Porphyromonas gulae lipopolysaccharide elicits inflammatory responses through toll-like receptor 2 and 4 in human gingivalis epithelial cells.

Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, has been associated with periodontal disease in companion animals and its virulence has been attributed to various factors, including lipopolysaccharide (LPS), protease and fimbriae. Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns, such as peptidoglycan, lipids, lipoproteins, nucleic acid and LPS. Following P. gulae infection, some inflammatory responses are dependent on both TLR2 and TLR4. In addition, a recent clinical study revealed that acute and persistent inflammatory responses enhance the expressions of TLR2 and TLR4 in the oral cavity. In this study, we investigated the interaction between P. gulae LPS and human gingivalis epithelial cells (Ca9-22 cells). P. gulae LPS was found to increase TLR2 and TLR4 mRNA expressions and protein productions, and enhanced inflammatory responses, such as COX2 , TNF-ɑ, IL-6 and IL-8. Stimulated Ca9-22 cells exhibited phosphorylation of ERK1/2 and p38, and their inhibitors diminished inflammatory responses, while knockdown of the TLR2 and/or TLR4 genes with small interfering RNA (siRNA) prevented inflammatory responses. Moreover, p38 and ERK1/2 phosphorylation was decreased in TLR2 and TLR4 gene knockdown cells. These findings suggest that P. gulae LPS activates p38 and ERK1/2 via TLR2 and TLR4, leading to inflammatory responses in human gingival epithelial cells.

Purification and characterization of a fimbrial protein from Porphyromonas salivosa ATCC 49407.

Periodontal disease is a significant problem in companion animals such as dogs and cats. However, there is little information available about fimbriae association of periodontal disease in companion animals. In this study, we have purified and characterized a fimbriae from Porphyromonas salivosa ATCC 49407. The molecular mass of this protein was approximately 60-kDa, as estimated by SDS-PAGE. Immunogold electron microscopy revealed that anti-60-kDa fimbrial serum bound to fimbria on the cell surface of P. salivosa ATCC 49407. However, fimbriae of P. gingivalis and P. gulae were not labeled with the same antibody. Immunoelectron-microscopic studies and immunoblot analysis revealed that antigenicity and molecular weight were distinct from previously reported Porphyromonas fimbrial proteins. The amino acid sequence of the N-terminal 15 residues of the 60-kDa fimbrillin protein revealed only 3 of 15 residues identical to other Porphyromonas species fimbrillin proteins. Thus, the N-terminal amino acid sequence of the 60-kDa fimbrillin protein of P. salivosa clearly differed from previously reported fimbrillin proteins. The level of adherence of the P. salivosa was 1.81%. It was confirmed that P. salivosa can adheres to human cells. These results suggest that the 60-kDa fimbriae of P. salivosa ATCC 49407 is a new type of fimbria and may have an important factor in the adherence host cells. We suggest that the surface structure of P. salivosa may have a role in the colonization of this organism in periodontal pockets in companion animals.

Identification of Porphyromonas isolates from clinical origin using MALDI-TOF Mass Spectrometry.

Despite the wide implementation of MALDI-TOF MS for the rapid and reliable identification of most microorganisms, some taxonomic groups such as the Porphyromonas genus remain largely untested. In this study we evaluated the performance of MALDI-TOF MS on this genus using a collection of 39 isolates sent for routine identification to our institution over a 16-year period. All of them were identified by DNA-sequencing analysis of the 16S rRNA gene plus the hsp60 gene when the previous one did not yield species-level assignment. MALDI-TOF MS provided correct identification at least at the genus level of 21/39 isolates (53.9%). Twelve isolates were correctly identified at the species level with a score value ≥ 2.0 and 9 more with score values < 2.0 and ≥ 1.7. The species most represented in the database (P. gingivalis and P. somerae) lay within this category. However, the species poorly represented in this database (P. asaccharolytica and P. uenonis) were mostly identified with lower scores (1.35-1.67) or remained unidentified by MALDI-TOF MS. The addition of two P. asaccharolytica reference spectra to our in-house library allowed 72.9% of genus-level identifications with 17/37 isolates (45.9%) identified with score values ≥ 2.0. Our results showed a high level of correlation between MALDI-TOF MS and DNA-based identification for Porphyromonas spp. strains at the species level, even with score values < 2.0. The reliability provided by MALDI-TOF MS increased when the database was fed with spectra from the species poorly represented in the commercial database.

Simultaneous Determination of Absolute Configuration and Quantity of Lipopeptides Using Chiral Liquid Chromatography/Mass Spectrometry and Diastereomeric Internal Standards.

Lipopeptides promote innate immune response and are related to disease pathology. To investigate the newly emerging roles of lipopeptides, accurate measurements of stereoisomers with multiple chiral centers are essential yet challenging. This work uses (3R)- and (3S)-(15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, abbreviated as l-serine-(R+S)-Lipid 654, to develop a method that combines chiral liquid chromatography, a diastereomeric mixture of isotopically labeled internal standards, and multiple reaction monitoring mass spectrometry. The new method allows for simultaneously determining the absolute configuration and quantity of stereoisomers of bacteria-derived lipopeptides. Total lipid extracts of nine evaluated bacteria strains had different amounts, but only the (R)-isoform of l-serine-Lipid 654. The developed method also allowed for the first quantitative analysis of hydrolysis of a nonphospholipid as a novel substrate of honey bee venom phospholipase A2.

First report on sepsis caused by Porphyromonas pogonae.

We report on a 62 year old patient who developed sepsis due to an infection caused by Porphyromonas pogonae, a recently described species of the bacterial genus Porphyromonas. This is the first case of an invasive infection with this pathogen.

Porphyromonas bronchialis sp. nov. Isolated from Intraoperative Bronchial Fluids of a Patient with Non-Small Cell Lung Cancer.

Porphyromonas strains, including Porphyromonas-like strains, have been isolated from oral and various other systemic infections. The characterization of such strains is a crucial issue, because such information contributes to both the taxonomy of anaerobic bacteria and the clinical aspects of infectious diseases. We previously isolated four Porphyromonas-like strains from intraoperative bronchial fluids of a patient with non-small cell lung cancer. This study aimed to characterize the genetic, biochemical and chemotaxonomic aspects of these isolates. Each strain only grew under anaerobic conditions and their colony morphology was convex, 0.1-1.0 mm in diameter, light gray, and slightly glistening colony, with no black or brown pigmentation on blood agar plates after five-day incubation. The pigmentation was helpful to differentiate the isolates from other Porphyromonas, as most of Porphyromonas species show the pigmentation. In the 16S rRNA gene phylogenetic analysis (98% sequence identity of isolates indicates the same species), the four isolates were closely related to one another (99.7-100.0%), but not related to Porphyromonas (P.) catoniae, the closest species (96.9%). In addition, the DNA-DNA hybridization data revealed less than 16% similarity values between a representative isolate and the P. catoniae, indicating that the strains were genetically independent. Biochemically, the isolates could be differentiated from closely related species, i.e., P. catoniae, P. gingivalis, P. gulae, and P. pogonae, with trypsin activity (negative only in the isolates) and leucine arylamidase activity (positive only in the isolates). We therefore propose a new species to include these isolates: Porphyromonas bronchialis sp. nov.

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro.

OBJECTIVE: To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7. SAMPLE POPULATION: Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot). PROCEDURES: Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O(2)and H(2)O(2), respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining. RESULTS: All bacteria produced at least 3 of the 4 SCFAs. Production of both O(2) and H(2)O(2) was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O(2) production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H(2)O(2) production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5. CONCLUSIONS AND CLINICAL RELEVANCE: Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.

Isolation and identification of a cytopathic activity in Tannerella forsythia.

Interactions between pathogens and host induce human disorders including periodontitis, disintegration of the tooth supporting tissues. Tannerella forsythia has been linked to the periodontitis and several cytopathic reagents have been found in the bacterium; however, its contribution to the disease remains unclear. Biochemical approach to explore the cytopathic effect revealed two distinct activities in T. forsythia (ATCC 43037) extract; one detaches adherent cells from substratum and another arrests cells at G2. An executor of former activity, forsythia detaching factor (FDF) was identified; its genomic sequence and peptidase activity revealed that FDF is a substantial form of putative PrtH; prtH gene was hypothetically identified directly from a DNA fragment of the bacterium and its native product has never been shown. Since FDF was found in the bacterial culture supernatant, its activity implies a contribution to the disintegration of tissues although the mechanism how FDF disturbs cellular anchors remains elusive.

Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources.

During studies on the bacteriology of appendicitis in children, we often isolated from inflamed and non-inflamed tissue samples, an unusual bile-resistant pigment-producing strictly anaerobic gram-negative rod. Phenotypically this organism resembles members of Bacteroides fragilis group of species, as it is resistant to bile and exhibits a special-potency-disk pattern (resistance to vancomycin, kanamycin and colistin) typical for the B. fragilis group. However, the production of brown pigment on media containing haemolysed blood and a cellular fatty acid composition dominated by iso-C15:0, suggests that the organism most closely resembles species of the genus Porphyromonas. However, the unidentified organism differs from porphyromonads by being bile-resistant and by not producing butyrate as a metabolic end-product. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism represents a distinct sub-line, associated with but distinct from, the miss-classified species Bacteroides putredinis. The clustering of the unidentified bacterium with Bacteroides putredinis was statistically significant, but they displayed > 4% sequence divergence with each other. Chromosomal DNA-DNA pairing studies further confirmed the separateness of the unidentified bacterium and Bacteroides putredinis. Based on phenotypic and phylogenetic considerations, it is proposed that Bacteroides putredinis and the unidentified bacterium from human sources be classified in a new genus Alistipes, as Alistipes putredinis comb. nov. and Alistipes finegoldii sp. nov., respectively. The type strain of Alistipes finegoldii is CCUG 46020(T) (= AHN243(T)).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proteomics: a new era in anaerobic microbiology.

Genome sequence data provide a framework for predicting potential microbial activities; however, the proteome content of the cell dictates its response to its environment. Microbiology is witnessing a major initiative to elucidate the nature of the proteome of large numbers of species. The tool driving the proteomic revolution is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. During the analysis process, proteins are ionized and separated on the basis of their mass-to-charge ratios, which results in a characteristic mass-spectral profile. Because of the dynamic nature of the cell and the large number of external parameters that could influence its mass-spectral profile, considerable work was needed initially to optimize sample analysis and obtain consistent and reproducible results. For many anaerobes that grow poorly or are nonreactive in most diagnostic systems, proteome analysis is likely to have a major impact on microbial diagnosis and the delineation of centers of diversity associated with infections.

Structural characterization of the O-antigenic polysaccharide chain of Porphyromonas circumdentaria NCTC 12469.

Structural studies were carried out on an O-antigenic polysaccharide moiety derived from Porphyromonas circumdentaria NCTC 12469, a reference strain of Porphyromonas species. The polysaccharide chain was composed of D-glucose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-galactosamine in a molar ratio of 1:2:1:1. On the basis of results from 1H- and 13C-NMR spectroscopic analyses including COSY, TOCSY, and HMQC experiments together with results of Smith degradation, methylation analysis, and partial acid hydrolysis, it is concluded that the polysaccharide chain has a pentasaccharide repeating unit of -->6)-beta-D-Glcp-(1-->6)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)-beta-D-GalpNAc-(1-->. The immunoreaction between P. circumdentaria LPS and the corresponding antiserum was strongly inhibited by the pentasaccharide fragment (Glc-Gal-Gal-GlcNAc-GalNAc) isolated from partial acid hydrolysis of the above polysaccharide, suggestive of O-antigen specific antibodies in the used antiserum.

Anaerobic bacterial extracts influence production of matrix metalloproteinases and their inhibitors by human dental pulp cells.

The role of human dental pulp (HDP) cells in extracellular matrix degradation in pulpitis is still unclear. In this study, the effects of sonicated bacterial extracts (SBEs) from Prevotella intermedia, Fusobacterium nucleatum, Porphyromonas endodontalis, and Porphyromonas gingivalis on the balance between the production of matrix metalloproteinases (MMPs) and that of their inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] by HDP cells were examined. HDP cells were treated with SBEs, and their culture media were later harvested. MMP activities and TIMP concentrations were determined by use of independent measurement strategies and sensitive ELISAs. The production of MMP-1 and MMP-2 was accelerated by all SBE. On the other hand, TIMP-1 production was slightly elevated; and TIMP-2 production was markedly inhibited by all of the extracts. SBEs derived from these anaerobic bacteria seemed to affect the acceleration of extracellular matrix degradation activity by HDP cells. These findings suggest that HDP cells stimulated by bacterial byproducts may be involved in the pathogenesis of pulpitis.

Inflammatory cytokine production and specific antibody responses to lipopolysaccharide from endodontopathic black-pigmented bacteria in patients with multilesional periapical periodontitis.

We examined the induction of the cytokines interleukin (IL)-1 beta, IL-6, and IL-8 by lipopolysaccharides (LPSs) from several species of possible endodontopathic black-pigmented bacteria. Studies were conducted in human whole blood cultures from six patients (two from each group) with differing numbers of periapical periodontitis lesions (i.e. patients with radiographically clear periapical lesions in 10 or more teeth (high-lesion group, n = 4), in one or two teeth (low-lesion group, n = 6), and six healthy volunteers with no periapical lesions (no lesion group)). LPS from Prevotella intermedia ATCC 25611, Porphyromonas gingivalis 381, and Prophyromonas endodontalis ATCC 27067 induced a higher IL-8 response in the subjects of the high-lesion group, compared with the subjects of the other two groups. To ascertain the degree of sensitization by test bacteria, we examined the reactivities of antibodies in serum and saliva from the subjects to different bacterial species. LPS from P. gingivalis reacted strongly with sera from the high-lesion group. Thus, LPS from black-pigmented bacteria may be involved in multilesional periapical periodontitis by inducing particular cytokines and/or humoral immune responses.

Cytotoxic effect of endodontic bacteria on periapical fibroblasts.

This study was conducted to investigate the effects of sonicated bacterial extracts (SBEs) from anaerobic Gram-negative bacteria on periapical fibroblast obtained from the apical portion of human periodontal ligaments. Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were chosen from among the endodontic bacteria isolated from root canals having a periapical lesion and compared in terms of their cytotoxicity. The purpose of this study was to examine which bacteria are involved in the development of periapical inflammation. The anaerobes were cultured under strict anaerobic conditions, and the bacterial cells were then harvested by centrifugation after incubation. The concentrated cell suspensions were sonicated and subsequently centrifuged. An SBE was made of each of the filtered supernatants. Each SBE was added to cultures of periapical fibroblasts. The cell growth and proliferation were measured by the MTT method after 3, 5, and 7 days. The SBEs from P. endodontalis, P. gingivalis, and F. nucleatum inhibited the growth of the fibroblasts, whereas the SBE from P. intermedia did not inhibit it. The SBEs from P. gingivalis and F. nucleatum inhibited the fibroblast growth more strongly than did the P. endodontalis, P. gingivalis, and F. nucleatum may participate in the development of periapical lesions.

Phospholipid molecular species distribution of Porphyromonas asaccharolytica ATCC 25260T: effects of temperature, culture age and pH.

The aim of this study was to determine the extent to which phospholipid molecular species profiles are affected by different environmental factors in Porphyromonas asaccharolytica ATCC 25260T. Phospholipids were analysed by Fast Atom Bombardment Mass Spectrometry (FAB-MS) in negative-ion mode. Under standard growth conditions (37 degrees C, pH 7.0, 48 h), the most intense high mass anions were m/z 653 and 662. The latter is consistent with the expected presence of PE (30:0). The only changes in profiles were quantitative. These were compared using the Pearson Coefficient of Linear Correlation. The r-values for initial pH comparisons ranged from 0.82 (pH 7.0 vs pH 6.0) to 1.00 (pH 5.0 vs pH 8.0), for incubation period, from 0.86 (48 vs 72 h) to 0.97 (96 vs 168 h), and for temperature, from 0.57 (40 vs 37 degrees C) to 0.96 (37 vs 36 degrees C). Differences were also seen when plates were incubated in anaerobe jars as opposed to an anaerobic work station (r = 0.75). It is concluded that it is essential to standardize growth parameters, and to use an anaerobe jar or an anaerobe work station, but not both.

Comparison of lipopolysaccharides from Bacteroides, Porphyromonas, Prevotella, Campylobacter and Wolinella spp. by tricine-SDS-PAGE.

Lipopolysaccharides (LPSs) of 11 bacterial strains from the type species of the genera Bacteroides (B. fragilis), Prevotella (Pr. melaninogenica), Porphyromonas (Po. gingivalis), Campylobacter (C. fetus subsp. fetus), and Wolinella (W. succinogenes), and from the type strains of B. distasonis, B. forsythus, B. ureolyticus, Po. levii, Po. macacae, and C. gracilis, were extracted with hot water-phenol (Westphal method). S-form LPSs, obtained from all organisms, were well resolved with tricine-sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and visualized by silver staining. Lipid A was not stained. Also profiles from LPS of Escherichia coli, serotypes 0111:B4 and 055:B5, could be distinguished. While W. succinogenes showed a relatively short S-form LPS on electrophoregrams, the other bacteria, including B. fragilis, exhibited long-ladder LPSs. Po. gingivalis displayed the largest number of bands and the longest O-chain. The long O-chain of this bacterium may be important for its virulence.

Stimulation of interleukin-1 beta production of human dental pulp cells by Porphyromonas endodontalis lipopolysaccharide.

IL-1 beta is synthesized as an inactive precursor, which is subsequently processed by IL-1 beta converting enzyme (ICE) and found extracellularly as a mature biologically active polypeptide. Also, IL-1 beta has been detected in necrotic and inflamed dental pulp. We examined the IL-1 beta production in human dental pulp (HDP) cells treated with lipopolysaccharide (LPS) from Porphyromonas endodontalis (P. e.) isolated from root canals and radicular cyst fluids. We demonstrated that P. e. LPS stimulated IL-1 beta release from HDP cells in a time- and dose-dependent manner. However, ICE activity was not increased by P. e. LPS. Northern blot hybridization analysis revealed that the IL-1 beta mRNA level in HDP cells was increased by P. e. LPS. These results suggest that stimulation of IL-1 beta release from HDP cells by P. e. LPS may have an important role in the progression of inflammation in pulpal and periapical disease.

Stimulation of interleukin-6 production of periodontal ligament cells by Porphyromonas endodontalis lipopolysaccharide.

Interleukin-6 (IL-6), which is a multifunctional cytokine, has important roles in acute and chronic inflammation and may also be implicated in bone resorption. We examined the IL-6 production in periodontal ligament (PDL) cells which were treated with lipopolysaccharide (LPS) from several oral inflammatory pathogens. The LPS from Porphyromonas endodontalis, which was isolated from infected root canals and radicular cyst fluids, was more potent than the LPS from any other periodontal organisms examined. P. endodontalis LPS stimulated IL-6 release from PDL cells in a time- and dose-dependent manner. Northern blot hybridization analysis revealed that the IL-6 mRNA level in PDL cells was increased by P. endodontalis LPS. These results suggest that stimulation of the IL-6 release of PDL cells by P. endodontalis LPS may have a role in the progression of inflammation and alveolar bone resorption in periodontal and periapical diseases.