We therefore investigated the characteristics of a panel of 13 P. intermedia-specific CD4+ T cells generated from the peripheral blood of a patient with chronic adult periodontitis. All 13 P. intermedia-specific CD4+ T cells recognized the antigens in the context of HLA-DR. The T cell clones were mainly classified as Th0, producing comparable amounts of interferon-gamma (IFN-gamma) and IL-4, and Th2, producing high amounts of IL-4 and almost no IFN-gamma. None of the P. intermedia-specific T cell clones recognized antigens of the periodontopathic bacteria Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis and of the autoantigens collagen and hsp. The reactivity profile of the T cell clones to size-fractionated cell envelope antigens of P. intermedia indicated that P. intermedia-specific CD4+ T cell clones recognize probably five different antigen specificities in the context of the MHC class II molecules, DR7 or DR15. These results suggest that a broad panel of cell-associated protein antigens play a role in the induction of P. intermedia-specific CD4+ T cell response.

Adult↗
PubMed2000

Elevated CTLA-4 expression on CD4 T cells from periodontitis patients stimulated with Porphyromonas gingivalis outer membrane antigen.

To characterize the T cell response to Porphyromonas gingivalis, we examined the expression of costimulatory molecules on T cells derived from adult periodontitis patients with high serum antibody titre to P. gingivalis. The expression of CD28, CTLA-4, CD40 ligand (CD40L) on CD4+ T cells was analysed by flow cytometry. IL-10 and transforming growth factor-beta (TGF-beta) mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent image analysis. Peripheral blood mononuclear cells (PBMC) derived from periodontitis patients showed higher proliferative responses to P. gingivalis outer membrane (OM) than those from healthy controls (P < 0.05). The percentage of CTLA-4+ cells within CD4+ T cells of patients was significantly higher than that of healthy controls after P. gingivalis OM stimulation (33.0% versus 11.9%, P < 0.01). There was no significant difference in the percentages of CD28+ cells and CD40L+ cells, and the percentage of CD40L+ cells was low in both groups even after stimulation. Stimulation of PBMC with P. gingivalis OM induced significantly higher IL-10 mRNA expression in periodontitis patients than in healthy controls (P < 0.05). The level of TGF-beta mRNA expression of patients tended to be higher than that of healthy controls, but there was no significant difference. To elucidate the functional role of CTLA-4, we further investigated the secondary proliferative response to P. gingivalis OM. Interestingly, P. gingivalis OM stimulation did not enhance antigen-specific secondary response. Anti-CTLA-4 MoAb had no effect on proliferation in the presence of P. gingivalis OM. CTLA-4Ig suppressed the proliferative response significantly (P < 0.01). These results suggest that T cell responses to P. gingivalis OM may be regulated by CTLA-4 that is expressed at the late phase of T cell activation, and, in part, immunosuppressive cytokines. Taken together, CTLA-4 may play a crucial role in the pathogenesis of chronic inflammatory periodontal disease.

Abatacept↗
PubMed2002

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model.

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti-F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.

Animals↗
PubMed1998

In vivo exposure to Porphyromonas gingivalis up-regulates nitric oxide but suppresses tumour necrosis factor-alpha production by cultured macrophages.

The present study was designed to test whether the functional response of mouse macrophages elicited by chronic exposure to bacteria will be different from that of cells elicited by a non-bacterial irritant. Macrophage elicitation was conducted by Porphyromonas gingivalis, a major periodontal pathogen, in comparison to a standard elicitation by thioglycollate (TG). We measured lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) secretion by the elicited macrophages, and the expression of inflammatory cytokines in the whole elicited cell population. In addition, we tested the response of TG-elicited macrophages to pretreatment with P. gingivalis LPS in vitro. Mouse peritoneal macrophages were harvested 4 days after intraperitoneal injection of TG or heat-killed P. gingivalis. TG-elicited macrophages produced undetectable levels of TNF-alpha and approximately 0.5 microM of NO. The stimulation of the macrophages with LPS resulted in the secretion of NO and TNF-alpha in a dose-dependent manner. The P. gingivalis-elicited macrophages produced basal levels of approximately 5 microM NO, but TNF-alpha was not detectable. LPS stimulation of these cells further increased the secretion of NO eightfold while TNF-alpha remained undetectable. The NO secretion by P. gingivalis-elicited cells was significantly higher than that by TG-elicited cells. Examination of cytokine expression in the whole elicited cell population revealed that both P. gingivalis-elicited cells and TG-elicited cells expressed messenger RNA for interleukin-2 (IL-2), TNF-alpha and interferon-gamma (IFN-gamma), but not for IL-4. IL-6 was expressed in P. gingivalis-elicited cells only. Pretreatment of TG-elicited macrophages with P. gingivalis LPS for 24 hr prior to a second LPS challenge resulted in down-regulation of TNF-alpha secretion and up-regulation of NO secretion, a response similar to that seen in P. gingivalis-elicited peritoneal macrophages. The results suggest that the in vivo exposure of resident macrophages to P. gingivalis induces functional changes in peritoneal macrophages. These changes might be due to the effect of P. gingivalis LPS.

Animals↗
PubMed2003

Induction of interleukin-8 gene expression by black-pigmented Bacteroides in human pulp fibroblasts and osteoblasts.

AIM: To investigate the effect of black-pigmented Bacteroides on the expression of interleukin (IL)-8 gene in human pulp fibroblasts and osteoblasts. METHODOLOGY: The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. RESULTS: Investigations of the time-dependence of IL-8 mRNA expression in black-pigmented Bacteroides-treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24-h incubation period. In addition, IL-8 mRNA gene expression was also found in human osteoblasts stimulated with black-pigmented Bacteroides. However, black-pigmented Bacteroides was found to be more effective in the induction of IL-8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P < 0.05). CONCLUSIONS: Black-pigmented Bacteroides are capable of amplifying the local immune response and promoting pulpal/periapical tissue inflammation by stimulating pulp fibroblasts and osteoblasts to express IL-8.

Analysis of Variance↗
PubMed2003

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets.

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.

Animal Feed↗
PubMed2002

CD14-mediated induction of interleukin-8 and monocyte chemoattractant protein-1 by a heat-resistant constituent of Porphyromonas gingivalis in endothelial cells.

Viable and inactivated Porphyromonas gingivalis dose-dependently induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion in human umbilical vein endothelial cells (HUVECs). The inactivated P. gingivalis, in comparison with viable bacteria, tended to enhance the production of both chemokines more strongly. The production of MCP-1 protein began increasing immediately after stimulation by P. gingivalis, and there was a nearly linear increase from 0 to 8 h of incubation, whereas IL-8 production showed a linear increase between 4 and 12 h of incubation. The IL-8 and MCP-1 mRNA expressions in HUVECs as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) or Quantikine mRNA colorimetric quantification kits were found to be enhanced by P. gingivalis. Furthermore, the time courses of IL-8 and MCP-1 mRNA expressions were in accordance with those of protein production. Addition of polymyxin B or boiling did not weaken the stimulatory effect of P. gingivalis, which inhibited the effect of Escherichia coli lipopolysaccharide (E. coli LPS) and tumour necrosis factor-alpha (TNF-alpha), respectively. In contrast, the induction of IL-8 and MCP-1 by P. gingivalis was significantly reduced by anti-CD14 antibody. Our results suggest that some heat-stable component of P. gingivalis, including LPS, may be responsible for the induction of IL-8 and MCP-1 in HUVECs by a CD14-dependent mechanism. These effects might be involved in the accumulation and activation of neutrophils and monocytes at an early stage of the periodontal pathogenesis.

Bacteroidaceae Infections↗
PubMed2003

Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus.

Flexibacter maritimus, a Gram-negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea-caged salmonids. For the development of serology based diagnostics, protective vaccines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O-specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disaccharide repeating unit built of 2-acetamido-3-O-acetyl-4-[(S)-2-hydroxyglutar-5-ylamido]-2,4,6-trideoxy-beta-glucose and 5-acetamido-7-[(S)-3-hydroxybutyramido]-8-amino-3,5,7,8,9-pentadeoxynonulopyranosonic acid (Sug) having the structure: The configuration of the C-2-C-7 fragment of the latter monosaccharide (B) was assigned beta-manno; however, the configuration at C-8 could not be established. NMR data indicate that the two monosaccharides have opposite absolute configurations. The repeating unit includes a linkage via a (S)-2-hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disaccharide beta-Sug-(2-3)-l-Rha, isolated from the products of the acidic methanolysis of the LPS.

Bacteroidaceae↗
PubMed1999

Ecology of genus Porphyromonas in canine periodontal disease.

Asaccharolytic pigmented Porphyromonas species, including P. endodontalis, P. gingivalis, P. circumdentaria and unclassified species, were isolated from the plaque of adult dogs, but not from any oral sites of puppies and adolescent dogs. With age-dependency, the proportion of Porphyromonas species in the flora of plaque increased. Isolation of the genus Porphyromonas was clearly associated with the progress of periodontol disease. We suggested that Porphyromonas is the exogenous organism and obligate pathogen for canine periodontal diseases.

Animals↗
PubMed2003

Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche.

At present, there is little information on the phylogenetic diversity of microbial species that inhabit the gastrointestinal tracts of wildlife. To increase understanding in this area, we initiated a characterization of the bacterial diversity in the digestive tracts of three wild African ruminant species namely eland (Taurotragus oryx), Thompson's gazelle (Gazella rufifrons) and Grant's gazelle (Gazella granti), together with a domesticated ruminant species, zebu cattle (Bos indicus), and a non-ruminant species, zebra (Equus quagga). Bacterial diversity was analysed by PCR amplification, sequencing and phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences. A total of 252 full-length 16S rDNA sequences averaging 1,500 base pairs (bp) in length, and an additional 27 partial sequences were obtained and subject to phylogenetic analysis. Using a 98% criterion for similarity, all except for one of the sequences were derived from distinct phylotypes. At least 24 distinct operational taxonomic units (OTU's) could be identified, with the majority of these sequences representing hitherto uncharacterized species and genera. The sequences were generally affiliated with four major bacterial phyla, the majority being members of the Firmicutes (low G+C Gram-positives) related to the genera Clostridium and Ruminococcus. By contrast, with earlier studies using 16S rDNA sequences to assess biodiversity in Bos taurus dairy cattle, Gram-negative bacteria in the Bacteroidales (Prevotella-Bacteroides group) were poorly represented. The lack of redundancy in the 16S rDNA dataset from the five African ungulate species, and the presence of novel sequences not previously described from the gastrointestinal tract of any animal species, highlights the level of diversity that exists in these ecosystems and raises the question as to the functional role of these species in the gastrointestinal tract.

Animals↗
PubMed1998

Large plasmids in ruminal strains of Selenomonas ruminantium.

The plasmid content of six different isolates of Selenomonas ruminantium from the rumen of sheep, cows or goats was examined by electron microscopy. In addition to small plasmids (< 12 kb) studied previously, all six strains contained at least one plasmid larger than 20 kb. Plasmid sizes of 1.4, 2.1, 2.4, 5.0, 6.2, 20.4, 20.8, 22.7, 23.3, 29.3, 30.7, 34.4 and 42.6 kb were estimated from contour length measurements. DNA-DNA hybridization experiments revealed homology among the large plasmids from five strains, while the 20.8 kb plasmid from a sixth isolate showed no apparent relationship with the plasmids of the other strains.

Animals↗