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Microbiology (Reading, England) Apr 2018Porphyromonas gingivalis produces hydrogen sulfide (H2S) from l-cysteine. However, the role of H2S produced by P. gingivalis in periodontal inflammation is unclear. In...
Porphyromonas gingivalis produces hydrogen sulfide (H2S) from l-cysteine. However, the role of H2S produced by P. gingivalis in periodontal inflammation is unclear. In this study, we identified the enzyme that catalyses H2S production from l-cysteine and analysed the role of H2S using a mouse abscess model. The enzyme identified was identical to methionine γ-lyase (PG0343), which produces methyl mercaptan (CH3SH) from l-methionine. Therefore, we analysed H2S and CH3SH production by P. gingivalis W83 and a PG0343-deletion mutant (ΔPG0343) with/without l-cysteine and/or l-methionine. The results indicated that CH3SH is produced constitutively irrespective of the presence of l-methionine, while H2S was greatly increased by both P. gingivalis W83 and ΔPG0343 in the presence of l-cysteine. In contrast, CH3SH production by ΔPG0343 was absent irrespective of the presence of l-methionine, and H2S production was eliminated in the absence of l-cysteine. Thus, CH3SH and H2S production involves different substrates, l-methionine or l-cysteine, respectively. Based on these characteristics, we analysed the roles of CH3SH and H2S in abscess formation in mice by P. gingivalis W83 and ΔPG0343. Abscess formation by P. gingivalis W83, but not ΔPG0343, differed significantly in the presence and absence of l-cysteine. In addition, the presence of l-methionine did not affect the size of abscesses generated by P. gingivalis W83 and ΔPG0343. Therefore, we conclude that H2S produced by P. gingivalis does not induce inflammation; however, H2S enhances inflammation caused by CH3SH. Thus, these results suggest the H2S produced by P. gingivalis plays a supportive role in inflammation caused by methionine γ-lyase.
Topics: Abscess; Animals; Bacterial Proteins; Bacteroidaceae Infections; Carbon-Sulfur Lyases; Cysteine; Disease Models, Animal; Female; Gene Deletion; Hydrogen Sulfide; Methionine; Mice, Inbred BALB C; Porphyromonas gingivalis; Sulfhydryl Compounds; Virulence
PubMed: 29488863
DOI: 10.1099/mic.0.000640 -
Proceedings of the National Academy of... Jan 2022Bacterial behavior and virulence during human infection is difficult to study and largely unknown, as our vast knowledge of infection microbiology is primarily derived...
Bacterial behavior and virulence during human infection is difficult to study and largely unknown, as our vast knowledge of infection microbiology is primarily derived from studies using in vitro and animal models. Here, we characterize the physiology of , a periodontal pathogen, in its native environment using 93 published metatranscriptomic datasets from periodontally healthy and diseased individuals. transcripts were more abundant in samples from periodontally diseased patients but only above 0.1% relative abundance in one-third of diseased samples. During human infection, highly expressed genes encoding virulence factors such as fimbriae and gingipains (proteases) and genes involved in growth and metabolism, indicating that is actively growing during disease. A quantitative framework for assessing the accuracy of model systems showed that 96% of genes were expressed similarly in periodontitis and in vitro midlogarithmic growth, while significantly fewer genes were expressed similarly in periodontitis and in vitro stationary phase cultures (72%) or in a murine abscess infection model (85%). This high conservation in gene expression between periodontitis and logarithmic laboratory growth is driven by overall low variance in gene expression, relative to other pathogens including and Together, this study presents strong evidence for the use of simple test tube growth as the gold standard model for studying biology, providing biological relevance for the thousands of laboratory experiments performed with logarithmic phase Furthermore, this work highlights the need to quantitatively assess the accuracy of model systems.
Topics: Animals; Bacteroidaceae Infections; Fimbriae, Bacterial; Gingipain Cysteine Endopeptidases; Humans; Laboratories; Mice; Periodontitis; Porphyromonas gingivalis; Transcriptome; Virulence; Virulence Factors
PubMed: 34992142
DOI: 10.1073/pnas.2116637119 -
Journal of Microbiology, Immunology,... Jun 2014The coaggregation of bacteria has been defined as one of the most important processes in the oral infection such as periodontitis. Prevotella oris and Porphyromonas...
BACKGROUND/PURPOSE
The coaggregation of bacteria has been defined as one of the most important processes in the oral infection such as periodontitis. Prevotella oris and Porphyromonas gingivalis, which are two of the periodontopathogens, are frequently detected in severe forms of periodontal diseases. However, the interaction between P. oris and P. gingivalis is still unknown. In this study, the coaggregation of P. oris with nine oral bacterial species including P. gingivalis was examined.
METHODS
All bacteria used in this study were cultured anaerobically and suspended in coaggregation buffer. Each cell suspension was mixed in a test tube and subjected to shaking at room temperature for 1 hour. Subsequently, the coaggregation values were scored. Furthermore, the effects of various chemical reagents, and heat, proteinase K, and serum treatment were examined.
RESULTS
In this study, P. oris coaggregated only with P. gingivalis. A heat-stable, nonproteinous component of P. oris and a heat-labile, proteinous component of P. gingivalis play important roles in this coaggregation. In addition, this coaggregation was inhibited by l-arginine, l-lysine, and Nα-p-tosyl-l-lysine. Therefore, it was considered that a cell surface protein on P. gingivalis, such as gingipain, may be involved in the coaggregation. Furthermore, the coaggregation was not inhibited by serum treatment.
CONCLUSION
This is the first report to describe the coaggregation of P. oris and P. gingivalis. Our study proposes the possibility that P. oris may promote the colonization of P. gingivalis in an early stage of biofilm formation. Furthermore, this coaggregation may contribute to the initiation and progression of periodontitis.
Topics: Animals; Biofilms; Coculture Techniques; Endopeptidase K; Horses; Hot Temperature; Humans; Microbial Interactions; Periodontitis; Porphyromonas gingivalis; Prevotella; Serum
PubMed: 23245806
DOI: 10.1016/j.jmii.2012.09.005 -
Journal of Clinical Microbiology Oct 2009Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains;...
Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains; however, the extent of variability remains unclear and regions of variability remain largely unidentified. To assess P. gingivalis strain diversity, we previously used heteroduplex analysis of the ribosomal operon intergenic spacer region (ISR) to type strains in clinical samples and identified 22 heteroduplex types. Additionally, we used ISR sequence analysis to determine the relatedness of P. gingivalis strains to one another and demonstrated a link between ISR sequence phylogeny and the disease-associated phenotype of the strains. In the current study, heteroduplex analysis of the ISR was used to determine the worldwide genetic variability and distribution of P. gingivalis, and microarray-based comparative genomic hybridization (CGH) analysis was used to more comprehensively examine the variability of major heteroduplex type strains by using the entire genome. Heteroduplex analysis of clinical samples from geographically diverse populations identified 6 predominant geographically widespread heteroduplex types (prevalence, > or = 5%) and 14 rare heteroduplex types (prevalence, <2%) which are found in one or a few locations. CGH analysis of the genomes of seven clinically prevalent heteroduplex type strains identified 133 genes from strain W83 that were divergent in at least one of the other strains. The relatedness of the strains to one another determined on the basis of genome content (microarray) analysis was highly similar to their relatedness determined on the basis of ISR sequence analysis, and a striking correlation between the genome contents and disease-associated phenotypes of the strains was observed.
Topics: Cluster Analysis; Comparative Genomic Hybridization; DNA, Bacterial; DNA, Ribosomal Spacer; Genetic Variation; Genotype; Geography; Heteroduplex Analysis; Humans; Periodontitis; Phylogeny; Porphyromonas gingivalis
PubMed: 19675220
DOI: 10.1128/JCM.00569-09 -
Scientific Reports Oct 2020Porphyromonas gingivalis is a causative agent in the onset and progression of periodontal disease. This study aims to investigate the effects of quercetin, a natural...
Porphyromonas gingivalis is a causative agent in the onset and progression of periodontal disease. This study aims to investigate the effects of quercetin, a natural plant product, on P. gingivalis virulence properties including gingipain, haemagglutinin and biofilm formation. Antimicrobial effects and morphological changes of quercetin on P. gingivalis were detected. The effects of quercetin on gingipains activities and hemolytic, hemagglutination activities were evaluated using chromogenic peptides and sheep erythrocytes. The biofilm biomass and metabolism with different concentrations of quercetin were assessed by the crystal violet and MTT assay. The structures and thickness of the biofilms were observed by confocal laser scanning microscopy. Bacterial cell surface properties including cell surface hydrophobicity and aggregation were also evaluated. The mRNA expression of virulence and iron/heme utilization was assessed using real time-PCR. Quercetin exhibited antimicrobial effects and damaged the cell structure. Quercetin can inhibit gingipains, hemolytic, hemagglutination activities and biofilm formation at sub-MIC concentrations. Molecular docking analysis further indicated that quercetin can interact with gingipains. The biofilm became sparser and thinner after quercetin treatment. Quercetin also modulate cell surface hydrophobicity and aggregation. Expression of the genes tested was down-regulated in the presence of quercetin. In conclusion, our study demonstrated that quercetin inhibited various virulence factors of P. gingivalis.
Topics: Anti-Bacterial Agents; Biofilms; Gingipain Cysteine Endopeptidases; Hemagglutinins; Humans; Microbial Sensitivity Tests; Microscopy, Confocal; Periodontal Diseases; Porphyromonas gingivalis; Quercetin; Virulence
PubMed: 33110205
DOI: 10.1038/s41598-020-74977-y -
Frontiers in Cellular and Infection... 2019has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two strains, one from a patient... (Comparative Study)
Comparative Study
has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both CP3 and H3 and conducted a comparative analysis regarding virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of and two of , H3 has no and only one copy of . Experimentally, this finding is related to lower hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with virulence.
Topics: Bacterial Proteins; Biofilms; Case-Control Studies; Cell Line; Chronic Periodontitis; Epithelial Cells; Fimbriae Proteins; Gene Expression Regulation, Bacterial; Gene Ontology; Genetic Variation; Genome, Bacterial; Genomics; Gingiva; Humans; Lectins; Molecular Sequence Annotation; Phenotype; Phylogeny; Porphyromonas gingivalis; Protein Isoforms; Sequence Analysis, DNA; Virulence; Virulence Factors
PubMed: 31355151
DOI: 10.3389/fcimb.2019.00246 -
Infection and Immunity Jun 2014The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tissue destruction,...
The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tissue destruction, loss of alveolar bone, periodontal pocket formation, and eventually, tooth loss. Potential virulence factors of P. gingivalis are peptidylarginine deiminase (PPAD), an enzyme modifying free or peptide-bound arginine to citrulline, and the bacterial proteases referred to as gingipains (Rgp and Kgp). Chemokines attract leukocytes during inflammation. However, posttranslational modification (PTM) of chemokines by proteases or human peptidylarginine deiminases may alter their biological activities. Since chemokine processing may be important in microbial defense mechanisms, we investigated whether PTM of chemokines by P. gingivalis enzymes occurs. Upon incubation of interleukin-8 (IL-8; CXCL8) with PPAD, only minor enzymatic citrullination was detected. In contrast, Rgp rapidly cleaved CXCL8 in vitro. Subsequently, different P. gingivalis strains were incubated with the chemokine CXCL8 or CXCL10 and their PTMs were investigated. No significant CXCL8 citrullination was detected for the tested strains. Interestingly, although considerable differences in the efficiency of CXCL8 degradation were observed with full cultures of various strains, similar rates of chemokine proteolysis were exerted by cell-free culture supernatants. Sequencing of CXCL8 incubated with supernatant or bacteria showed that CXCL8 is processed into its more potent forms consisting of amino acids 6 to 77 and amino acids 9 to 77 (the 6-77 and 9-77 forms, respectively). In contrast, CXCL10 was entirely and rapidly degraded by P. gingivalis, with no transient chemokine forms being observed. In conclusion, this study demonstrates PTM of CXCL8 and CXCL10 by gingipains of P. gingivalis and that strain differences may particularly affect the activity of these bacterial membrane-associated proteases.
Topics: Cells, Cultured; Chemokine CCL3; Chemokine CXCL10; Citrulline; Hydrolases; Interleukin-8; Porphyromonas gingivalis; Protein-Arginine Deiminases; Proteolysis
PubMed: 24686061
DOI: 10.1128/IAI.01624-14 -
Infection and Immunity Mar 1993Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and...
Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Base Sequence; Cloning, Molecular; Female; Fimbriae Proteins; Fimbriae, Bacterial; Lymphocyte Activation; Mice; Mice, Inbred CBA; Molecular Sequence Data; Molecular Weight; Oligodeoxyribonucleotides; Porphyromonas gingivalis; Recombinant Proteins
PubMed: 8094377
DOI: 10.1128/iai.61.3.1040-1047.1993 -
In vitro anti‑bacterial activity of diosgenin on Porphyromonas gingivalis and Prevotella intermedia.Molecular Medicine Reports Dec 2020Diosgenin (Dios), a natural steroidal sapogenin, is a bioactive compound extracted from dietary fenugreek seeds. It has a wide range of applications, exhibiting...
Diosgenin (Dios), a natural steroidal sapogenin, is a bioactive compound extracted from dietary fenugreek seeds. It has a wide range of applications, exhibiting anti‑oxidant, anti‑inflammatory and anti‑cancer activities. However, whether the extracts have beneficial effects on periodontal pathogens has so far remained elusive. The aim of the present study was to investigate the anti‑bacterial effects of Dios on Porphyromonas gingivalis (P. gingivalis) and Prevotella intermedia (P. intermedia) in vitro. The anti‑microbial effect of Dios on P. gingivalis and P. intermedia was assessed by a direct contact test (DCT) and the Cell Counting Kit (CCK)‑8 assay at 60, 90 and 120 min. In addition, counting of colony‑forming units (CFU) and live/dead cell staining were used to evaluate the anti‑bacterial effects. The results of the DCT and CCK‑8 assays indicated that Dios had beneficial dose‑dependent inhibitory effects on P. gingivalis and P. intermedia. The CFU counting results also indicated that Dios had dose‑dependent anti‑bacterial effects on P. gingivalis and P. intermedia. Of note, Dios had significant anti‑bacterial effects on the biofilms of P. gingivalis and P. intermedia in vitro as visualized by the live/dead cell staining method. In conclusion, the present results demonstrated that Dios had a marked anti‑bacterial activity against P. gingivalis and P. intermedia in vitro, both in suspension and on biofilms. The present study highlighted the potential applications of Dios as a novel natural agent to prevent and treat periodontitis through its anti‑bacterial effects.
Topics: Anti-Bacterial Agents; Biofilms; China; Diosgenin; Microbial Sensitivity Tests; Periodontitis; Porphyromonas gingivalis; Prevotella intermedia
PubMed: 33174005
DOI: 10.3892/mmr.2020.11620 -
NPJ Biofilms and Microbiomes Jan 2020In order to persist, bacteria need to adjust their physiological state in response to external and internal cues. External stimuli are often referred to as stressors....
In order to persist, bacteria need to adjust their physiological state in response to external and internal cues. External stimuli are often referred to as stressors. The stringent response, mediated by the alarmone (p)ppGpp, is central to the stress response in many bacteria; yet, there is limited knowledge regarding the role of (p)ppGpp signaling in bacteria belonging to the phylum Bacteroidetes. Like its counterparts in the gut (e.g., Bacteroides thetaiotaomicron and Bacteroides fragilis), Porphyromonas gingivalis persists in close association with its human host. Given the potential for numerous perturbations in the oral cavity, and the fact that P. gingivalis can enter and replicate within host cells, we hypothesized that (p)ppGpp is a key signaling molecule for stress adaptation and persistence. Here, we show that accumulation of ppGpp in P. gingivalis is governed by two homologous enzymes, designated Rel, and RshB, and that ppGpp signaling affects growth rate, survival, biofilm formation, production of outer membrane vesicles, and expression of genes encoding type IX secretion structural and cargo proteins. Overall, our findings provide a potential mechanism by which biofilm formation and virulence of P. gingivalis are integrated via ppGpp signaling, a regulatory mechanism central to bacterial survival in dynamic environments.
Topics: Animals; Bacterial Proteins; Bacteroidaceae Infections; Biofilms; Disease Models, Animal; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Lepidoptera; Porphyromonas gingivalis; Signal Transduction; Stress, Physiological; Survival Analysis; Virulence
PubMed: 32005827
DOI: 10.1038/s41522-020-0115-4