Science.gov

Sample records for actinomycetemcomitans porphyromonas gingivalis

  1. Detection of antimicrobial activity of banana peel (Musa paradisiaca L.) on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Kapadia, Suraj Premal; Pudakalkatti, Pushpa S.; Shivanaikar, Sachin

    2015-01-01

    Introduction and Aim: Banana is used widely because of its nutritional values. In past, there are studies that show banana plant parts, and their fruits can be used to treat the human diseases. Banana peel is a part of banana fruit that also has the antibacterial activity against microorganisms but has not been studied extensively. Since, there are no studies that relate the antibacterial activity of banana peel against periodontal pathogens. Hence, the aim of this study is to determine the antimicrobial activity of banana peel extract on Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Material and Methods: Standard strains of P. gingivalis and A. actinomycetemcomitans were used in this study which was obtained from the in-house bacterial bank of Department of Molecular Biology and Immunology at Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre. The banana peel extract was prepared, and the antibacterial activity was assessed using well agar diffusion method and minimum inhibitory concentration was assessed using serial broth dilution method. Results: In the current study, both the tested microorganisms showed antibacterial activity. In well diffusion method, P. gingivalis and A. actinomycetemcomitans showed 15 mm and 12 mm inhibition zone against an alcoholic extract of banana peel, respectively. In serial broth dilution method P. gingivalis and A. actinomycetemcomitans were sensitive until 31.25 μg/ml dilutions. Conclusion: From results of the study, it is suggested that an alcoholic extract of banana peel has antimicrobial activity against P. gingivalis and A. actinomycetemcomitans. PMID:26681854

  2. Antibacterial Effect of an Herbal Product Persica on Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans: An In-Vitro Study

    PubMed Central

    Jelvehgaran Esfahani, Zahra; Kadkhoda, Zeinab; Eshraghi, Seyed Saeed; Salehi Surmaghi, Mohammad Hossein

    2014-01-01

    Objective: The plant Salvadora persica is used for oral hygiene in many parts of the world. It has been suggested that it has antibacterial properties, in addition to its ability to mechanically remove plaques. The aim of this study was to assess the antimicrobial activity of the herbal product Persica containing Salvadora persica against periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in vitro. Materials and Methods: Fifty patients with moderate and severe periodontitis were recruited. Using paper points, subgingival plaque samples were taken from pockets with attachment loss ≥ 3mm. The samples were subjected to microbial culture to yield P. gingivalis and A. actinomycetemcomitans. The ditch plate method was used for antimicrobial susceptibility testing of the bacteria to Persica compared to chlorhexidine and distilled water. The growth inhibition zones of microorganisms around the ditches were measured in millimeters. The data were analyzed using SPSS 16. Freidman test and Wilcoxon signed ranks test with Bonferroni adjustment were used for analysis of variance with 5% significance level. P<0.05 for main comparisons and P< 0.017 for multiple comparisons were considered statistically significant. Results: P. gingivalis was sensitive to chlorhexidine and persica. There was a significant difference (P=0.001) between antimicrobial activity of chlorhexidine (mean 28.733mm, SD 5.216) and Persica (mean 16.333mm, SD 5.259) compared to water against P. gingivalis. There was a significant difference (P< 0.001) between the antimicrobial activity of chlorhexidine (24.045mm, SD 3.897) and Persica (0.545mm, SD 2.558) with respect to A. actinomycetemcomitans. There was no significant difference (P=0.317) between the antimicrobial activity of Persica and water against A. actinomycetemcomitans. Conclusion: The herbal product Persica had significant antimicrobial activity against P. gingivalis and negligible antimicrobial activity against A

  3. The survival rate of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides forsythus following 4 randomized treatment modalities.

    PubMed

    Shiloah, J; Patters, M R; Dean, J W; Bland, P; Toledo, G

    1997-08-01

    The overall goal of this clinical study was to determine the short-term anti-infective effects of four randomized treatment modalities on Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Bacteroides forsythus (Bf) and determine the effects of bacterial survival on treatment outcomes in patients with adult periodontitis. Twelve adult patients requiring therapy for moderate periodontitis were selected for this study. All patients had at least one tooth in each quadrant that had an inflamed pocket of probing depth > or =5 mm with probing attachment loss that harbored at least one of the following three periodontal pathogens: Aa, Pg, or Bf. The number of target organisms per site was determined pre-operatively, at 1 week, and 1 month and 3 months postoperatively utilizing DNA probes. One quadrant in each patient was randomly assigned to each one of the following four treatment groups: 1) scaling and root planing (SRP group); 2) pocket reduction through osseous surgery and apically-positioned flap (OS group); 3) modified Widman flap (MWF group); and 4) modified Widman flap and topical application of saturated citric acid at pH 1 for 3 minutes (CA group). The 4 treatment modalities were performed in one appointment. No postoperative antibiotics were used. Patients were instructed to supplement their daily oral hygiene with chlorohexidine oral rinse during the study. The results of this investigation indicated that: 1) none of the treatment modalities was effective in eliminating the target species; 2) the incidence of infected sites for all groups was 100% preoperatively; 62.5%, 33.3%, and 31.3% at 1 week, and 1 and 3 months postoperatively, respectively; 3) these infected sites lost 1.1 +/- 0.4 mm of probing attachment compared to gain of 0.0 +/- 0.3 mm for uninfected sites; 4) the infected sites had higher plaque and bleeding on probing 0.9 +/- 0.3, 73 +/- 12%, respectively, compared to 0.3 +/- 0.1 and 30 +/- 8% for the uninfected sites

  4. Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis.

    PubMed Central

    Choi, J I; Ha, M H; Kim, J H; Kim, S J

    1996-01-01

    The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP). PMID:8926092

  5. Analytical performance of an immunologic-based periodontal bacterial test for simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.

    PubMed

    Snyder, B; Ryerson, C C; Corona, H; Grogan, E A; Reynolds, H S; Contestable, P B; Boyer, B P; Mayer, J; Mangan, T; Norkus, N; Zambon, J J; Genco, R J

    1996-05-01

    The analytical performance of a membrane-based immunoassay for the simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (including Prevotella nigrescens) was investigated. Positive reactions were observed for 71 of 71 reference strains and recent oral isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. No cross-reactivity was observed with 39 other common oral and environmental species. The specificity of the test was unaffected by the presence of potential oral interferents including whole blood, white blood cells, mucin, saliva, toothpastes, and oral rinses. A proficiency test by dental professionals using a standardized set of unknown simulated samples yielded a sensitivity of 97% (116/120) and a 100% specificity (240/ 240). An additional group including dental professionals and high school students was shown to be 99% proficient (1385/1397) in distinguishing proper from improper test function when processing control samples with normal test devices and devices with simulated error conditions. Comparisons to a culture standard for 104 subgingival plaque samples collected from 26 adult periodontitis patients yielded > 98% specificity for each of the test bacteria. In addition, the detection threshold for the test was determined to be equivalent to 10(4) cultivable test bacteria when compared to the culture standard. The data indicate that this membrane immunoassay is a valid and easy-to-use method for the detection of A. actinomycetemcomitans, P. gingivalis, and P. intermedia in subgingival plaque, at levels above the detection threshold of the test.

  6. Evaluation of chemical composition and efficacy of Chinese propolis extract on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Agarwal, Garima; Vemanaradhya, Gayathri G.; Mehta, Dhoom S.

    2012-01-01

    Background: Propolis as a natural remedy has maintained its popularity over long periods of time. The aim of this study was to determine the chemical composition in terms of total phenolic compounds and flavonoids present in Chinese propolis and to carry out an in vitro evaluation of its antimicrobial activity and the minimal inhibitory concentrations for Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: From the ethanolic extract of propolis (EEP), total phenol content was determined by the Folin–Ciocalteau method, flavones and flavonols by the modified aluminum chloride colorimetric method, and flavanones by the 2.4-dinitrophenylhydrazine (2,4-DNP) method. Agar well diffusion assay was used to evaluate the antimicrobial potential of propolis against Pg and Aa. The minimum inhibitory concentration of propolis against the two bacteria was determined using serial tube dilution technique. Results: The total concentration of phenol in the EEP was 19.44%, flavones and flavonols 2.616%, and flavanones 16.176%. The inhibitory zone depicting antimicrobial activity ranged from 18 to 25 mm for Pg and from 12 to 14 mm for Aa. The concentration range of Chinese propolis that is sensitive to inhibit the growth of Pg was 0.1–0.0125 μg/ml and for Aa it was 0.1–0.025 μg/ml. Conclusion: These data suggest that Chinese propolis has potent antimicrobial activity against the two periodontopathogens, suggesting its possible use as a natural alternative to the widely used synthetic antibiotics for periodontal therapy. PMID:23293477

  7. Determination of antibacterial activity of green coffee bean extract on periodontogenic bacteria like Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Bharath, Nagaraj; Sowmya, Nagur Karibasappa; Mehta, Dhoom Singh

    2015-01-01

    Background: The aim of this study was to evaluate the antibacterial activity of pure green coffee bean extract on periodonto pathogenic bacteria Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were used to assess the antibacterial effect of pure green coffee bean extract against periodonto pathogenic bacteria by micro dilution method and culture method, respectively. Results: MIC values of Pg, Pi and Aa were 0.2 μg/ml whereas Fn showed sensitive at concentration of 3.125 μg/ml. MBC values mirrors the values same as that of MIC. Conclusion: Antimicrobial activity of pure green coffee bean extract against Pg, Pi, Fn and Aa suggests that it could be recommended as an adjunct to mechanical therapy in the management of periodontal disease. PMID:26097349

  8. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo. PMID:27594851

  9. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo.

  10. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population.

    PubMed

    Torrungruang, Kitti; Jitpakdeebordin, Supawadee; Charatkulangkun, Orawan; Gleebbua, Yingampa

    2015-01-01

    Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7-3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2-23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4-9.1) and 2.2 (95%CI 1.5-3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td and Pi

  11. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population

    PubMed Central

    Torrungruang, Kitti; Jitpakdeebordin, Supawadee; Charatkulangkun, Orawan; Gleebbua, Yingampa

    2015-01-01

    Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7–3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2–23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4–9.1) and 2.2 (95%CI 1.5–3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td

  12. Putative respiratory chain of Porphyromonas gingivalis.

    PubMed

    Meuric, Vincent; Rouillon, Astrid; Chandad, Fatiha; Bonnaure-Mallet, Martine

    2010-05-01

    The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

  13. Prevalence of fimA genotypes of Porphyromonas gingivalis and other periodontal bacteria in a Spanish population with chronic periodontitis

    PubMed Central

    Puig-Silla, Miriam; Dasí-Fernánde, Francisco; Montiel-Company, José-María

    2012-01-01

    Objectives: The aim of this study was to determine the prevalence of the different fimA genotypes of Porphyromonas gingivalis in adult Spanish patients with chronic periodontitis, patients with gingivitis and periodontally healthy subjects, and the relationship between these genotypes and other periodontopathogenic bacteria. Study design: Samples of subgingival plaque were taken from 86 patients (33 with chronic periodontitis, 16 with gingivitis, and 37 periodontally healthy) in the course of a full periodontal examination. PCR was employed to determine the presence of the 6 fimA genotypes of Porphyromonas gingivalis (I-V and Ib) and of Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola. Results: Porphyromonas gingivalis fimA genotypes II and Ib were present in significantly higher percentages in periodontal patients (39.4% and 12.1% respectively) than in healthy or gingivitis subjects. The prevalence of Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis fimA genotype IV was significantly higher in the group that presented bleeding greater than 30%. A positive correlation was found between Porphyromonas gingivalis fimA genotype IV and Treponema denticola. Conclusions: A strong association between Porphyromonas gingivalis fimA genotypes II and Ib and chronic periodontitis exists in the Spanish population. The most prevalent genotype in periodontal patients is II. Key words:Periodontitis, Porphyromonas gingivalis, fimA genotype, periodontal bacteria, polymerase chain reaction. PMID:22549664

  14. Major neutrophil functions subverted by Porphyromonas gingivalis.

    PubMed

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil-P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  15. Major neutrophil functions subverted by Porphyromonas gingivalis

    PubMed Central

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil–P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  16. Arginine deiminase inhibits Porphyromonas gingivalis surface attachment

    PubMed Central

    Cugini, Carla; Stephens, Danielle N.; Nguyen, Daniel; Kantarci, Alpdogan

    2013-01-01

    The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity. PMID:23242802

  17. Porphyromonas gingivalis causing brain abscess in patient with recurrent periodontitis.

    PubMed

    Rae Yoo, Jeong; Taek Heo, Sang; Kim, Miyeon; Lee, Chang Sub; Kim, Young Ree

    2016-06-01

    We report an extremely rare case of Porphyromonas gingivalis causing brain abscess in a patient with recurrent periodontitis. The patient presented with right-sided homonymous hemianopsia and right hemiparesis. Emergent surgical drainage was performed and antibiotics were administered. P. gingivalis was identified from the anaerobic culture of the abscess. The clinical course of the patient improved with full recovery of the neurologic deficit. PMID:27085200

  18. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

  19. Treponema denticola improves adhesive capacities of Porphyromonas gingivalis.

    PubMed

    Meuric, V; Martin, B; Guyodo, H; Rouillon, A; Tamanai-Shacoori, Z; Barloy-Hubler, F; Bonnaure-Mallet, M

    2013-02-01

    Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg-gingipain A (RgpA), Lys-gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time-dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain-containing proteins. PMID:23194417

  20. Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis.

    PubMed Central

    Grenier, D; Michaud, J

    1993-01-01

    The aim of the present study was to evaluate the ability of Porphyromonas gingivalis to degrade hyaluronic acid. No hyaluronidase activity was detected using a turbidimetric method, whereas a standard plate assay showed a positive reaction for P. gingivalis. We postulated that the high proteolytic activity of P. gingivalis may account for this observation. A modified plate assay was designed to avoid false-positive reactions caused by proteolytic bacteria. The new assay, based on the formation of a water-insoluble salt between hyaluronic acid and the polyanion cetylpyridinium chloride, indicated that P. gingivalis does not have hyaluronidase activity. By this modified plate method, it was found that among 24 different oral bacterial species tested, Propionibacterium acnes and Prevotella oris were the only species that possess hyaluronidase activity. Images PMID:8394379

  1. Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

    PubMed Central

    Zhu, Ying; Dashper, Stuart G.; Chen, Yu-Yen; Crawford, Simon; Slakeski, Nada; Reynolds, Eric C.

    2013-01-01

    Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola. PMID:23990979

  2. Presence of Porphyromonas gingivalis in gingival squamous cell carcinoma

    PubMed Central

    Katz, Joseph; Onate, Mairelys D; Pauley, Kaleb M; Bhattacharyya, Indraneel; Cha, Seunghee

    2011-01-01

    Periodontal disease has been recently linked to a variety of systemic conditions such as diabetes, cardiovascular disease, preterm delivery, and oral cancer. The most common bacteria associated with periodontal disease, Porphyromonas gingivalis (P. gingivalis) has not yet been studied in the malignant gingival tissues. The objective of this study was to investigate the presence of P. gingivalis in specimens from squamous cell carcinoma patients. We have performed immunohistochemical staining to investigate the presence of P. gingivalis and Streptococcus gordonii (S. gordonii), a non invasive oral bacteria, in paraffin embedded samples of gingival squamous cell carcinoma (n=10) and normal gingiva (n=5). Staining for P. gingivalis revealed the presence of the bacteria in normal gingival tissues and gingival carcinoma, with higher levels (more than 33%, P<0.05) detected in the carcinoma samples. The staining intensity was also significantly enhanced in the malignant tissue by 2 folds (P<0.023) compared to specimens stained for the non-invasive S. gordonii. P. gingivalis is abundantly present in malignant oral epithelium suggesting a potential association of the bacteria with gingival squamous cell carcinoma. PMID:22010579

  3. Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis

    PubMed Central

    2012-01-01

    Background Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. Results In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups ‘D’ (cell division), ‘I’ (lipid transport and metabolism) and ‘J’ (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions. Conclusions A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis

  4. Porphyromonas gingivalis infection of oral epithelium inhibits neutrophil transepithelial migration.

    PubMed Central

    Madianos, P N; Papapanou, P N; Sandros, J

    1997-01-01

    Periodontal diseases are inflammatory disorders caused by microorganisms of dental plaque that colonize the gingival sulcus and, subsequently, the periodontal pocket. As in other mucosal infections, the host response to plaque bacteria is characterized by an influx of polymorphonuclear leukocytes (PMNs) to the gingival crevice. Neutrophil migration through the epithelial lining of the gingival pocket is thought to be the first line of defense against plaque bacteria. In order to model this phenomenon in vitro, we used the oral epithelial cell line KB and human PMNs in the Transwell system and examined the impact of Porphyromonas gingivalis-epithelial cell interactions on subsequent PMN transepithelial migration. We demonstrate here that P. gingivalis infection of oral epithelial cells failed to trigger transmigration of PMNs. Furthermore, it significantly inhibited neutrophil transmigration actively induced by stimuli such as N-formylmethionyl leucyl phenylalanine, interleukin-8 (IL-8), and the intestinal pathogen enterotoxigenic Escherichia coli. The ability of P. gingivalis to block PMN transmigration was strongly positively correlated with the ability to adhere to and invade epithelial cells. In addition, P. gingivalis attenuated the production of IL-8 and the expression of intercellular adhesion molecule 1 by epithelial cells. The ability of P. gingivalis to block neutrophil migration across an intact epithelial barrier may critically impair the potential of the host to confront the bacterial challenge and thus may play an important role in the pathogenesis of periodontal disease. PMID:9316996

  5. Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles

    PubMed Central

    Meka, Archana; Bakthavatchalu, Vasudevan; Sathishkumar, Sabapathi; Lopez, M. Cecilia; Verma, Raj K.; Wallet, Shannon M.; Bhattacharyya, Indraneel; Boyce, Brendan F.; Handfield, Martin; Lamont, Richard J.; Baker, Henry V.; Ebersole, Jeffrey L.; Lakshmyya, Kesavalu N.

    2010-01-01

    Introduction Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. The objectives of this investigation were to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption. Methods P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and analyzed for transcript profiles using Murine GeneChip® arrays to provide a molecular profile of the events that occur following infection of these tissues. Results After P. gingivalis infection, 5517 and 1900 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P ≤ 0.05) and up-regulated. Biological pathways significantly impacted by P. gingivalis infection in tissues and calvarial bone included cell adhesion (immune system) molecules, Toll-like receptors, B cell receptor signaling, TGF-β cytokine family receptor signaling, and MHC class II antigen processing pathways resulting in proinflammatory, chemotactic effects, T cell stimulation, and down regulation of antiviral and T cell chemotactic effects. P. gingivalis-induced inflammation activated osteoclasts, leading to local bone resorption. Conclusion This is the first in vivo evidence that localized P. gingivalis infection differentially induces transcription of a broad array of host genes that differed between inflamed soft tissues and calvarial bone. PMID:20331794

  6. Porphyromonas gingivalis downregulates the immune response of fibroblasts

    PubMed Central

    2013-01-01

    Background Porphyromonas gingivalis is a key pathogen in periodontitis, an inflammatory disease leading to destruction of bone and tooth-supporting tissue. P. gingivalis possesses a number of pathogenic properties to enhance growth and survival, including proteolytic gingipains. Accumulating data shows that gingipains are involved in the regulation of host inflammatory responses. The aim of this study was to determine if P. gingivalis infection modulates the inflammatory response of fibroblasts, including the release of chemokines and cytokines. Human gingival fibroblasts or primary dermal fibroblasts were pre-stimulated with tumor-necrosis factor-α (TNF- α) and cocultured with P. gingivalis. Gingipain inhibitors were used to explore the effect of gingipains. CXCL8 levels were determined with ELISA and the relative levels of various inflammatory mediators were determined by a cytokine assay. Results TNF-α-triggered CXCL8 levels were completely abolished by viable P. gingivalis, whereas heat-killed P. gingivalis did not suppress CXCL8. Accumulation of CXCL8 was partially restored by an arginine-gingipain inhibitor. Furthermore, fibroblasts produced several inflammatory mediators, notably chemokines, all of which were suppressed by viable P. gingivalis. Conclusion These findings provide evidence that fibroblast-derived inflammatory signals are modulated by heat-instable gingipains, whereby the bacteria can escape killing by the host immune system and promote its own growth and establishment. In addition, we show that fibroblasts are important mediators of inflammation in response to infection and thereby play a crucial role in determining the nature and magnitude of the invasion of immune cells. PMID:23841502

  7. Benzamidine derivatives inhibit the virulence of Porphyromonas gingivalis

    PubMed Central

    Fröhlich, Esther; Kantyka, Tomasz; Plaza, Karolina; Schmidt, Karl-Hermann; Pfister, Wolfgang; Potempa, Jan; Eick, Sigrun

    2012-01-01

    SUMMARY We have previously shown that benzamidine-type compoundscan inhibit the activity of arginine-specific cysteine proteinases (gingipainsHRgpA and RgpB); well-known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important period on to pathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, as pentamidine, which is a 20-fold less efficient inhibitor of gingipainthan 2,6-bis-(4-amidinobenzyl)-cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine-derived compounds other than Arg-gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5-1-2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine-Sepharose column to identify proteins interacting withbenzamidine. In addition to HRgpA and RgpB the analysis revealed heat-shock protein GroELas another ligand forbenzamidine. To better understand the effect ofbenzamidine-derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine-derived compounds caused significant increasesin GroEL, both at the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also bypentamidine, a relatively week gingipain inhibitor. This effect may depend not only ongingipain inhibition but also oninteraction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment. PMID:23279840

  8. Increased levels of Porphyromonas gingivalis are associated with ischemic and hemorrhagic cerebrovascular disease in humans: an in vivo study

    PubMed Central

    GHIZONI, Janaina Salomon; TAVEIRA, Luís Antônio de Assis; GARLET, Gustavo Pompermaier; GHIZONI, Marcos Flávio; PEREIRA, Jefferson Ricardo; DIONÍSIO, Thiago José; BROZOSKI, Daniel Thomas; SANTOS, Carlos Ferreira; SANT'ANA, Adriana Campos Passanezi

    2012-01-01

    Objective: This study investigated the role of periodontal disease in the development of stroke or cerebral infarction in patients by evaluating the clinical periodontal conditions and the subgingival levels of periodontopathogens. Material and Methods: Twenty patients with ischemic (I-CVA) or hemorrhagic (H-CVA) cerebrovascular episodes (test group) and 60 systemically healthy patients (control group) were evaluated for: probing depth, clinical attachment level, bleeding on probing and plaque index. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were both identified and quantified in subgingival plaque samples by conventional and real-time PCR, respectively. Results: The test group showed a significant increase in each of the following parameters: pocket depth, clinical attachment loss, bleeding on probing, plaque index and number of missing teeth when compared to control values (p<0.05, unpaired t-test). Likewise, the test group had increased numbers of sites that were contaminated with P. gingivalis (60%x10%; p<0.001; chi-squared test) and displayed greater prevalence of periodontal disease, with an odds ratio of 48.06 (95% CI: 5.96-387.72; p<0.001). Notably, a positive correlation between probing depth and the levels of P. gingivalis in ischemic stroke was found (r=0.60; p=0.03; Spearman's rank correlation coefficient test). A. actinomycetemcomitans DNA was not detected in any of the groups by conventional or real-time PCR. Conclusions: Stroke patients had deeper pockets, more severe attachment loss, increased bleeding on probing, increased plaque indexes, and in their pockets harbored increased levels of P. gingivalis. These findings suggest that periodontal disease is a risk factor for the development of cerebral hemorrhage or infarction. Early treatment of periodontitis may counteract the development of cerebrovascular episodes. PMID:22437687

  9. Initial serum antibody titer to Porphyromonas gingivalis influences development of antibody avidity and success of therapy for chronic periodontitis.

    PubMed Central

    Mooney, J; Adonogianaki, E; Riggio, M P; Takahashi, K; Haerian, A; Kinane, D F

    1995-01-01

    This study assessed the effect of periodontal therapy on specific serum antibody concentration, expressed as titer, and antibody binding strength, expressed as relative avidity. The immune responses to Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were investigated. Antibody titer was assayed by enzyme-linked immunosorbent assay (ELISA) and relative avidity was measured by thiocyanate elution in 17 adult periodontitis patients before and after therapy. Immunoglobulin G (IgG) avidities (expressed as thiocyanate molarity) to P. gingivalis increased from 1.01 to 1.38 M (P = 0.05) and IgA titers (expressed as ELISA units [EU]) increased from 89 to 237 EU (P = 0.012). There were no significant changes in avidity to A. actinomycetemcomitans, but the titer of all three immunoglobulin classes increased significantly (P < 0.03). More specifically, when patients were divided into subgroups which had originally been either IgG seropositive (i.e., having an IgG titer to this organism > 2 times the control median) or seronegative for P. gingivalis, only patients who were initially seropositive showed a significant increase in antibody avidity (P = 0.026; mean difference, 0.69 M). Patients who were originally seropositive in terms of IgG and IgA titer to P. gingivalis had demonstrably better treatment outcomes in terms of a reduced number of deep pockets and sites which bled on probing (P < 0.05). These findings suggest that periodontal therapy affects the magnitude and quality of the humoral immune response to suspected periodontopathogens, that this effect is dependent on initial serostatus, and that initial serostatus may have a bearing on treatment outcome. PMID:7642270

  10. Isolation and characterization of a minor fimbria from Porphyromonas gingivalis.

    PubMed Central

    Hamada, N; Sojar, H T; Cho, M I; Genco, R J

    1996-01-01

    We have discovered two distinctly different fimbriae expressed by the same Porphyromonas gingivalis strain. The construction of a fimA mutant of P. gingivalis ATCC 33277 has previously been reported by N. Hamada et al. (Infect. Immun. 62:1696-1704, 1994). Expression of fimbriae on the surface of the fimA mutant and the wild-type strain, ATCC 33277, were investigated by electron microscopy. The wild-type strain produced long fimbrial structures extending from the cell surface, whereas those structures were not observed on the fimA mutant. However, short fimbrial structures were seen on the surface of the fimA mutant. The short fimbrial protein was purified from the fimA mutant by selective protein precipitation and chromatography on DEAE Sepharose CL-6B. We have found that the second fimbrial structure of P. gingivalis ATCC 33277 is distinct from the 41-kDa (43-kDa) major fimbrial protein (FimA). We provisionally call this protein minor fimbriae. The molecular mass of the minor fimbriae is 67 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions after boiling at 100 degrees C. The component shows a ladder-like pattern at 80 degrees C under nonreducing conditions, suggesting a tendency to aggregate or polymerize. In immunoblotting analysis, anti-minor fimbria serum reacted with both the 100 degrees C- and the 80 degrees C-treated minor fimbriae. The anti-minor fimbria serum also reacts with the same-molecular-size fimbrial preparation from the wild-type strain. Immunogold electron microscopy showed that the anti-minor fimbria serum bound to the minor fimbria on the cell surface of the wild-type strain. This is the first report on the identification of the minor fimbria produced by P. gingivalis. These results suggest that the minor fimbriae appearing on the fimA mutant strain are produced together with numerous long major fimbriae on the wild-type strain. Moreover, the minor fimbriae are different in size and

  11. Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis.

    PubMed Central

    Chandad, F; Mayrand, D; Grenier, D; Hinode, D; Mouton, C

    1996-01-01

    To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate. PMID:8641806

  12. Porphyromonas gingivalis FDC381 multiplies and persists within human oral epithelial cells in vitro.

    PubMed Central

    Madianos, P N; Papapanou, P N; Nannmark, U; Dahlén, G; Sandros, J

    1996-01-01

    Porphyromonas gingivalis FDC381 replication and persistence within KB epithelial cells in vitro were studied by means of an antibiotic protection assay and electron microscopy. Intracellular counts decreased during the first 24 h; showed a threefold increase during the second day, indicating intracellular multiplication; and after 8 days declined to levels approximating 40% of the initial invasion. The ability of P. gingivalis to persist and multiply within epithelial cells may constitute a pathogenic mechanism in periodontal disease. PMID:8550223

  13. Characterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue.

    PubMed

    Zhang, Lianyi; Butler, Catherine A; Khan, Hasnah S G; Dashper, Stuart G; Seers, Christine A; Veith, Paul D; Zhang, Jian-Guo; Reynolds, Eric C

    2016-01-01

    PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10(-11) M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10(-10) M and Kd2 ≤ 6.0 x 10(-8) M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner.

  14. Characterisation of the Porphyromonas gingivalis Manganese Transport Regulator Orthologue

    PubMed Central

    Dashper, Stuart G.; Seers, Christine A.; Veith, Paul D.; Zhang, Jian-Guo; Reynolds, Eric C.

    2016-01-01

    PgMntR is a predicted member of the DtxR family of transcriptional repressors responsive to manganese in the anaerobic periodontal pathogen Porphyromonas gingivalis. Our bioinformatic analyses predicted that PgMntR had divalent metal binding site(s) with elements of both manganous and ferrous ion specificity and that PgMntR has unusual twin C-terminal FeoA domains. We produced recombinant PgMntR and four variants to probe the specificity of metal binding and its impact on protein structure and DNA binding. PgMntR dimerised in the absence of a divalent transition metal cation. PgMntR bound three Mn(II) per monomer with an overall dissociation constant Kd 2.0 x 10−11 M at pH 7.5. PgMntR also bound two Fe(II) with distinct binding affinities, Kd1 2.5 x 10−10 M and Kd2 ≤ 6.0 x 10−8 M at pH 6.8. Two of the metal binding sites may form a binuclear centre with two bound Mn2+ being bridged by Cys108 but this centre provided only one site for Fe2+. Binding of Fe2+ or Mn2+ did not have a marked effect on the PgMntR secondary structure. Apo-PgMntR had a distinct affinity for the promoter region of the gene encoding the only known P. gingivalis manganese transporter, FB2. Mn2+ increased the DNA binding affinity of PgMntR whilst Fe2+ destabilised the protein-DNA complex in vitro. PgMntR did not bind the promoter DNA of the gene encoding the characterised iron transporter FB1. The C-terminal FeoA domain was shown to be essential for PgMntR structure/function, as its removal caused the introduction of an intramolecular disulfide bond and abolished the binding of Mn2+ and DNA. These data indicate that PgMntR is a novel member of the DtxR family that may function as a transcriptional repressor switch to specifically regulate manganese transport and homeostasis in an iron-dependent manner. PMID:27007570

  15. Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells.

    PubMed Central

    Weinberg, A; Belton, C M; Park, Y; Lamont, R J

    1997-01-01

    Porphyromonas gingivalis is a periodontal pathogen capable of invading primary cultures of normal human gingival epithelial cells (NHGEC). Involvement of P. gingivalis fimbriae in the invasion process was examined. Purified P. gingivalis 33277 fimbriae blocked invasion of this organism into NHGEC in a dose-dependent manner. DPG3, a P. gingivalis fimbria-deficient mutant, was impaired in its invasion capability approximately eightfold compared to its parent, strain 381. However, adherence of the mutant was only 50% reduced compared to the parent. Biotin labeling of NHGEC surface proteins revealed that two fimbriated strains, but not DPG3, bound a 48-kDa NHGEC protein. Adhesin-receptor interactions, such as fimbriae binding to a 48-kDa NHGEC surface receptor, may trigger activation of eukaryotic proteins involved in signal transduction and/or provoke the generation of surface P. gingivalis molecules required for internalization. PMID:8975930

  16. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis

    PubMed Central

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

  17. Comparative genomics and proteomics of 13 Porphyromonas gingivalis strains.

    PubMed

    Chen, Tsute; Siddiqui, Huma; Olsen, Ingar

    2015-01-01

    At the current time, genome sequences of a total of 13 Porphyromonas gingivalis strains are available, including five completed genomes (strains ATCC 33277, HG66, TDC60, JCVISC001, and W83) and eight high-coverage draft sequences (F0185, F0566, F0568, F0569, F0570, SJD2, W4087, and W50) that are assembled into fewer than 300 contigs. This study compared these genomes at both nucleotide and protein sequence levels in order to understand their phylogenetic and functional relatedness. There are four copies of 16S rRNA gene sequences in each of the strains of ATCC 33277, HG66, TDC60, and W83 and one copy in the other nine genomes. These 25 16S rRNA sequences represent only 13 unique sequences. The five copies in W83 and W50 are identical and the three copies in HG66 are identical to the four copies in ATCC 33277, suggesting close evolutionary lineage between W83 and W50, as well as HG66 and ATCC 33277. Genome-wide comparison based on "Rapid Annotation using Subsystem Technology" (RAST) also showed that for the overall biological functions of the genomes, W83 is closer to W50, and HG66 to ATCC33277, than to other genomes. The comparison of the RAST subsystems identified biological functions that are unique to individual, shared by some, or by all genomes. Functions unique to individual genomes include: a tetracycline resistance protein TetQ, DNA metabolism gene YcfH, and DNA repair gene exonuclease SbcC (only in SJD2); very-short-patch mismatch repair endonuclease and a phage packaging terminase similar to Bacteroides phage B124-14 (in W4087); an internalin similar to a Listeria surface virulence protein (W83); a Type I restriction-modification system (F0569); an iron acquisition/heme transport protein (F0566); colicin I receptor and carbamoylputrescine amidase (W50); L-serine dehydratase (TDC60); and spermidine synthase and ribokinase (JCVISC001). The results also identified biological functions that are missing in individual or several genomes. For example, JCVISC001

  18. The peptidylarginine deiminase gene is a conserved feature of Porphyromonas gingivalis

    PubMed Central

    Gabarrini, Giorgio; de Smit, Menke; Westra, Johanna; Brouwer, Elisabeth; Vissink, Arjan; Zhou, Kai; A. Rossen, John W.; Stobernack, Tim; van Dijl, Jan Maarten; Jan van Winkelhoff, Arie

    2015-01-01

    Periodontitis is an infective process that ultimately leads to destruction of the soft and hard tissues that support the teeth (the periodontium). Periodontitis has been proposed as a candidate risk factor for development of the autoimmune disease rheumatoid arthritis (RA). Porphyromonas gingivalis, a major periodontal pathogen, is the only known prokaryote expressing a peptidyl arginine deiminase (PAD) enzyme necessary for protein citrullination. Antibodies to citrullinated proteins (anti-citrullinated protein antibodies, ACPA) are highly specific for RA and precede disease onset. Objective of this study was to assess P. gingivalis PAD (PPAD) gene expression and citrullination patterns in representative samples of P. gingivalis clinical isolates derived from periodontitis patients with and without RA and in related microbes of the Porphyromonas genus. Our findings indicate that PPAD is omnipresent in P. gingivalis, but absent in related species. No significant differences were found in the composition and expression of the PPAD gene of P. gingivalis regardless of the presence of RA or periodontal disease phenotypes. From this study it can be concluded that if P. gingivalis plays a role in RA, it is unlikely to originate from a variation in PPAD gene expression. PMID:26403779

  19. Humoral immune response to an antigen from Porphyromonas gingivalis 381 in periodontal disease.

    PubMed Central

    Kurihara, H; Nishimura, F; Nakamura, T; Nakagawa, M; Tanimoto, I; Nomura, Y; Kokeguchi, S; Kato, K; Murayama, Y

    1991-01-01

    The humoral immune responses of patients with periodontitis were evaluated to characterize the host response to Porphyromonas gingivalis. A sonic extract of P. gingivalis 381 from whole cells was fractionated by gel chromatography and ion-exchange chromatography. The fractionated extracts were evaluated by Western blot (immunoblot) analyses with patient sera. A dominant antigen was identified from the sonic extract with an apparent molecular mass of 53 kDa. The 53-kDa protein antigen (Ag53) was purified by affinity chromatography by using a monoclonal antibody. Ag53 was detected on the vesicle surface of P. gingivalis 381 by immunoelectron microscopy by using the monoclonal antibody and was detected as a major protein in the outer membrane and in vesicles by Western blot analysis. Monoclonal antibody cross-reactivity to Ag53 in the sonic extracts of P. gingivalis ATCC 33277, P. gingivalis 1021, and Porphyromonas endodontalis ATCC 35406 was revealed. Seventy-seven patients with periodontitis were examined for their responses to Ag53. Serum immunoglobulin G (IgG) from 54 patients reacted strongly to Ag53; however, serum IgG from the remaining 23 patients did not exhibit detectable reactivity at all to Ag53, even though the patients had high serum IgG titers to the sonic extract. Ag53 is a new marker that represents an interesting aspect of the humoral immune response to P. gingivalis in patients with periodontitis. Images PMID:1855992

  20. Inhibitory Effect of Enterococcus faecium WB2000 on Volatile Sulfur Compound Production by Porphyromonas gingivalis

    PubMed Central

    Higuchi, Takuya; Nakajima, Masato; Fujimoto, Akie; Hanioka, Takashi; Hirofuji, Takao

    2016-01-01

    Volatile sulfur compounds (VSCs) produced by oral anaerobes are the major compounds responsible for oral malodor. Enterococcus faecium WB2000 is recognized as an antiplaque probiotic bacterium. In this study, the effect of E. faecium WB2000 on VSC production by Porphyromonas gingivalis was evaluated, and the mechanism of inhibition of oral malodor was investigated. P. gingivalis ATCC 33277 was cultured in the presence of four lactic acid bacteria, including E. faecium WB2000. Subsequently, P. gingivalis ATCC 33277, W50, W83, and two clinical isolates were cultured in the presence or absence of E. faecium WB2000, and the emission of VSCs from spent culture medium was measured by gas chromatography. The number of P. gingivalis ATCC 33277 in mixed culture with E. faecium WB2000 decreased at 6 h, and the rate of decrease was higher than that in mixed cultures with the other lactic acid bacteria. The numbers of five P. gingivalis strains decreased at similar rates in mixed culture with E. faecium WB2000. The concentration of methyl mercaptan was lower in spent culture medium from P. gingivalis and E. faecium WB2000 cultures compared with that from P. gingivalis alone. Therefore, E. faecium WB2000 may reduce oral malodor by inhibiting the growth of P. gingivalis and neutralizing methyl mercaptan. PMID:27799940

  1. Lipid raft-dependent uptake, signaling, and intracellular fate of Porphyromonas gingivalis in mouse macrophages

    PubMed Central

    Wang, Min; Hajishengallis, George

    2009-01-01

    Summary Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-β-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signaling and entry platforms, which determine its intracellular fate to the pathogen’s own advantage. PMID:18547335

  2. Porphyromonas gingivalis Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells

    PubMed Central

    Tada, Hiroyuki; Matsuyama, Takashi; Nishioka, Takashi; Hagiwara, Makoto; Kiyoura, Yusuke; Shimauchi, Hidetoshi; Matsushita, Kenji

    2016-01-01

    The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that P. gingivalis increased IL-33 expression in the cytoplasm of human gingival epithelial cells in vitro. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from P. gingivalis did not increase IL-33 expression. Specific inhibitors of P. gingivalis proteases (gingipains) suppressed IL-33 mRNA induction by P. gingivalis and the P. gingivalis gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by P. gingivalis. These results indicate that the PAR-2/IL-33 axis is promoted by P. gingivalis infection in human gingival epithelial cells through a gingipain-dependent mechanism. PMID:27058037

  3. Pyocycanin, a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung

    PubMed Central

    Benedyk, Malgorzata; Byrne, Dominic P.; Glowczyk, Izabela; Potempa, Jan; Olczak, Mariusz; Olczak, Teresa; Smalley, John W.

    2015-01-01

    Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain

  4. Porphyromonas gingivalis Lipopolysaccharide Induced Proliferation and Activation of Natural Killer Cells in Vivo.

    PubMed

    Wang, Yuhua; Zhang, Wei; Xu, Li; Jin, Jun-O

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) promoted different innate immune activation than that promoted by Escherichia coli (E. coli) LPS. In this study, we examined the effect of P. gingivalis LPS on the proliferation and activation of natural killer (NK) cells in vivo and compared that function with that of E. coli LPS. Administration of P. gingivalis LPS to C57BL/6 mice induced stronger proliferation of NK cells in the spleen and submandibular lymph nodes (sLNs) and increased the number of circulating NK cells in blood compared to those treated with E. coli LPS. However, P. gingivalis LPS did not induce interferon-gamma (IFN-γ) production and CD69 expression in the spleen and sLN NK cells in vivo, and this was attributed to the minimal activation of the spleen and sLN dendritic cells (DCs), including low levels of co-stimulatory molecule expression and pro-inflammatory cytokine production. Furthermore, P. gingivalis LPS-treated NK cells showed less cytotoxic activity against Yac-1 target cells than E. coli LPS-treated NK cells. Hence, these data demonstrated that P. gingivalis LPS promoted limited activation of spleen and sLN NK cells in vivo, and this may play a role in the chronic inflammatory state observed in periodontal disease. PMID:27548133

  5. Green tea epigallocatechin-3-gallate attenuates Porphyromonas gingivalis-induced atherosclerosis.

    PubMed

    Cai, Yu; Kurita-Ochiai, Tomoko; Hashizume, Tomomi; Yamamoto, Masafumi

    2013-02-01

    The purpose of this study was to determine whether epigallocatechin-3-gallate (EGCG) ameliorates Porphyromonas gingivalis-induced atherosclerosis. EGCG is a polyphenol extract from green tea with health benefits and P. gingivalis is shown here to accelerate atheroma formation in a murine model. Apolipoprotein E knockout mice were administered EGCG or vehicle in drinking water; they were then fed high-fat diets and injected with P. gingivalis three times a week for 3 weeks. Mice were then killed at 15 weeks. Atherosclerotic plaques in the proximal aorta were determined by Oil Red O staining. Atherosclerosis risk factors in serum, liver or aorta were analysed using cytokine antibody arrays, enzyme-linked immunosorbent assay and real-time PCR. Atherosclerotic lesion areas of the aortic sinus caused by P. gingivalis infection decreased in EGCG-treated groups, wherein EGCG reduced the production of C-reactive protein, monocyte chemoattractant protein-1, and oxidized low-density lipoprotein (LDL), and slightly lowered LDL/very LDL cholesterol in P. gingivalis-challenged mice serum. Furthermore, the increase in CCL2, MMP-9, ICAM-1, HSP60, CD44, LOX-1, NOX-4, p22phox and iNOS gene expression levels in the aorta of P. gingivalis-challenged mice were reduced in EGCG-treated mice. However, HO-1 mRNA levels were elevated by EGCG treatment, suggesting that EGCG, as a natural substance, inhibits P. gingivalis-induced atherosclerosis through anti-inflammatory and antioxidative effects. PMID:23620122

  6. Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms

    PubMed Central

    Huang, George T.-J.; Kim, Daniel; Lee, Jonathan K.-H.; Kuramitsu, Howard K.; Haake, Susan Kinder

    2001-01-01

    Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8

  7. Lactobacillus rhamnosus could inhibit Porphyromonas gingivalis derived CXCL8 attenuation

    PubMed Central

    Mendi, Ayşegül; Köse, Sevil; Uçkan, Duygu; Akca, Gülçin; Yilmaz, Derviş; Aral, Levent; Gültekin, Sibel Elif; Eroğlu, Tamer; Kiliç, Emine; Uçkan, Sina

    2016-01-01

    ABSTRACT An increasing body of evidence suggests that the use of probiotic bacteria is a promising intervention approach for the treatment of inflammatory diseases with a polymicrobial etiology. P. gingivalis has been noted to have a different way of interacting with the innate immune response of the host compared to other pathogenic bacteria, which is a recognized feature that inhibits CXCL8 expression. Objective The aim of the study was to determine if P. gingivalis infection modulates the inflammatory response of gingival stromal stem cells (G-MSSCs), including the release of CXCL8, and the expression of TLRs and if immunomodulatory L. rhamnosus ATCC9595 could prevent CXCL8 inhibition in experimental inflammation. Material and Methods G-MSSCs were pretreated with L. rhamnosus ATCC9595 and then stimulated with P. gingivalis ATCC33277. CXCL8 and IL-10 levels were investigated with ELISA and the TLR-4 and 2 were determined through flow cytometer analysis. Results CXCL8 was suppressed by P. gingivalis and L. rhamnosus ATCC9595, whereas incubation with both strains did not abolish CXCL8. L. rhamnosus ATCC9595 scaled down the expression of TLR4 and induced TLR2 expression when exposed to P. gingivalis stimulation (p<0.01). Conclusions These findings provide evidence that L. rhamnosus ATCC9595 can modulate the inflammatory signals and could introduce P. gingivalis to immune systems by inducing CXCL8 secretion. PMID:27008259

  8. Draft Genome Sequence of Low-Passage Clinical Isolate Porphyromonas gingivalis MP4-504.

    PubMed

    To, Thao T; Liu, Quanhui; Watling, Michael; Bumgarner, Roger E; Darveau, Richard P; McLean, Jeffrey S

    2016-04-07

    We present the draft genome ofPorphyromonas gingivalisMP4-504, a low-passage clinical isolate obtained from a periodontitis patient. The genome is composed of 92 contigs for a length of 2,373,453 bp and a G+C of 48.3%. ThetraA-Qconjugative transfer locus is genetically distinct from W83 but highly similar to ATCC 33277.

  9. Draft Genome Sequence of Low-Passage Clinical Isolate Porphyromonas gingivalis MP4-504

    PubMed Central

    Liu, Quanhui; Watling, Michael; Bumgarner, Roger E.; Darveau, Richard P.

    2016-01-01

    We present the draft genome of Porphyromonas gingivalis MP4-504, a low-passage clinical isolate obtained from a periodontitis patient. The genome is composed of 92 contigs for a length of 2,373,453 bp and a G+C of 48.3%. The traA-Q conjugative transfer locus is genetically distinct from W83 but highly similar to ATCC 33277. PMID:27056232

  10. Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

    PubMed Central

    Cutler, C W; Kalmar, J R; Arnold, R R

    1991-01-01

    No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone. PMID:2037370

  11. Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

    PubMed Central

    Goulbourne, P A; Ellen, R P

    1991-01-01

    Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species. Images PMID:1679428

  12. The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine.

    PubMed

    Nylander, M; Lindahl, T L; Bengtsson, T; Grenegård, M

    2008-08-01

    Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct

  13. Altered antigenic profiling and infectivity of Porphyromonas gingivalis in smokers and non-smokers with periodontitis

    PubMed Central

    Zeller, Iris; Hutcherson, Justin A.; Lamont, Richard J.; Demuth, Donald R.; Gumus, Pinar; Nizam, Nejat; Buduneli, Nurcan; Scott, David A.

    2014-01-01

    Background Cigarette smokers are more susceptible to periodontal diseases and are more likely to be infected with Porphyromonas gingivalis than non-smokers. Furthermore, smoking is known to alter the expression of P. gingivalis surface components and to compromise IgG generation. The aim of this study was to evaluate whether IgG response to P. gingivalis is suppressed in smokers in vivo and whether previously established in vitro tobacco-induced phenotypic P. gingivalis changes would be reflected in vivo. Methods We examined the humoral response to several P. gingivalis strains as well as specific tobacco-regulated outer membrane proteins (FimA and RagB) by ELISA in biochemically-validated (salivary cotinine) smokers and non-smokers with chronic (CP, n = 13) or aggressive (AP, n = 20) periodontitis. We also monitored the local and systemic presence of P. gingivalis DNA by PCR. Results Smoking was associated with decreased total IgG responses against clinical (10512, 5607, and 10208C; all p < 0.05) but not laboratory (ATCC 33277, W83) P. gingivalis strains. Smoking did not influence IgG produced against specific cell surface proteins, although a non-significant pattern towards increased total FimA-specific IgG in CP subjects, but not AP subjects, was observed. Seropositive smokers were more likely to be infected orally and systemically with P. gingivalis (p < 0.001), as determined by 16S RNA analysis. Conclusions Smoking alters the humoral response against P. gingivalis, strengthening the evidence that mechanisms of periodontal disease progression in smokers may differ from non-smokers with the same disease classification. PMID:24147843

  14. Abrogation of neuraminidase reduces biofilm formation, capsule biosynthesis, and virulence of Porphyromonas gingivalis.

    PubMed

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping; Li, Chunhao

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection.

  15. Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis.

    PubMed

    Benedyk, Małgorzata; Mydel, Piotr Mateusz; Delaleu, Nicolas; Płaza, Karolina; Gawron, Katarzyna; Milewska, Aleksandra; Maresz, Katarzyna; Koziel, Joanna; Pyrc, Krzysztof; Potempa, Jan

    2016-01-01

    Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia. PMID:26613585

  16. Antimicrobial Efficacy of Various Essential Oils at Varying Concentrations against Periopathogen Porphyromonas gingivalis

    PubMed Central

    Grover, Harpreet Singh; Deswal, Himanshu; Agarwal, Preeti

    2016-01-01

    Introduction Porphyromonas gingivalis (P.gingivalis) is a notorious perio-pathogen with the ability to evade host defense mechanism and invade into the periodontal tissues. Many antimicrobial agents have been tested that curb its growth, although these agents tend to produce side effects such as antibiotic resistance and opportunistic infections. Therefore search for naturally occurring anti-microbials with lesser side effects is the need of the hour. Aim The aim of this study was to substantiate the antimicrobial activity of various essential oils; eucalyptus oil, chamomile oil, tea tree oil and turmeric oil against P. gingivalis. Materials and Methods Pure cultures of P. gingivalis were grown on selective blood agar. Antimicrobial efficacy of various concentrations of essential oils (0%, 25%, 50% and 100%) was assessed via disc diffusion test. Zone of inhibition were measured around disc after 48 hours in millimeters. Results Zones of inhibition were directly proportional to the concentration of essential oils tested. At 100% concentration all the tested oils possess antimicrobial activity against P.gingivalis with eucalyptus oil being most effective followed by tea tree oil, chamomile oil and turmeric oil. Conclusion All essential oils tested were effective against P.gingivalis. After testing for their clinical safety they could be developed into local agents to prevent and treat periodontitis. PMID:27790572

  17. Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

    PubMed Central

    Li, Chen; Kurniyati; Hu, Bo; Bian, Jiang; Sun, Jianlan; Zhang, Weiyan; Liu, Jun; Pan, Yaping

    2012-01-01

    The oral bacterium Porphyromonas gingivalis is a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide. P. gingivalis exhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence of P. gingivalis remain elusive. In this report, we found that P. gingivalis encodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed that PG0352 is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPg is an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that the PG0352 deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type, in vitro studies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement. In vivo studies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPg is an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity of P. gingivalis, and it can potentially serve as a new target for developing therapeutic agents against P. gingivalis infection. PMID:22025518

  18. Proteomic and transcriptional analysis of interaction between oral microbiota Porphyromonas gingivalis and Streptococcus oralis.

    PubMed

    Maeda, Kazuhiko; Nagata, Hideki; Ojima, Miki; Amano, Atsuo

    2015-01-01

    Porphyromonas gingivalis, a major periodontal pathogen, forms biofilm with other oral bacteria such as streptococci. Here, by using shotgun proteomics, we examined the molecular basis of mixed-biofilm formation by P. gingivalis with Streptococcus oralis. We identified a total of 593 bacterial proteins in the biofilm. Compared to the expression profile in the P. gingivalis monobiofilm, the expression of three proteins was induced and that of 31 proteins was suppressed in the mixed biofilm. Additionally, the expression of two S. oralis proteins was increased, while that of two proteins was decreased in the mixed biofilm, as compared to its monotypic profile. mRNA expression analysis of selected genes using a quantitative reverse transcription polymerase chain reaction confirmed the proteomics data, which included overexpression of P. gingivalis FimA and S. oralis glyceraldehyde-3-phosphate dehydrogenase in association with the biofilm. The results also indicated that S. oralis regulates the transcriptional activity of P. gingivalis luxS to influence autoinducer-2-dependent signaling. These findings suggest that several functional molecules are involved in biofilm formation between P. gingivalis and S. oralis.

  19. Transglutaminase 2 is essential for adherence of Porphyromonas gingivalis to host cells

    PubMed Central

    Boisvert, Heike; Lorand, Laszlo; Duncan, Margaret J.

    2014-01-01

    Porphyromonas gingivalis is the major causative agent of periodontitis, and it may also be involved in the development of systemic diseases (atherosclerosis, rheumatoid arthritis). P. gingivalis is found on and within oral and gingival epithelial cells following binding to surface components of host cells, which serve as receptors for the bacterium. Evidence is presented in this study that shows that transglutaminase 2 (TG2) plays a critical role in the adherence of P. gingivalis to host cells. Studies of confocal microscopy indicate colocalization of P. gingivalis with TG2 on the surface of HEp-2 epithelial cells, with clusters of TG2 seen at bacterial attachment sites. By silencing the expression of TG2 with siRNA in HEp-2 cells, P. gingivalis association was greatly diminished. The bacterium does not bind well to a mouse fibroblast cell line that produces low amounts of surface TG2, but binding can be restored by introduction of TG2 expressed on a plasmid. TG2 can form very tight complexes with fibronectin (FN), and the complementary binding sites of the two proteins are known. A synthetic peptide that mimics the main FN-binding sequence of TG2 blocks the formation of TG2–FN complexes and is highly effective in inhibiting adherence of P. gingivalis to host cells. These findings provide evidence of a role for cell-surface TG2 in bacterial attachment and subsequent internalization. PMID:24706840

  20. Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

    PubMed

    Aguilera, O; Andrés, M T; Heath, J; Fierro, J F; Douglas, C W

    1998-05-01

    The antimicrobial effect of lactoferrin (apoLf) on the oral, black-pigmented anaerobes Porphyromonas gingivalis, Prevotella intermedia and P. nitrescens has been studied. ApoLf did not kill any of these species but it did inhibit the growth of P. gingivalis, while iron-saturated Lf (FeLf) had no effect. The other two species were unaffected by apoLf. This growth inhibitory effect of apoLf could not be explained on the basis of chelation of inorganic iron, since growth of P. gingivalis occurred in the presence of ethylenediamine di-o-hydroxyphenylacetic acid provided haemin was added. Both apoLf and FeLf reduced haemin uptake by all three species and caused the release of cell-bound haemin in a dose-dependent manner. In addition, haemin reduced the binding of both apoLf and FeLf to P. intermedia and P. nigrescens but stimulated the binding of Lf by P. gingivalis. These data suggest that Lf forms complexes with haemin in solution and competes for the binding of haemin to certain cell receptors, possibly lipopolysaccharides, but this is not sufficient to inhibit growth of the bacteria. P. gingivalis appears to bind Lf-haemin complexes, as well as haemin alone, which may facilitate access of the Lf to the outer and cytoplasmic membranes of P. gingivalis, so disrupting function.

  1. Susceptibility of Porphyromonas gingivalis and Streptococcus mutans to Antibacterial Effect from Mammea americana.

    PubMed

    Herrera Herrera, Alejandra; Franco Ospina, Luis; Fang, Luis; Díaz Caballero, Antonio

    2014-01-01

    The development of periodontal disease and dental caries is influenced by several factors, such as microorganisms of bacterial biofilm or commensal bacteria in the mouth. These microorganisms trigger inflammatory and immune responses in the host. Currently, medicinal plants are treatment options for these oral diseases. Mammea americana extracts have reported antimicrobial effects against several microorganisms. Nevertheless, this effect is unknown against oral bacteria. Therefore, the aim of this study was to evaluate the antibacterial effect of M. americana extract against Porphyromonas gingivalis and Streptococcus mutans. For this, an experimental study was conducted. Ethanolic extract was obtained from seeds of M. americana (one oil phase and one ethanolic phase). The strains of Porphyromonas gingivalis ATCC 33277 and Streptococcus mutans ATCC 25175 were exposed to this extract to evaluate its antibacterial effect. Antibacterial activity was observed with the two phases of M. americana extract on P. gingivalis and S. mutans with lower MICs (minimum inhibitory concentration). Also, bactericidal and bacteriostatic activity was detected against S. mutans, depending on the concentration of the extract, while on M. americana extract presented only bacteriostatic activity against P. gingivalis. These findings provide important and promising information allowing for further exploration in the future.

  2. Susceptibility of Porphyromonas gingivalis and Streptococcus mutans to Antibacterial Effect from Mammea americana

    PubMed Central

    Herrera Herrera, Alejandra; Franco Ospina, Luis; Fang, Luis; Díaz Caballero, Antonio

    2014-01-01

    The development of periodontal disease and dental caries is influenced by several factors, such as microorganisms of bacterial biofilm or commensal bacteria in the mouth. These microorganisms trigger inflammatory and immune responses in the host. Currently, medicinal plants are treatment options for these oral diseases. Mammea americana extracts have reported antimicrobial effects against several microorganisms. Nevertheless, this effect is unknown against oral bacteria. Therefore, the aim of this study was to evaluate the antibacterial effect of M. americana extract against Porphyromonas gingivalis and Streptococcus mutans. For this, an experimental study was conducted. Ethanolic extract was obtained from seeds of M. americana (one oil phase and one ethanolic phase). The strains of Porphyromonas gingivalis ATCC 33277 and Streptococcus mutans ATCC 25175 were exposed to this extract to evaluate its antibacterial effect. Antibacterial activity was observed with the two phases of M. americana extract on P. gingivalis and S. mutans with lower MICs (minimum inhibitory concentration). Also, bactericidal and bacteriostatic activity was detected against S. mutans, depending on the concentration of the extract, while on M. americana extract presented only bacteriostatic activity against P. gingivalis. These findings provide important and promising information allowing for further exploration in the future. PMID:24864137

  3. Green tea epigallocatechin-3-gallate alleviates Porphyromonas gingivalis-induced periodontitis in mice.

    PubMed

    Cai, Yu; Chen, ZhiBin; Liu, Hao; Xuan, Yan; Wang, XiaoXuan; Luan, QingXian

    2015-12-01

    Porphyromonas gingivalis causes inflammation, and leads to the periodontitis in gingival tissue damage and bone resorption. Epigallocatechin-3-gallate (EGCG) is a major polyphenol extract from green tea with plenty of pharmacological functions. The aim of this study was to determine whether continuous oral intake of EGCG would alleviate P. gingivalis-induced periodontitis. Eight-week BALB/c mice were administered with EGCG (0.02%) or vehicle in drinking water. They were fed normal food and orally infected with P. gingivalis every 2days, up to a total of 20 times, and then sacrificed at 15weeks of age. The P. gingivalis-challenged group markedly increased alveolar bone resorption of the maxillae in BALB/c mice by Micro-CT detection, and administration of EGCG resulted in a significant reduction in bone loss. Inflammation cytokine antibody array and enzyme linked immunosorbent assay revealed that some inflammatory mediators in serum were increased by P. gingivalis infection, but were lowered after EGCG treatment. High positive areas of IL-17 and IL-1β in the gingival tissue were observed in the P. gingivalis-challenged mice, and were reduced by EGCG treatment. Real-time polymerase chain reaction (PCR) analyses also showed the expressions of IL-1β, IL-6, IL-17, IL-23, TNF-α and other mediators in gingival tissue were higher in P. gingivalis-challenged mice, and were down-regulated with EGCG treatment, except IL-23. Our results suggest that EGCG, as a natural healthy substance, probably alleviates P. gingivalis-induced periodontitis by anti-inflammatory effect.

  4. A Major Fimbrilin Variant of Mfa1 Fimbriae in Porphyromonas gingivalis.

    PubMed

    Nagano, K; Hasegawa, Y; Yoshida, Y; Yoshimura, F

    2015-08-01

    The periodontal pathogen Porphyromonas gingivalis is known to express 2 distinct types of fimbriae: FimA and Mfa1 fimbriae. However, we previously reported that fimbria-like structures were found in a P. gingivalis strain in which neither FimA nor Mfa1 fimbriae were detected. In this study, we identified a major protein in the bacterial lysates of the strain, which has been reported as the 53-kDa major outer membrane protein of P. gingivalis (53K protein) and subsequently reported as a major fimbrilin of a novel-type fimbria. Sequencing of the chromosomal DNA of the strain showed that the 53k gene (encoding the 53K protein) was located at a locus corresponding to the mfa1 gene (encoding the Mfa1 protein, which is a major fimbrilin of Mfa1 fimbriae) of the ATCC 33277 type strain. However, the 53K and Mfa1 proteins showed a low amino acid sequence homology and different antigenicity. The 53K protein was detected in 34 of 84 (41%) P. gingivalis strains, while the Mfa1 protein was detected in 44% of the strains. No strain expressed both 53K and Mfa1 proteins. Additionally, fimbriae were normally expressed in mutants in which the 53k and mfa1 genes were interchanged. These results indicate that the 53K protein is another major fimbrilin of Mfa1 fimbriae in P. gingivalis. PMID:26001707

  5. Porphyromonas gingivalis Accelerates Inflammatory Atherosclerosis in the Innominate Artery of ApoE Deficient Mice

    PubMed Central

    Hayashi, Chie; Viereck, Jason; Hua, Ning; Phinikaridou, Alkystis; Madrigal, Andres G.; Gibson, Frank C.; Hamilton, James A.; Genco, Caroline A.

    2011-01-01

    Objective Studies in humans support a role for the oral pathogen Porphyromonas gingivalis in the development of inflammatory atherosclerosis. The goal of this study was to determine if P. gingivalis infection accelerates inflammation and atherosclerosis in the innominate artery of mice, an artery which has been reported to exhibit many features of human atherosclerotic disease, including plaque rupture. Methods and Results Apolipoprotein E-deficient (ApoE−/−) mice were orally infected with P. gingivalis, and Magnetic Resonance Imaging (MRI) was used to monitor the progression of atherosclerosis in live mice. P. gingivalis infected mice exhibited a statistically significant increase in atherosclerotic plaque in the innominate artery as compared to uninfected mice. Polarized light microscopy and immunohistochemistry revealed that the innominate arteries of infected mice had increased lipids, macrophages and T cells as compared to uninfected mice. Increases in plaque, total cholesterol esters and cholesterol monohydrate crystals, macrophages, and T cells were prevented by immunization with heat-killed P. gingivalis prior to pathogen exposure. Conclusions These are the first studies to demonstrate progression of inflammatory plaque accumulation in the innominate arteries by in-vivo MRI analysis following pathogen exposure, and to document protection from plaque progression in the innominate artery via immunization. PMID:21251656

  6. Multilocus Sequence Typing of Porphyromonas gingivalis Strains from Different Geographic Origins

    PubMed Central

    Enersen, Morten; Olsen, Ingar; van Winkelhoff, Arie J.; Caugant, Dominique A.

    2006-01-01

    Porphyromonas gingivalis is an important periodontal pathogen that can be isolated from both active and inactive periodontal lesions. Apparently, differences in virulence between P. gingivalis strains exist, but the mechanisms underlying these differences are not yet fully understood. To obtain more information about pathogenicity and virulence of P. gingivalis, it is relevant to assess the genetic population structure of the species and to examine the occurrence of putative virulence factors against the genetic background. Presently, multilocus sequence typing (MLST) is the best method for analyzing bacterial population structures. Forty P. gingivalis strains from worldwide sources were analyzed by MLST. Internal 310- to 420-bp DNA fragments of the eight ubiquitous chromosomal genes, ftsQ, hagB, gdpxJ, pepO, mcmA, recA, pga, and nah, were amplified by PCR and then sequenced. The number of alleles at individual loci ranged from 2 to 19, and a total of 33 allelic profiles, or sequence types (STs), were identified. Nucleotide variation between alleles was located at one or a few sites. Identical or similar STs were found in isolates from different geographic regions. Our results showed signs of a clonal population structure with a level of recombination not as high as that previously suggested for the species. We also found that P. gingivalis isolates from individual patients were genetically heterogeneous. PMID:16390944

  7. Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis.

    PubMed

    Tagawa, Junpei; Inoue, Tetsuyoshi; Naito, Mariko; Sato, Keiko; Kuwahara, Tomomi; Nakayama, Masaaki; Nakayama, Koji; Yamashiro, Takashi; Ohara, Naoya

    2014-10-01

    We report here the construction of a plasmid vector designed for the efficient electrotransformation of the periodontal pathogen Porphyromonas gingivalis. The novel Escherichia coli-Bacteroides/P. gingivalis shuttle vector, designated pTIO-1, is based on the 11.0-kb E. coli-Bacteroides conjugative shuttle vector, pVAL-1 (a pB8-51 derivative). To construct pTIO-1, the pB8-51 origin of replication and erythromycin resistance determinant of pVAL-1 were cloned into the E. coli cloning vector pBluescript II SK(-) and non-functional regions were deleted. pTIO-1 has an almost complete multiple cloning site from pBluescript II SK(-). The size of pTIO-1 is 4.5kb, which is convenient for routine gene manipulation. pTIO-1 was introduced into P. gingivalis via electroporation, and erythromycin-resistant transformants carrying pTIO-1 were obtained. We characterized the transformation efficiency, copy number, host range, stability, and insert size capacity of pTIO-1. An efficient plasmid electrotransformation of P. gingivalis will facilitate functional analysis and expression of P. gingivalis genes, including the virulence factors of this bacterium.

  8. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis.

    PubMed

    Lam, Roselind S; O'Brien-Simpson, Neil M; Holden, James A; Lenzo, Jason C; Fong, Shao B; Reynolds, Eric C

    2016-01-01

    Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis. PMID:27383471

  9. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis

    PubMed Central

    Lenzo, Jason C.; Fong, Shao B.; Reynolds, Eric C.

    2016-01-01

    Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis. PMID:27383471

  10. Characterization of the α- and β-Mannosidases of Porphyromonas gingivalis

    PubMed Central

    Aduse-Opoku, Joseph; Hashim, Ahmed; Paramonov, Nikolay; Curtis, Michael A.

    2013-01-01

    Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1→3 and α-1→2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1→6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated. PMID:24056103

  11. Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

    PubMed Central

    Rosenberg, M; Buivids, I A; Ellen, R P

    1991-01-01

    Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable

  12. Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

    SciTech Connect

    Nakajima, Yoshitaka; Ito, Kiyoshi Xu, Yue; Yamada, Nozomi; Onohara, Yuko; Ito, Takashi; Yoshimoto, Tadashi

    2005-12-01

    P. gingivalis prolyl tripeptidyl aminopeptidase has been crystallized by the vapour-diffusion method. Diffraction data have been collected and processed to 2.1 Å resolution. A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 149.4, c = 159.7 Å. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a V{sub M} value of 3.14 Å{sup 3} Da{sup −1}. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation at the BL5 station of the Photon Factory.

  13. Porphyromonas gingivalis: An Overview of Periodontopathic Pathogen below the Gum Line

    PubMed Central

    How, Kah Yan; Song, Keang Peng; Chan, Kok Gan

    2016-01-01

    Periodontal disease represents a group of oral inflammatory infections initiated by oral pathogens which exist as a complex biofilms on the tooth surface and cause destruction to tooth supporting tissues. The severity of this disease ranges from mild and reversible inflammation of the gingiva (gingivitis) to chronic destruction of connective tissues, the formation of periodontal pocket and ultimately result in loss of teeth. While human subgingival plaque harbors more than 500 bacterial species, considerable research has shown that Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is the major etiologic agent which contributes to chronic periodontitis. This black-pigmented bacterium produces a myriad of virulence factors that cause destruction to periodontal tissues either directly or indirectly by modulating the host inflammatory response. Here, this review provides an overview of P. gingivalis and how its virulence factors contribute to the pathogenesis with other microbiome consortium in oral cavity. PMID:26903954

  14. Porphyromonas gingivalis: An Overview of Periodontopathic Pathogen below the Gum Line.

    PubMed

    How, Kah Yan; Song, Keang Peng; Chan, Kok Gan

    2016-01-01

    Periodontal disease represents a group of oral inflammatory infections initiated by oral pathogens which exist as a complex biofilms on the tooth surface and cause destruction to tooth supporting tissues. The severity of this disease ranges from mild and reversible inflammation of the gingiva (gingivitis) to chronic destruction of connective tissues, the formation of periodontal pocket and ultimately result in loss of teeth. While human subgingival plaque harbors more than 500 bacterial species, considerable research has shown that Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is the major etiologic agent which contributes to chronic periodontitis. This black-pigmented bacterium produces a myriad of virulence factors that cause destruction to periodontal tissues either directly or indirectly by modulating the host inflammatory response. Here, this review provides an overview of P. gingivalis and how its virulence factors contribute to the pathogenesis with other microbiome consortium in oral cavity. PMID:26903954

  15. The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis

    PubMed Central

    2010-01-01

    Background The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss. Results Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others. Conclusion Analyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized. PMID:20920246

  16. Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis.

    PubMed

    Fujimura, Setsuo; Hirai, Kaname; Shibata, Yukinaga

    2002-03-19

    A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases. PMID:12007665

  17. Synthesis of sulfonamides with effective inhibitory action against Porphyromonas gingivalis γ-carbonic anhydrase.

    PubMed

    Ceruso, Mariangela; Del Prete, Sonia; AlOthman, Zeid; Osman, Sameh M; Scozzafava, Andrea; Capasso, Clemente; Supuran, Claudiu T

    2014-08-15

    New benzenesulfonamides incorporating GABA or N-α-acetyl-L-lysine scaffolds as well as guanidine functionalities as water solubilizing moieties were obtained, using 4-aminoethyl/methyl-benzenesulfonamide and metanilamide/sulfanilamide as zinc-binding motives. The new compounds were medium potency inhibitors of the widespread cytosolic human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II and more effective inhibitors (KIs low nanomolar range) of the bacterial γ-CA from the oral pathogen Porphyromonas gingivalis. These sulfonamides may be useful tools for understanding the physiological role of bacterial CAs in pathogenesis of some infectious disease. PMID:25011913

  18. Photodynamic destruction of Porphyromonas gingivalis induced by delta-aminolaevulinic acid

    NASA Astrophysics Data System (ADS)

    Sieron, Aleksander; Wiczkowski, Andrzej; Adamek, Mariusz; Dyla, Lucja; Mazur, Sebastian; Wierucka-Mlynarczyk, Beata

    2004-09-01

    Photodynamic therapy (PDT) is one of a novel modalities which has recently been exploited to eradicate various microorganisms. In our study we have evaluated bactericidal efficacy of PDT in the presence of 5-δ aminolaevulinic acid (ALA). Porphyromonas gingivalis were incubated with increasing concentration of ALA and subsequently irradiated by progressive light doses. Complete killing effect was obtained for bacteria irradiated with 25J/cm2 in ALA solution final concentration of 1mM, 5mM, 10mM. Statistical analysis has revealed ALA concentration to be a major factor responsible for eradication of bacteria. The latter may be attributable to the known ALA dark toxicity.

  19. Isolation and characterization of a cloned Porphyromonas gingivalis hemagglutinin from an avirulent strain of Salmonella typhimurium.

    PubMed Central

    Dusek, D M; Progulske-Fox, A; Whitlock, J; Brown, T A

    1993-01-01

    Identification of surface macromolecules of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications for studying host-parasite interactions as well as for potential vaccine development. The objective of this study was to determine whether a cloned, P. gingivalis hemagglutinin gene could be expressed in an intact form in an avirulent Salmonella typhimurium vaccine construct and to characterize the recombinant protein. The recombinant protein was purified from the vaccine strain, characterized, and tested for biological activity as a competitive inhibitor of hemagglutination. Cells of S. typhimurium SL3261/pST7 grown in Luria broth were broken by sonic disruption and fractionated. The purified recombinant protein was found to inhibit hemagglutination of erythrocytes by whole P. gingivalis cells. The same purified protein was analyzed for its N-terminal amino acid sequence and amino acid composition and found to match that predicted from the nucleotide sequence of the cloned gene. These results indicate that a surface macromolecule of P. gingivalis can be expressed in an intact and biologically active form in a Salmonella carrier strain. Images PMID:8381773

  20. In Situ Anabolic Activity of Periodontal Pathogens Porphyromonas gingivalis and Filifactor alocis in Chronic Periodontitis.

    PubMed

    Spooner, Ralee; Weigel, Kris M; Harrison, Peter L; Lee, KyuLim; Cangelosi, Gerard A; Yilmaz, Özlem

    2016-01-01

    Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis. PMID:27642101

  1. Comparison of inherently essential genes of Porphyromonas gingivalis identified in two transposon-sequencing libraries.

    PubMed

    Hutcherson, J A; Gogeneni, H; Yoder-Himes, D; Hendrickson, E L; Hackett, M; Whiteley, M; Lamont, R J; Scott, D A

    2016-08-01

    Porphyromonas gingivalis is a Gram-negative anaerobe and keystone periodontal pathogen. A mariner transposon insertion mutant library has recently been used to define 463 genes as putatively essential for the in vitro growth of P. gingivalis ATCC 33277 in planktonic culture (Library 1). We have independently generated a transposon insertion mutant library (Library 2) for the same P. gingivalis strain and herein compare genes that are putatively essential for in vitro growth in complex media, as defined by both libraries. In all, 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in fundamentally important metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis, and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat-shock protein homologues, sigma factors, enzymes with proteolytic activity, and the majority of sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon-sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets.

  2. Mechanisms of Resistance of Porphyromonas gingivalis to Killing by Serum Complement

    PubMed Central

    Slaney, Jennifer M.; Gallagher, Alexandra; Aduse-Opoku, Joseph; Pell, Keith; Curtis, Michael A.

    2006-01-01

    The complement system plays an important role in the host defense against infection, and the formation of the terminal complement complex on the bacterial surface has been shown to be particularly important in killing of gram-negative bacteria. The gram-negative periodontal pathogen Porphyromonas gingivalis is resistant to complement killing, and possible mechanisms suggested for this resistance include protease production and capsule formation. In this study, P. gingivalis Arg- and Lys-gingipain deletion mutants and polysaccharide synthesis deletion mutants have been used to investigate these hypotheses. When Arg- and Lys-gingipain protease mutants were incubated in 20% normal human serum, deposition of complement components on the cell surface was significantly increased compared to that for the wild-type organism. However, despite the increased deposition, the protease mutants maintained resistance to killing and their viability was equal to that seen with heat-inactivated serum. Similar data were obtained when the wild-type organism was treated with gingipain protease inhibitors. K-antigen expression mutants were also resistant to killing. However, mutants which no longer synthesized a surface anionic polysaccharide (APS) (a phosphorylated branched mannan) were extremely sensitive to serum killing. These mutants lack the organized dense glycan surface layer present on the parent strain on the basis of electron microscopy. We conclude that the production of APS at the surface of P. gingivalis rather than Arg- and Lys-gingipain synthesis is the principal mechanism of serum resistance in P. gingivalis. PMID:16926430

  3. In Situ Anabolic Activity of Periodontal Pathogens Porphyromonas gingivalis and Filifactor alocis in Chronic Periodontitis

    PubMed Central

    Spooner, Ralee; Weigel, Kris M.; Harrison, Peter L.; Lee, KyuLim; Cangelosi, Gerard A.; Yilmaz, Özlem

    2016-01-01

    Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis. PMID:27642101

  4. Effect of Porphyromonas gingivalis on epithelial cell MMP-9 type IV collagenase production.

    PubMed Central

    Fravalo, P; Ménard, C; Bonnaure-Mallet, M

    1996-01-01

    Porphyromonas gingivalis is reportedly capable of stimulating the expression of host cell matrix metalloproteinases (MMP), contributing to tissue destruction. However, the impact of this bacterium on specific molecules remains to be determined. In this study, we evaluate the effect of P. gingivalis on regulation of MMP-9 expression in human gingival epithelial cells (HGEC). Various inocula of P. gingivalis were added to cultures of HGEC. The effects of live bacteria, heat-killed bacteria, and outer membrane extract were analyzed. MMP-9 secretion by HGEC was evaluated by enzyme-linked immunosorbent assay. For inocula smaller than one bacterium per cell, the quantity of MMP-9 secreted by HGEC was increased in comparison to control conditions. For inocula from 2.5 to 250 bacteria per cell, an inhibition of MMP-9 secretion in a dose-response fashion was observed, with a maximum reduction (ranging from 80 to 95% in five experiments) at 50 bacteria per cell. Gelatin zymograms confirmed the decrease in MMP-9 secretion. A band of 83 kDa, corresponding to activated enzyme, was present for inocula of 0.5 to 50 bacteria. Inhibition took place without any alteration of epithelial cell viability. Heat-killed bacteria and outer membrane extract also provoked proenzyme activation but did not inhibit MMP-9 secretion. These results demonstrate a direct effect of P. gingivalis on HGEC, suggesting a specific action on the collagen renewal process at the interface between the epithelium and connective tissue. PMID:8945530

  5. Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

    SciTech Connect

    Svintradze, David V.; Peterson, Darrell L.; Collazo-Santiago, Evys A.; Lewis, Janina P.; Wright, H. Tonie

    2013-10-01

    Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.

  6. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils

    PubMed Central

    Chen, Yang; Cheng, Xiao-fan; Qiu, Jia-ying; Xu, Yan; Sun, Ying

    2016-01-01

    Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)–tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells. PMID:27536946

  7. Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity.

    PubMed

    Bielecka, Ewa; Scavenius, Carsten; Kantyka, Tomasz; Jusko, Monika; Mizgalska, Danuta; Szmigielski, Borys; Potempa, Barbara; Enghild, Jan J; Prossnitz, Eric R; Blom, Anna M; Potempa, Jan

    2014-11-21

    Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis. PMID:25324545

  8. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils.

    PubMed

    Zhu, Xiang-Qing; Lu, Wei; Chen, Yang; Cheng, Xiao-Fan; Qiu, Jia-Ying; Xu, Yan; Sun, Ying

    2016-01-01

    Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells. PMID:27536946

  9. Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

    PubMed Central

    Burmistrz, Michał; Dudek, Bartosz; Staniec, Dominika; Rodriguez Martinez, Jose Ignacio; Bochtler, Matthias; Potempa, Jan

    2015-01-01

    ABSTRACT The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3′ end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3′ terminus by the appropriate PAM element. IMPORTANCE The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial

  10. Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

    PubMed Central

    Goulas, Theodoros; Mizgalska, Danuta; Garcia-Ferrer, Irene; Kantyka, Tomasz; Guevara, Tibisay; Szmigielski, Borys; Sroka, Aneta; Millán, Claudia; Usón, Isabel; Veillard, Florian; Potempa, Barbara; Mydel, Piotr; Solà, Maria; Potempa, Jan; Gomis-Rüth, F. Xavier

    2015-01-01

    Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer’s disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a “Michaelis loop” that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants. PMID:26132828

  11. PERIODONTAL DISEASE AND BONE PATHOGENESIS: THE CROSSTALK BETWEEN CYTOKINES AND PORPHYROMONAS GINGIVALIS.

    PubMed

    Ballini, A; Cantore, S; Farronato, D; Cirulli, N; Inchingolo, F; Papa, F; Malcangi, G; Inchingolo, A D; Dipalma, G; Sardaro, N; Lippolis, R; Santacroce, L; Coscia, M F; Pettini, F; De Vito, D; Scacco, S

    2015-01-01

    Periodontal disease is the most frequent cause of tooth loss among adults. It is defined as a plaque-induced inflammation of the periodontal tissues that results in a loss of support of the affected teeth. This process is characterized by destruction of the periodontal attachment apparatus, increased bone resorption with loss of crestal alveolar bone, apical migration of the epithelial attachment, and formation of periodontal pockets. Although the presence of periodontal pathogens such as Porphyromonas gingivalis is a prerequisite, the progression of periodontal disease is dependent on the host response to pathogenic bacteria that colonize the tooth surface. Nowadays, a growing body of literature has accumulated to investigate the association between bone diseases, periodontal pathogens and periodontal diseases. The integration of pathogen-associated molecular patterns from microorganisms with their surface receptors in the immune cells, induces the production of several cytokines and chemokines that present either a pro- and/or anti-inflammatory role and the activation of mechanisms of controlling this and the related disease, such as osteoporosis and rheumatoid arthritis. This review focuses on the evidence and significance of bone host cell invasion by Porphyromonas gingivalis in the pathogenesis of bone disorders, as well as the different lines of evidence supporting the role of cytokines in bone diseases. PMID:26122214

  12. Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

    PubMed

    Kitano, Taiichi; Mikami, Yoshikazu; Iwase, Takashi; Asano, Masatake; Komiyama, Kazuo

    2016-01-01

    Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

  13. Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis

    SciTech Connect

    Li, N.; Yun, P.; Nadkarni, M.A.; Ghadikolaee, N.B.; Nguyen, K.A.; Lee, M.; Hunter, N.; Collyer, C.A.

    2010-08-27

    Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel {beta}-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

  14. Genetic structure of populations of Porphyromonas gingivalis associated with periodontitis and other oral infections.

    PubMed Central

    Loos, B G; Dyer, D W; Whittam, T S; Selander, R K

    1993-01-01

    One hundred isolates of the oral pathogenic bacterium Porphyromonas gingivalis were genetically characterized by determining the electrophoretic mobilities of 16 metabolic enzymes and the presence or absence of catalase activity. A total of 78 distinct electrophoretic types (ETs), representing multilocus genotypes, were identified, and cluster analysis placed them in three major phylogenetic divisions. Division I (71 ETs) included all 88 human isolates examined, most of which had been recovered from patients with periodontitis, together with 4 monkey isolates. The strains in division II (four ETs) and division III (three ETs) are strongly differentiated from those in division I and apparently represent two previously unclassified (cryptic) species. The mean genetic diversity per enzyme locus among the 92 isolates of division I (P. gingivalis, strict sense) was 0.321, and the strains were distributed among 14 phylogenetic clusters and single-ET lineages. The population structure is basically clonal, with some clonal genotypes being widespread, and even global, in distribution. There was no evidence of association between specific genetic lineages or clusters of ETs and the type of disease (periodontitis or root canal infections), invasive potential, serogroup, or fimbrial restriction fragment length polymorphism group. The finding that dental patients are infected by strains of a wide variety of chromosomal genotypes suggests that interstrain variation in pathogenicity is small. On the basis of the observed genetic structure of natural populations of P. gingivalis, we hypothesize that the role of this microorganism in the pathogenesis of periodontitis and other dental infections is largely opportunistic. PMID:8380281

  15. Effects of temperature stress on expression of fimbriae and superoxide dismutase by Porphyromonas gingivalis.

    PubMed Central

    Amano, A; Sharma, A; Sojar, H T; Kuramitsu, H K; Genco, R J

    1994-01-01

    We examined the biosynthesis of fimbriae and superoxide dismutase (SOD) produced by the periodontopathic bacterium Porphyromonas gingivalis in response to elevated temperature. P. gingivalis 2561, grown at 37 degrees C to mid-logarithmic phase, was subsequently incubated at 39, 41, and 43 degrees C, respectively, to stationary phase. There was no difference in the growth of cells at 37 and 39 degrees C. However, at 39 degrees C there was a 54% reduction in the amount of fimbrillin (fimbriae) as well as decreased expression of mRNA for fimA. On the other hand, under the same conditions, a more than twofold increase in the amount of SOD activity, as well as in the levels of SOD mRNA, was observed. Moreover, cells cultured for 20 h at 39 degrees C showed an 86% decrease of fimbrillin protein and a threefold increase in SOD activity. These observations suggest that P. gingivalis may undergo alterations in its virulence and susceptibility to host immune responses as a result of the elevated temperatures found in inflamed periodontal pockets. Images PMID:7927742

  16. Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis

    PubMed Central

    Gomes-Filho, I. S.; Meyer, R.; Olczak, T.; Xavier, M. T.; Trindade, S. C.

    2016-01-01

    Periodontitis is a multifactorial disease, with participation of bacterial, environmental, and host factors. It results from synergistic and dysbiotic multispecies microorganisms, critical “keystone pathogens,” affecting the whole bacterial community. The purpose of this study was to review the role of Porphyromonas gingivalis in the immunopathogenesis of chronic periodontitis, with special attention paid to HmuY. The host response during periodontitis involves the innate and adaptive immune system, leading to chronic inflammation and progressive destruction of tooth-supporting tissues. In this proinflammatory process, the ability of P. gingivalis to evade the host immune response and access nutrients in the microenvironment is directly related to its survival, proliferation, and infection. Furthermore, heme is an essential nutrient for development of these bacteria, and HmuY is responsible for its capture from host heme-binding proteins. The inflammatory potential of P. gingivalis HmuY has been shown, including induction of high levels of proinflammatory cytokines and CCL2, decreased levels of IL-8, and increased levels of anti-HmuY IgG and IgG1 antibodies in individuals with chronic periodontitis. Therefore, the HmuY protein might be a promising target for therapeutic strategies and for development of diagnostic methods in chronic periodontitis, especially in the case of patients with chronic periodontitis not responding to treatment, monitoring, and maintenance therapy. PMID:27403039

  17. Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis

    PubMed Central

    Shoji, Mikio; Yukitake, Hideharu; Sato, Keiko; Shibata, Yasuko; Naito, Mariko; Aduse-Opoku, Joseph; Abiko, Yoshimitsu; Curtis, Michael A; Nakayama, Koji

    2013-01-01

    The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN_2005, which encodes a hypothetical protein of P. gingivalis ATCC 33277. We found that (i) LPSs purified from the PGN_2005 mutant were shorter than those of the wild type; (ii) the PGN_2005 protein was located in the inner membrane fraction; and (iii) the PGN_2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant. These results indicate that the PGN_2005 protein, which was designated WzzP, is a functional homolog of the Wzz protein in P. gingivalis. Comparison of amino acid sequences among WzzP and conventional Wzz proteins indicated that WzzP had an additional fragment at the C-terminal region. In addition, we determined that the PGN_1896 and PGN_1233 proteins and the PGN_1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein involved in LPS biosynthesis, respectively. PMID:23509024

  18. Role of Porphyromonas gingivalis HmuY in Immunopathogenesis of Chronic Periodontitis.

    PubMed

    Carvalho-Filho, P C; Gomes-Filho, I S; Meyer, R; Olczak, T; Xavier, M T; Trindade, S C

    2016-01-01

    Periodontitis is a multifactorial disease, with participation of bacterial, environmental, and host factors. It results from synergistic and dysbiotic multispecies microorganisms, critical "keystone pathogens," affecting the whole bacterial community. The purpose of this study was to review the role of Porphyromonas gingivalis in the immunopathogenesis of chronic periodontitis, with special attention paid to HmuY. The host response during periodontitis involves the innate and adaptive immune system, leading to chronic inflammation and progressive destruction of tooth-supporting tissues. In this proinflammatory process, the ability of P. gingivalis to evade the host immune response and access nutrients in the microenvironment is directly related to its survival, proliferation, and infection. Furthermore, heme is an essential nutrient for development of these bacteria, and HmuY is responsible for its capture from host heme-binding proteins. The inflammatory potential of P. gingivalis HmuY has been shown, including induction of high levels of proinflammatory cytokines and CCL2, decreased levels of IL-8, and increased levels of anti-HmuY IgG and IgG1 antibodies in individuals with chronic periodontitis. Therefore, the HmuY protein might be a promising target for therapeutic strategies and for development of diagnostic methods in chronic periodontitis, especially in the case of patients with chronic periodontitis not responding to treatment, monitoring, and maintenance therapy. PMID:27403039

  19. Bactericidal effect of a 405-nm diode laser on Porphyromonas gingivalis

    NASA Astrophysics Data System (ADS)

    Kotoku, Y.; Kato, J.; Akashi, G.; Hirai, Y.; Ishihara, K.

    2009-05-01

    The study was conducted to determine the effect of 405-nm diode laser irradiation on periodontopathic bacteria such as Porphyromonas gingivalis in vitro. A diluted suspension of P. gingivalis was irradiated directly with a 405-nm diode laser under conditions of 100 mW-10 sec, 100 mW-20 sec, 200 mW-5 sec, 200 mW-10 sec, 200 mW-20 sec, 400 mW-5 sec, 400 mW-10 sec, and 400 mW-20 sec. The energy density ranged from 2.0 to 16.0 J/cm2. The irradiated bacterial suspension was spread on a blood agar plate and growth of the colonies was examined after an anaerobic culture for 7 days. Bacterial growth was inhibited under all irradiation conditions, but the bactericidal effect of the 405-nm diode laser depended on the energy density. More than 97% of bacterial growth was inhibited with irradiation at an energy density > 4.0 J/cm2. The mechanism of the bactericidal effect is photochemical, rather than photothermal. These findings suggest that a 405-nm diode laser has a high bactericidal effect on P. gingivalis.

  20. Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: an in vitro study.

    PubMed

    Feuerstein, Osnat; Persman, Nir; Weiss, Ervin I

    2004-01-01

    The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400-500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz-tungsten-halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16-62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159-212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens. PMID:15623322

  1. Antibacterial activity against Porphyromonas gingivalis and biological characteristics of antibacterial stainless steel.

    PubMed

    Zhang, Dan; Ren, Ling; Zhang, Yang; Xue, Nan; Yang, Ke; Zhong, Ming

    2013-05-01

    To evaluate the possibility of an alternative to the traditional orthodontic stainless steel implants, the antibacterial activity against Porphyromonas gingivalis (P. gingivalis) and the related cytotoxicity of a type 304 Cu bearing antibacterial stainless steel were studied. The results indicated that the antibacterial stainless steel showed excellent antibacterial property against P. gingivalis, compared with the control steel (a purchased medical grade 304 stainless steel). Compared to the control steel, there were fewer bacteria on the surface of the antibacterial stainless steel, with significant difference in morphology. The cytotoxicities of the antibacterial stainless steel to both MG-63 and KB cells were all grade 1, the same as those of the control steel. There were no significant differences in the apoptosis rates on MG-63 and KB cells between the antibacterial stainless steel and the control steel. This study demonstrates that the antibacterial stainless steel is possible to reduce the incidence of implant-related infections and can be a more suitable material for the micro-implant than the conventional stainless steel in orthodontic treatment.

  2. Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis

    PubMed Central

    Maekawa, Tomoki; Krauss, Jennifer L.; Abe, Toshiharu; Jotwani, Ravi; Triantafilou, Martha; Triantafilou, Kathy; Hashim, Ahmed; Hoch, Shifra; Curtis, Michael A.; Nussbaum, Gabriel; Lambris, John D.; Hajishengallis, George

    2014-01-01

    SUMMARY Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides ‘bystander’ protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR, and can contribute to the persistence of microbial communities that drive dysbiotic diseases. PMID:24922578

  3. Arg-Gingipain A DNA Vaccine Induces Protective Immunity against Infection by Porphyromonas gingivalis in a Murine Model

    PubMed Central

    Yonezawa, Hideo; Ishihara, Kazuyuki; Okuda, Katsuji

    2001-01-01

    Arginine-specific cysteine proteinases (RgpA and RgpB) produced by the periodontal pathogen Porphyromonas gingivalis are suspected virulence factors and are involved in interrupting host defense mechanisms as well as in penetrating and destroying periodontal connective tissues. To induce a protective immune response against P. gingivalis, we constructed an rgpA DNA vaccine. BALB/c mice were immunized intradermally by Gene Gun with plasmid DNA carrying rgpA. Antibody responses against P. gingivalis were determined by an enzyme-linked immunosorbent assay. The rgpA DNA vaccine induced high levels of serum antibodies against P. gingivalis. Sera from the rgpA DNA vaccine-immunized mice diminished the proteolytic activity of RgpA and RgpB and inhibited the binding of P. gingivalis to a type I collagen sponge. Moreover, the sera effectively reduced the hemagglutination of P. gingivalis, indicating that the hemagglutinin activity of the organism is associated with RgpA. We found with a murine abscess model that mice immunized with the rgpA DNA vaccine were resistant to an invasive P. gingivalis W50 challenge. These results suggest that the rgpA DNA vaccine induced specific antibodies against the enzyme and that this vaccine could confer protective immunity against P. gingivalis infection. PMID:11292699

  4. Gingipains from Porphyromonas gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells.

    PubMed

    Sheets, Shaun M; Potempa, Jan; Travis, James; Casiano, Carlos A; Fletcher, Hansel M

    2005-03-01

    The presence of Porphyromonas gingivalis in the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells and human microvascular endothelial cells to gingipain-active extracellular protein preparations and/or purified gingipains from P. gingivalis. Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin beta1 was observed in immunoblots of cell lysates. There was a direct correlation between the kinetics of cleavage of N- and VE-cadherin and loss of cell adhesion properties. Loss of cell adhesion, as well as N- and VE-cadherin and integrin beta1 cleavage, could be inhibited or significantly delayed by preincubation of P. gingivalis W83 gingipain-active extracellular extracts with the cysteine protease inhibitor Nalpha-p-tosyl-l-lysine chloromethylketone. Furthermore, purified gingipains also induced endothelial cell detachment and apoptosis. Apoptosis-associated events, including annexin V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase and topoisomerase I (Topo I), were observed in endothelial cells after detachment. All of the effects observed were correlated with the different levels of cysteine-dependent proteolytic activity of the extracts tested. Taken together, these results indicate that gingipains from P. gingivalis can alter cell adhesion molecules and induce endothelial cell death, which could have implications for the pathogenicity of this organism. PMID:15731052

  5. Erythritol alters microstructure and metabolomic profiles of biofilm composed of Streptococcus gordonii and Porphyromonas gingivalis.

    PubMed

    Hashino, E; Kuboniwa, M; Alghamdi, S A; Yamaguchi, M; Yamamoto, R; Cho, H; Amano, A

    2013-12-01

    The effects of sugar alcohols such as erythritol, xylitol, and sorbitol on periodontopathic biofilm are poorly understood, though they have often been reported to be non-cariogenic sweeteners. In the present study, we evaluated the efficacy of sugar alcohols for inhibiting periodontopathic biofilm formation using a heterotypic biofilm model composed of an oral inhabitant Streptococcus gordonii and a periodontal pathogen Porphyromonas gingivalis. Confocal microscopic observations showed that the most effective reagent to reduce P. gingivalis accumulation onto an S. gordonii substratum was erythritol, as compared with xylitol and sorbitol. In addition, erythritol moderately suppressed S. gordonii monotypic biofilm formation. To examine the inhibitory effects of erythritol, we analyzed the metabolomic profiles of erythritol-treated P. gingivalis and S. gordonii cells. Metabolome analyses using capillary electrophoresis time-of-flight mass spectrometry revealed that a number of nucleic intermediates and constituents of the extracellular matrix, such as nucleotide sugars, were decreased by erythritol in a dose-dependent manner. Next, comparative analyses of metabolites of erythritol- and sorbitol-treated cells were performed using both organisms to determine the erythritol-specific effects. In P. gingivalis, all detected dipeptides, including Glu-Glu, Ser-Glu, Tyr-Glu, Ala-Ala and Thr-Asp, were significantly decreased by erythritol, whereas they tended to be increased by sorbitol. Meanwhile, sorbitol promoted trehalose 6-phosphate accumulation in S. gordonii cells. These results suggest that erythritol has inhibitory effects on dual species biofilm development via several pathways, including suppression of growth resulting from DNA and RNA depletion, attenuated extracellular matrix production, and alterations of dipeptide acquisition and amino acid metabolism.

  6. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  7. Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis *

    PubMed Central

    Ohara-Nemoto, Yuko; Rouf, Shakh M. A.; Naito, Mariko; Yanase, Amie; Tetsuo, Fumi; Ono, Toshio; Kobayakawa, Takeshi; Shimoyama, Yu; Kimura, Shigenobu; Nakayama, Koji; Saiki, Keitarou; Konishi, Kiyoshi; Nemoto, Takayuki K.

    2014-01-01

    Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 μm and 11.02 μm−1 s−1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea. PMID:24398682

  8. Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis.

    PubMed

    Schifferle, R E; Wilson, M E; Levine, M J; Genco, R J

    1993-07-01

    We previously reported that hot aqueous phenol extraction of Porphyromonas gingivalis yields a preparation containing both lipopolysaccharide (LPS) and an antigenically distinct capsular polysaccharide (PS). In the present study, we examined the capacity of phenol-water extracts from a number of strains of P. gingivalis to activate human serum complement. Anticomplementary activity of extracts from two invasive and two noninvasive strains of P. gingivalis was assessed in a sheep erythrocyte hemolytic assay and in an alternative pathway-selective rabbit erythrocyte hemolytic assay. In the sheep erythrocyte assay, extracts from noninvasive strains were found to exhibit greater anticomplementary activity than extracts derived from invasive strains. A phenol-water extract from invasive strain ATCC 53977 was further resolved into its LPS and PS fractions. Whereas isolated LPS from this strain exhibited strong anticomplementary activity, the PS fraction was only weakly active. Phenol-water extracts from three of four strains were found to be potent activators of the alternative pathway, with extracts from the two noninvasive strains being most active. The extract from the remaining strain (ATCC 53977) was a poor activator of the alternative pathway. Further analysis of this extract revealed, however, that the LPS fraction was a potent activator of the alternative pathway, although the PS fraction exhibited negligible activity. The results of this study indicate that phenol-water extracts of invasive and noninvasive strains of P. gingivalis differ in their respective anticomplementary activities, with invasive strains being less active. Although extracts from both invasive and noninvasive strains activated the alternative pathway, this activity appears to be attributable to the LPS, rather than the PS, component.

  9. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, amino-terminal sequence, and antigenic heterogeneity.

    PubMed Central

    Lee, J Y; Sojar, H T; Bedi, G S; Genco, R J

    1991-01-01

    Bacterial fimbriae mediate cell adhesion and are important in colonization. Fimbrial proteins from strains of Porphyromonas (Bacteroides) gingivalis isolated from different individuals were compared for their size, amino-terminal sequence, and antigenic diversity. Two major protein components of the crude fimbrial preparations differed in apparent molecular mass, ranging from 41 to 49 kDa for the fimbrillin monomer and from 61 to 78 kDa for the other major protein. The amino-terminal sequence of the antigenically related group of proteins of the fimbrillin monomer in the 41- to 49-kDa range showed significant homology; however, minor sequence heterogeneity was observed, mainly in residues 4 to 6. One of the observed amino-terminal sequences, AFGVGDDESKVAKLTVMVYNG, resembled the deduced sequence of P. gingivalis 381 (D.P. Dickinson, M. K. Kubiniec, F. Yoshimura, and R.J. Genco, J. Bacteriol. 170:1658-1665, 1988). Fimbriae from all the strains of P. gingivalis showing this sequence contained a fimbrillin monomer of 43 kDa and showed a strong reaction with both polyclonal and monoclonal antibodies directed to the fimbriae from P. gingivalis 2561 (381). Fimbriae from strains showing amino-terminal sequence variations in residues 4 to 6 (i.e., substitution of VGD with either E or NAG) were more diverse in their molecular sizes. Most of these variant fimbriae showed weak reactions with the polyclonal antibodies and no reaction with the monoclonal antibodies induced to the fimbriae of strain 2561. No correlation could be established between the molecular size and immunological reactivity of the fimbrillin monomer of P. gingivalis strains. Strains 9-14K-1 and HG 564 not only showed markedly different sequences from the other three amino-terminal sequences but also did not react with either polyclonal or monoclonal antibodies to the fimbriae of strain 2561. Strains W50, W83, and AJW 5 failed to show any immunological reactivity with the antibodies to fimbrillin or fimbriae

  10. Honey – a potential agent against Porphyromonas gingivalis: an in vitro study

    PubMed Central

    2014-01-01

    Background Honey has been discussed as a therapeutic option in wound healing since ancient time. It might be also an alternative to the commonly used antimicrobials in periodontitis treatment. The in-vitro study was aimed to determine the antimicrobial efficacy against Porphyromonas gingivalis as a major periodontopathogen. Methods One Manuka and one domestic beekeeper honey have been selected for the study. As a screening, MICs of the honeys against 20 P. gingivalis strains were determined. Contents of methylglyoxal and hydrogen peroxide as the potential antimicrobial compounds were determined. These components (up to 100 mg/l), propolis (up to 200 mg/l) as well as the two honeys (up to 10% w/v) were tested against four P. gingivalis strains in planktonic growth and in a single-species biofilm. Results 2% of Manuka honey inhibited the growth of 50% of the planktonic P. gingivalis, the respective MIC50 of the German beekeeper honey was 5%. Manuka honey contained 1.87 mg/kg hydrogen peroxide and the domestic honey 3.74 mg/kg. The amount of methylglyoxal was found to be 2 mg/kg in the domestic honey and 982 mg/kg in the Manuka honey. MICs for hydrogen peroxide were 10 mg/l - 100 mg/l, for methylglyoxal 5 – 20 mg/l, and for propolis 20 mg/l – 200 mg/l. 10% of both types of honey inhibited the formation of P. gingivalis biofilms and reduced the numbers of viable bacteria within 42 h-old biofilms. Neither a total prevention of biofilm formation nor a complete eradication of a 42 h-old biofilm by any of the tested compounds and the honeys were found. Conclusions Honey acts antibacterial against P. gingivalis. The observed pronounced effects of Manuka honey against planktonic bacteria but not within biofilm can be attributed to methylglyoxal as the characteristic antimicrobial component. PMID:24666777

  11. Porphyromonas gingivalis HmuY stimulates expression of Bcl-2 and Fas by human CD3+ T cells

    PubMed Central

    2013-01-01

    Background Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. Results CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). Conclusion The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein. PMID:24025186

  12. A Two-Component System Regulates Hemin Acquisition in Porphyromonas gingivalis

    PubMed Central

    Scott, Jodie C.; Klein, Brian A.; Duran-Pinedo, Ana; Hu, Linden; Duncan, Margaret J.

    2013-01-01

    Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with infection of the periodontia. The organism has a small number of two-component signal transduction systems, and after comparing genome sequences of strains W83 and ATCC 33277 we discovered that the latter was mutant in histidine kinase (PGN_0752), while the cognate response regulator (PGN_0753) remained intact. Microarray-based transcriptional profiling and ChIP-seq assays were carried out with an ATCC 33277 transconjugant containing the functional histidine kinase from strain W83 (PG0719). The data showed that the regulon of this signal transduction system contained genes that were involved in hemin acquisition, including gingipains, at least three transport systems, as well as being self-regulated. Direct regulation by the response regulator was confirmed by electrophoretic mobility shift assays. In addition, the system appears to be activated by hemin and the regulator acts as both an activator and repressor. PMID:24039921

  13. Intraspecies Variability Affects Heterotypic Biofilms of Porphyromonas gingivalis and Prevotella intermedia: Evidences of Strain-Dependence Biofilm Modulation by Physical Contact and by Released Soluble Factors.

    PubMed

    Barbosa, Graziela Murta; Colombo, Andrea Vieira; Rodrigues, Paulo Henrique; Simionato, Maria Regina Lorenzetti

    2015-01-01

    It is well known that strain and virulence diversity exist within the population structure of Porphyromonas gingivalis. In the present study we investigate intra- and inter-species variability in biofilm formation of Porphyromonas gingivalis and partners Prevotella intermedia and Prevotella nigrescens. All strains tested showed similar hydrophobicity, except for P. gingivalis W83 which has roughly half of the hydrophobicity of P. gingivalis ATCC33277. An intraspecies variability in coaggregation of P. gingivalis with P. intermedia was also found. The association P. gingivalis W83/P. intermedia 17 produced the thickest biofilm and strain 17 was prevalent. In a two-compartment system P. gingivalis W83 stimulates an increase in biomass of strain 17 and the latter did not stimulate the growth of P. gingivalis W83. In addition, P. gingivalis W83 also stimulates the growth of P. intermedia ATCC25611 although strain W83 was prevalent in the association with P. intermedia ATCC25611. P. gingivalis ATCC33277 was prevalent in both associations with P. intermedia and both strains of P. intermedia stimulate the growth of P. gingivalis ATCC33277. FISH images also showed variability in biofilm structure. Thus, the outcome of the association P. gingivalis/P. intermedia seems to be strain-dependent, and both soluble factors and physical contact are relevant. The association P. gingivalis-P. nigrescens ATCC33563 produced larger biomass than each monotypic biofilm, and P. gingivalis was favored in consortia, while no differences were found in the two-compartment system. Therefore, in consortia P. gingivalis-P. nigrescens physical contact seems to favor P. gingivalis growth. The intraspecies variability found in our study suggests strain-dependence in ability of microorganisms to recognize molecules in other bacteria which may further elucidate the dysbiosis event during periodontitis development giving additional explanation for periodontal bacteria, such as P. gingivalis and P

  14. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

    PubMed Central

    Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.

    2015-01-01

    Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169

  15. Diagnostic evaluation of a nanobody with picomolar affinity towards the protease RgpB from Porphyromonas gingivalis

    PubMed Central

    Skottrup, Peter Durand; Leonard, Paul; Kaczmarek, Jakub Zbigniew; Veillard, Florian; Enghild, Jan J.; O’Kennedy, Richard; Sroka, Aneta; Clausen, Rasmus Prætorius; Potempa, Jan; Riise, Erik

    2011-01-01

    Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases, termed gingipains, and in this study we have utilized the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naïve camel nanobody library and used phage display to select one nanobody towards RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB as it did not bind to the homologous gingipain HRgpA. This indicated a presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted, soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection. PMID:21569755

  16. Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways

    PubMed Central

    Dawson, Deborah V.; Blanchette, Derek R.; Drake, David R.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    ABSTRACT Lipids endogenous to skin and mucosal surfaces exhibit potent antimicrobial activity against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Our previous work demonstrated the antimicrobial activity of the fatty acid sapienic acid (C16:1Δ6) against P. gingivalis and found that sapienic acid treatment alters both protein and lipid composition from those in controls. In this study, we further examined whole-cell protein differences between sapienic acid-treated bacteria and untreated controls, and we utilized open-source functional association and annotation programs to explore potential mechanisms for the antimicrobial activity of sapienic acid. Our analyses indicated that sapienic acid treatment induces a unique stress response in P. gingivalis resulting in differential expression of proteins involved in a variety of metabolic pathways. This network of differentially regulated proteins was enriched in protein-protein interactions (P = 2.98 × 10−8), including six KEGG pathways (P value ranges, 2.30 × 10−5 to 0.05) and four Gene Ontology (GO) molecular functions (P value ranges, 0.02 to 0.04), with multiple suggestive enriched relationships in KEGG pathways and GO molecular functions. Upregulated metabolic pathways suggest increases in energy production, lipid metabolism, iron acquisition and processing, and respiration. Combined with a suggested preferential metabolism of serine, which is necessary for fatty acid biosynthesis, these data support our previous findings that the site of sapienic acid antimicrobial activity is likely at the bacterial membrane. IMPORTANCE P. gingivalis is an important opportunistic pathogen implicated in periodontitis. Affecting nearly 50% of the population, periodontitis is treatable, but the resulting damage is irreversible and eventually progresses to tooth loss. There is a great need for natural products that can be used to treat and/or prevent the overgrowth of

  17. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  18. microRNAs responsive to A. actinomycetemcomitans and P. gingivalis LPS modulate expression of genes regulating innate immunity in human macrophages

    PubMed Central

    Naqvi, Afsar R.; Fordham, Jezrom B.; Khan, Asma; Nares, Salvador

    2013-01-01

    microRNAs (miRNA) are a class of small noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including lipopolysaccharide (LPS). Here we examined the early (4h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) or environmentally modified LPS obtained from Pg grown in cigarette smoke extract. Predicted miRNA-gene target interactions for LPS-responsive miR-29b and let-7f were confirmed using dual-luciferase assays and by transfection experiments using miRNA mimics and inhibitors. Convergent and divergent miRNA profiles were observed in treated samples where differences in miRNA levels related to the type, concentration and incubation times of LPS challenge. Dual-luciferase experiments revealed miR-29b targeting of IL-6Rα and IFN-γ inducible protein 30 (IFI30) and let-7f targeting of suppressor of cytokine signaling 4 (SOCS4) and Thrombospondin-1 (TSP-1). Transfection experiments confirmed miR-29b and let-7f modulation of IL-6R and SOCS4 protein expression levels, respectively. Thus, we demonstrate convergent/divergent miRNA responses to wild type and its environmentally-modified LPS and demonstrate miRNA targeting of key genes linked to inflammation and immunity. Our data indicate that these LPS-responsive miRNAs may play a key role in fine-tuning the host response to periodontal pathogens. PMID:24062196

  19. Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

    PubMed Central

    Cheng, Wan‐Chien; van Asten, Saskia D.; Burns, Lachrissa A.; Evans, Hayley G.; Walter, Gina J.; Hashim, Ahmed; Hughes, Francis J.

    2016-01-01

    The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17+ CD4+ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4+ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17+ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17+ CD4+ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4+ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4+ T cells, a process that appears enhanced in patients with PD. PMID:27334899

  20. Identification of Small-Molecule Inhibitors against Meso-2, 6-Diaminopimelate Dehydrogenase from Porphyromonas gingivalis

    PubMed Central

    Stone, Victoria N.; Parikh, Hardik I.; El-rami, Fadi; Ge, Xiuchun; Chen, Weihau; Zhang, Yan; Kellogg, Glen E.; Xu, Ping

    2015-01-01

    Species-specific antimicrobial therapy has the potential to combat the increasing threat of antibiotic resistance and alteration of the human microbiome. We therefore set out to demonstrate the beginning of a pathogen-selective drug discovery method using the periodontal pathogen Porphyromonas gingivalis as a model. Through our knowledge of metabolic networks and essential genes we identified a “druggable” essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. We adopted a high-throughput virtual screen method on the ZINC chemical library to select a group of potential small-molecule inhibitors. Meso-diaminopimelate dehydrogenase from P. gingivalis was first expressed and purified in Escherichia coli then characterized for enzymatic inhibitor screening studies. Several inhibitors with similar structural scaffolds containing a sulfonamide core and aromatic substituents showed dose-dependent inhibition. These compounds were further assayed showing reasonable whole-cell activity and the inhibition mechanism was determined. We conclude that the establishment of this target and screening strategy provides a model for the future development of new antimicrobials. PMID:26544875

  1. Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces.

    PubMed Central

    Tokuda, M; Duncan, M; Cho, M I; Kuramitsu, H K

    1996-01-01

    Cysteine proteases, including Arg-gingipain of Porphyromonas gingivalis, have been implicated as important virulence factors in periodontal diseases. These enzymes are also involved in the hemagglutinating activity of the organisms. In order to determine the role of proteases in the colonization of the gingival margin, we have compared the attachment properties of P. gingivalis 381 with those of its Arg-gingipain-defective mutant, G-102. Interactions with gram-positive bacteria, human oral epithelial cells, extracellular matrix proteins, and type I collagen were evaluated. In all cases, mutant G-102 was deficient in attachment relative to the parental strain. The mutant's defects could be explained, in part, by the weak autoaggregation displayed by the mutant, which appeared to result from altered fimbrial expression. Both Western blot (immunoblot) and Northern (RNA) blot analyses indicated reduced expression of the major 43-kDa fimbrillin subunit in the mutant. These results suggest that Arg-gingipain may play both direct and indirect roles in the colonization of the gingival margin. In addition, fimbriae may play a direct role in interacting with some host surfaces. PMID:8926070

  2. Role of Arg-Gingipain A in Virulence of Porphyromonas gingivalis

    PubMed Central

    Tokuda, Masayuki; Karunakaran, Thonthi; Duncan, Margaret; Hamada, Nobushiro; Kuramitsu, Howard

    1998-01-01

    In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37°C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism. PMID:9488409

  3. The chronicles of Porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay

    PubMed Central

    Yilmaz, Özlem

    2009-01-01

    The microbiota of the human oral mucosa consists of a myriad of bacterial species that normally exist in commensal harmony with the host. Porphyromonas gingivalis, an aetiological agent in severe forms of periodontitis (a chronic inflammatory disease), is a prominent component of the oral microbiome and a successful colonizer of the oral epithelium. This Gram-negative anaerobe can also exist within the host epithelium without the existence of overt disease. Gingival epithelial cells, the outer lining of the gingival mucosa, which function as an important part of the innate immune system, are among the first host cells colonized by P. gingivalis. This review describes recent studies implicating the co-existence and intracellular adaptation of the organism in these target host cells. Specifically, recent findings on the putative mechanisms of persistence, intercellular dissemination and opportunism are highlighted. These new findings may also represent an original and valuable model for mechanistic characterization of other successful host-adapted, self-limiting, persistent intracellular bacteria in human epithelial tissues. PMID:18832296

  4. Acute Toxicity and the Effect of Andrographolide on Porphyromonas gingivalis-Induced Hyperlipidemia in Rats

    PubMed Central

    Al-Bayaty, Fouad; Al-Obaidi, Mazen M. Jamil; Abdulla, Mahmood A.

    2013-01-01

    The aim of the current study is to evaluate the effect of andrographolide on hyperlipidemia induced by Porphyromonas gingivalis in rats. Thirty male Sprague Dawley (SD) rats were divided into five groups as follows: group 1 (vehicle) and four experimental groups (groups 2, 3, 4, and 5) were challenged orally with P. gingivalis ATCC 33277 (0.2 mL of 1.5 ×1012 bacterial cells/mL in 2% carboxymethylcellulose (CMC) with phosphate-buffered saline (PBS)) five times a week for one month to induce hyperlipidemia. Then, group 3 received a standard oral treatment with simvastatin 100 mg/kg, and groups 4 and 5 received oral treatment with andrographolide 20 mg/kg and 10 mg/kg, respectively, for another month. The results showed that total cholesterol (TC), low-density lipoprotein (LDL-C), and triglycerides (TG) were reduced significantly in groups treated with andrographolide. The malondialdehyde (MDA) level was low in treated groups, while antioxidant enzymes, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were significantly increased in these groups (P < 0.05). Liver tissues of the groups treated with andrographolide reduce the accumulation of lipid droplets in hepatic tissue cells. An acute toxicity test did not show any toxicological symptoms in rats. PMID:23844365

  5. Tetratricopeptide repeat protein-associated proteins contribute to the virulence of Porphyromonas gingivalis.

    PubMed

    Kondo, Yoshio; Ohara, Naoya; Sato, Keiko; Yoshimura, Mamiko; Yukitake, Hideharu; Naito, Mariko; Fujiwara, Taku; Nakayama, Koji

    2010-06-01

    Porphyromonas gingivalis is one of the most etiologically important microorganisms in periodontal disease. We found in a previous study that PG1385 (TprA) protein, a tetratricopeptide repeat (TPR) protein, was upregulated in P. gingivalis wild-type cells placed in a mouse subcutaneous chamber and that a tprA mutant was clearly less virulent in the mouse subcutaneous abscess model (M. Yoshimura et al., Oral Microbiol. Immunol. 23:413-418, 2008). In the present study, we investigated the gene expression profile of tprA mutant cells placed in a mouse subcutaneous chamber and found that 9 genes, including PG2102 (tapA), PG2101 (tapB), and PG2100 (tapC) genes, were downregulated in the tprA mutant compared with those in the wild type. Expression of a cluster of tapA, tapB, and tapC genes of the mutant was also downregulated in an in vitro culture with enriched brain heart infusion medium. The TprA protein has three TPR motifs known as a protein-protein interaction module. Yeast two-hybrid system analysis and in vitro protein binding assays with immunoprecipitation and surface plasmon resonance detection revealed that the TprA protein could bind to TapA and TapB proteins. TprA and TapB proteins were located in the periplasmic space, whereas TapA, which appeared to be one of the C-terminal domain family proteins, was located at the outer membrane. We constructed tapA, tapB, and tapC single mutants and a tapA-tapB-tapC deletion mutant. In the mouse subcutaneous infection experiment, all of the mutants were less virulent than the wild type. These results suggest that TprA, TapA, TapB, and TapC are cooperatively involved in P. gingivalis virulence. PMID:20351137

  6. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    SciTech Connect

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  7. Insights into the antiatherogenic molecular mechanisms of andrographolide against Porphyromonas gingivalis-induced atherosclerosis in rabbits.

    PubMed

    Al Batran, Rami; Al-Bayaty, Fouad; Al-Obaidi, Mazen M Jamil; Ashrafi, Amer

    2014-12-01

    Atherosclerosis is the commonest and most important vascular disease. Andrographolide (AND) is the main bioactive component of the medicinal plant Andrographis paniculata and is used in traditional medicine. This study was aimed to evaluate the antiatherogenic effect of AND against atherosclerosis induced by Porphyromonas gingivalis in White New Zealand rabbits. Thirty rabbits were divided into five groups as follows: G1, normal group; G2-5, were orally challenged with P. gingivalis five times a week over 12 weeks; G2, atherogenic control group; G3, standard group treated with atorvastatin (AV) 5 mg/kg; and G4 and G5, treatment groups treated with AND 10 and 20 mg/kg, respectively over 12 weeks. Serums were subjected to antioxidant enzymatic and anti-inflammatory activities, and the aorta was subjected to histological analyses. Groups treated with AND showed a significant reversal of liver and renal biochemical changes, compared with the atherogenic control group. In the same groups, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total glutathione (GSH) levels in serum were significantly increased (P < 0.05), and lipid peroxidation (malondialdehyde (MDA)) levels were significantly decreased (P < 0.05), respectively. Furthermore, treated groups with AV and AND showed significant decrease in the level of VCAM-1 and ICAM-1 compared with the atherogenic control group. In aortic homogenate, the level of nitrotyrosine was significantly increased, while the level of MCP1 was significantly decreased in AV and AND groups compared with the atherogenic control group. In addition, staining the aorta with Sudan IV showed a reduction in intimal thickening plaque in AV and AND groups compared with the atherogenic control group. AND has showed an antiatherogenic property as well as the capability to reduce lipid, liver, and kidney biomarkers in atherogenic serum that prevents atherosclerosis complications caused by P. gingivalis.

  8. Expression, Purification and Characterization of Enoyl-ACP Reductase II, FabK, from Porphyromonas gingivalis

    PubMed Central

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2012-01-01

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the U.S. population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP+ during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution. PMID:22820244

  9. Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.

    PubMed

    Wunsch, Christopher M; Lewis, Janina P

    2015-12-17

    Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth. The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal

  10. Genetic Exchange of Fimbrial Alleles Exemplifies the Adaptive Virulence Strategy of Porphyromonas gingivalis

    PubMed Central

    Kerr, Jennifer E.; Abramian, Jared R.; Dao, Doan-Hieu V.; Rigney, Todd W.; Fritz, Jamie; Pham, Tan; Gay, Isabel; Parthasarathy, Kavitha; Wang, Bing-yan; Zhang, Wenjian; Tribble, Gena D.

    2014-01-01

    Porphyromonas gingivalis is a gram–negative anaerobic bacterium, a member of the human oral microbiome, and a proposed “keystone” pathogen in the development of chronic periodontitis, an inflammatory disease of the gingiva. P. gingivalis is a genetically diverse species, and is able to exchange chromosomal DNA between strains by natural competence and conjugation. In this study, we investigate the role of horizontal DNA transfer as an adaptive process to modify behavior, using the major fimbriae as our model system, due to their critical role in mediating interactions with the host environment. We show that P. gingivalis is able to exchange fimbrial allele types I and IV into four distinct strain backgrounds via natural competence. In all recombinants, we detected a complete exchange of the entire fimA allele, and the rate of exchange varies between the different strain backgrounds. In addition, gene exchange within other regions of the fimbrial genetic locus was identified. To measure the biological implications of these allele swaps we compared three genotypes of fimA in an isogenic background, strain ATCC 33277. We demonstrate that exchange of fimbrial allele type results in profound phenotypic changes, including the quantity of fimbriae elaborated, membrane blebbing, auto-aggregation and other virulence-associated phenotypes. Replacement of the type I allele with either the type III or IV allele resulted in increased invasion of gingival fibroblast cells relative to the isogenic parent strain. While genetic variability is known to impact host-microbiome interactions, this is the first study to quantitatively assess the adaptive effect of exchanging genes within the pan genome cloud. This is significant as it presents a potential mechanism by which opportunistic pathogens may acquire the traits necessary to modify host-microbial interactions. PMID:24626479

  11. High In Vitro Antibacterial Activity of Pac-525 against Porphyromonas gingivalis Biofilms Cultured on Titanium

    PubMed Central

    Li, Ji-yin; Wang, Xue-jin; Wang, Li-na; Ying, Xiao-xia; Ren, Xiang; Liu, Hui-ying; Xu, Li; Ma, Guo-wu

    2015-01-01

    In order to investigate the potential of short antimicrobial peptides (AMPs) as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, including Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-old P. gingivalis biofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM). The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, for P. gingivalis and 0.0078 mg/mL and 0.0156 mg/mL, respectively, for F. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants. PMID:25710035

  12. Identification of signaling pathways in macrophage exposed to Porphyromonas gingivalis or to its purified cell wall components.

    PubMed

    Zhou, Qingde; Amar, Salomon

    2007-12-01

    Porphyromonas gingivalis (P. gingivalis) can trigger an inflammatory condition leading to the destruction of periodontal tissues. However P. gingivalis LPS and its fimbriae (FimA) play different roles compared with the live bacteria in the context of intracellular molecule induction and cytokine secretion. To elucidate whether this difference results from different signaling pathways in host immune response to P. gingivalis, its LPS, or its FimA, we examined gene expression profile of human macrophages exposed to P. gingivalis, its LPS, or its FimA. A comparison of gene expression resulted in the identification of three distinct groups of expressed genes. Furthermore, computer-assisted promoter analysis of a subset of each group of differentially regulated genes revealed four putative transcriptional regulation models that associate with transcription factors NFkappaB, IRF7, and KLF4. Using gene knockout mice and siRNA to silence mouse genes, we showed that both TLR2 and TLR7 are essential for the induction of NFkappaB-containing genes and NFkappaB-IFN-sensitive response element (ISRE) cocontaining genes by either P. gingivalis or its purified components. The gene induction via either TLR2 or TLR7 is dependent on both MyD88 and p38 MAPK. However, the unique induction of IFN-beta by P. gingivalis LPS requires TLR7 and IFNalphabetaR cosignaling, and the induction of ISRE-containing gene is dependent on the activation of IFN-beta autocrine loop. Taken together, these data demonstrate that P. gingivalis and its components induce NFkappaB-containing genes through either TLR2- or TLR7-MyD88-p38 MAPK pathway, while P. gingivalis LPS uniquely induces ISRE-containing genes, which requires IFNalphabetaR signaling involving IRF7, KLF4, and pY701 STAT1. PMID:18025224

  13. Porphyromonas gingivalis Lipopolysaccharide Weakly Activates M1 and M2 Polarized Mouse Macrophages but Induces Inflammatory Cytokines

    PubMed Central

    Holden, James A.; Attard, Troy J.; Laughton, Katrina M.; Mansell, Ashley; O'Brien-Simpson, Neil M.

    2014-01-01

    Porphyromonas gingivalis is associated with chronic periodontitis, an inflammatory disease of the tooth's supporting tissues. Macrophages are important in chronic inflammatory conditions, infiltrating tissue and becoming polarized to an M1 or M2 phenotype. As responses to stimuli differ between these phenotypes, we investigated the effect of P. gingivalis lipopolysaccharide (LPS) on M1 and M2 macrophages. M1 and M2 polarized macrophages were produced from murine bone marrow macrophages (BMMϕ) primed with gamma interferon (IFN-γ) or interleukin-4 (IL-4), respectively, and incubated with a low or high dose of P. gingivalis LPS or control TLR2 and TLR4 ligands. In M1-Mϕ, the high dose of P. gingivalis LPS (10 μg/ml) significantly increased the expression of CD40, CD86, inducible nitric oxide synthase, and nitric oxide secretion. The low dose of P. gingivalis LPS (10 ng/ml) did not induce costimulatory or antibacterial molecules but did increase the secretion of IL-1α, IL-6, IL-12p40, IL-12p70, and tumor necrosis factor alpha (TNF-α). P. gingivalis LPS marginally increased the expression of CD206 and YM-1, but it did enhance arginase expression by M2-Mϕ. Furthermore, the secretion of the chemokines KC, RANTES, eotaxin, and MCP-1 from M1, M2, and nonpolarized Mϕ was enhanced by P. gingivalis LPS. TLR2/4 knockout macrophages combined with the TLR activation assays indicated that TLR2 is the main activating receptor for P. gingivalis LPS and whole cells. In conclusion, although P. gingivalis LPS weakly activated M1-Mϕ or M2-Mϕ compared to control TLR ligands, it induced the secretion of inflammatory cytokines, particularly TNF-α from M1-Mϕ and IL-10 from M2-Mϕ, as well as chemotactic chemokines from polarized macrophages. PMID:25047849

  14. Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism

    PubMed Central

    Chen, Dina; Seers, Christine A.; Mitchell, Helen A.; Chen, Yu-Yen; Glew, Michelle D.; Dashper, Stuart G.; Reynolds, Eric C.

    2015-01-01

    The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria. PMID:26340749

  15. A Dual Role for P2X7 Receptor during Porphyromonas gingivalis Infection

    PubMed Central

    Ramos-Junior, E.S.; Morandini, A.C.; Almeida-da-Silva, C.L.C.; Franco, E.J.; Potempa, J.; Nguyen, K.A.; Oliveira, A.C.; Zamboni, D.S.; Ojcius, D.M.; Scharfstein, J.

    2015-01-01

    Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1β and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1β secretion by means of its fimbriae in a purinergic P2X7 receptor–dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1β processing and secretion by P. gingivalis–infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1β secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1β from P. gingivalis–infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1β to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1β secretion but also for intracellular pro-IL-1β processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7-/- mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis. PMID:26152185

  16. Assessment of outer membrane vesicles of periodontopathic bacterium Porphyromonas gingivalis as possible mucosal immunogen.

    PubMed

    Nakao, Ryoma; Hasegawa, Hideki; Dongying, Bai; Ohnishi, Makoto; Senpuku, Hidenobu

    2016-08-31

    Periodontitis is the most prevalent infectious disease and related to oral and systemic health, therefore novel prophylaxis to prevent the disease is highly desirable. Here, we assessed the outer membrane vesicles (OMVs) of a keystone periodontal pathogen, Porphyromonas gingivalis, as a candidate mucosal immunogen and adjuvant for a periodontitis vaccine. The structural and functional stability of OMVs, demonstrated by proteinase K resistance and ability to withstand long-term storage, are considered advantageous for carrying the OMV components into the host immune system. Intranasal vaccination of OMVs in mice elicited production of P. gingivalis-specific antibodies in blood and saliva by OMVs in a dose-dependent manner, which was dramatically enhanced by addition of a TLR3 agonist, Poly(I:C). Serum samples from mice immunized with OMVs plus Poly(I:C) adjuvant [OMV+Poly(I:C)] showed significant inhibition of gingipain proteolytic activity of not only the vaccine strain, but also heterologous strains. The viability of P. gingivalis was also decreased by preincubation with OMV+Poly(I:C)-immunized sera, while the killing effect was partially blocked by heat-inactivation of the sera. Saliva samples from mice immunized with OMV+Poly(I:C) enhanced bacterial agglutination of both the vaccine and heterologous strains. In an oral infection mouse model, the numbers of P. gingivalis in the oral cavity were significantly decreased in mice intranasally immunized with OMV+Poly(I:C) as compared to mock (only Poly[I:C])-immunized mice. The high levels of serum IgG (including IgG1 and IgG2a) and salivary S-IgA were elicited in mice intranasally immunized with OMV+Poly(I:C), which were maintained for at least 28 and 18weeks, respectively, after immunization. An experiment examining the accumulation of OMVs after intranasal immunization in proximal organs and an intracerebral injection experiment confirmed the safety of OMVs. Based on our results, we propose that intranasal

  17. Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Chen, Dina; Seers, Christine A; Mitchell, Helen A; Chen, Yu-Yen; Glew, Michelle D; Dashper, Stuart G; Reynolds, Eric C

    2015-09-01

    The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.

  18. Porphyromonas gingivalis Peptidylarginine Deiminase, a Key Contributor in the Pathogenesis of Experimental Periodontal Disease and Experimental Arthritis

    PubMed Central

    Gully, Neville; Bright, Richard; Marino, Victor; Marchant, Ceilidh; Cantley, Melissa; Haynes, David; Butler, Catherine; Dashper, Stuart; Reynolds, Eric; Bartold, Mark

    2014-01-01

    Objectives To investigate the suggested role of Porphyromonas gingivalis peptidylarginine deiminase (PAD) in the relationship between the aetiology of periodontal disease and experimentally induced arthritis and the possible association between these two conditions. Methods A genetically modified PAD-deficient strain of P. gingivalis W50 was produced. The effect of this strain, compared to the wild type, in an established murine model for experimental periodontitis and experimental arthritis was assessed. Experimental periodontitis was induced following oral inoculation with the PAD-deficient and wild type strains of P. gingivalis. Experimental arthritis was induced via the collagen antibody induction process and was monitored by assessment of paw swelling and micro-CT analysis of the radio-carpal joints. Experimental periodontitis was monitored by micro CT scans of the mandible and histological assessment of the periodontal tissues around the mandibular molars. Serum levels of anti-citrullinated protein antibodies (ACPA) and P. gingivalis were assessed by ELISA. Results The development of experimental periodontitis was significantly reduced in the presence of the PAD-deficient P. gingivalis strain. When experimental arthritis was induced in the presence of the PAD-deficient strain there was less paw swelling, less erosive bone damage to the joints and reduced serum ACPA levels when compared to the wild type P. gingivalis inoculated group. Conclusion This study has demonstrated that a PAD-deficient strain of P. gingivalis was associated with significantly reduced periodontal inflammation. In addition the extent of experimental arthritis was significantly reduced in animals exposed to prior induction of periodontal disease through oral inoculation of the PAD-deficient strain versus the wild type. This adds further evidence to the potential role for P. gingivalis and its PAD in the pathogenesis of periodontitis and exacerbation of arthritis. Further studies are now

  19. NOX1/2 activation in human gingival fibroblasts by Fusobacterium nucleatum facilitates attachment of Porphyromonas gingivalis.

    PubMed

    Ahn, Sun Hee; Song, Ji-Eun; Kim, Suhee; Cho, Sung-Hyun; Lim, Yun Kyong; Kook, Joong-Ki; Kook, Min-Suk; Lee, Tae-Hoon

    2016-08-01

    Periodontal diseases are infectious polymicrobial inflammatory diseases that lead to destruction of the periodontal ligament, gingiva, and alveolar bone. Sequential colonization of a broad range of bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis, is an important phenomenon in this disease model. F. nucleatum is a facultative anaerobic species thought to be a key mediator of dental plaque maturation due to its extensive coaggregation with other oral bacteria, while P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting various virulence factors. The formation of a bacterial complex by these two species is central to the pathogenesis of periodontal disease. Reactive oxygen species (ROS) are produced during bacterial infections and are involved in intracellular signaling. However, the impact of oral bacteria-induced ROS on the ecology of F. nucleatum and P. gingivalis has yet to be clarified. In the present study, we investigated ROS production induced in primary human oral cells by F. nucleatum and P. gingivalis and its effect on the formation of their bacterial complexes and further host cell apoptosis. We found that in primary human gingival fibroblasts (GFs), two NADPH oxidase isoforms, NOX1 and NOX2, were activated in response to F. nucleatum infection but not P. gingivalis infection. Accordingly, increased NADPH oxidase activity and production of superoxide anion were observed in GFs after F. nucleatum infection, but not after P. gingivalis infection. Interestingly, in NOX1, NOX2, or NOX1/NOX2 knockdown cells, the number of P. gingivalis decreased when the cells were coinfected with F. nucleatum. A similar pattern of host cell apoptosis was observed. This implies that F. nucleatum contributes to attachment of P. gingivalis by triggering activation of NADPH oxidase in host cells, which may provide an environment more favorable to strict anaerobic bacteria and have a subsequent effect on apoptosis of

  20. Development and evaluation of a saliva-based chair-side diagnostic for the detection of Porphyromonas gingivalis.

    PubMed

    O'Brien-Simpson, Neil M; Burgess, Kate; Brammar, Gail C; Darby, Ivan B; Reynolds, Eric C

    2015-01-01

    Porphyromonas gingivalis is a key pathogen in the polymicrobial biofilm that is associated with the oral disease chronic periodontitis. A number of studies have shown that in humans the level of P. gingivalis in the polymicrobial biofilm is positively correlated with disease progression. The aim of this study was to develop a P. gingivalis diagnostic that has high specificity and sensitivity for P. gingivalis using a range of laboratory and clinical isolates and then compare the efficacy of the diagnostic with RTPCR using samples from chronic periodontitis patients and age- and sex-matched healthy controls. Key parameters for the kit were to use saliva as the biological fluid as this is a most convenient medium for chair-side sampling and to give a positive reading for the reported threshold for detection of 5×10(5) P. gingivalis cells/mL that indicates disease progression. We initially screened a range of monoclonal antibodies for recognition of the P. gingivalis conserved virulence factor RgpA-Kgp complex and identified two mAbs that could be used in a capture and detection ELISA system. These mAbs were used to formulate and manufacture the GC P. gingivalis saliva diagnostic kit used in the study. To validate the saliva kit, saliva (P. gingivalis free) was spiked with known concentrations of viable P. gingivalis whole cells of W50, 381, A7A1-28, and ATCC 33277; P. gingivalis clinical isolates; P. gingivalis vesicles; and the secreted form of the RgpA-Kgp complex. Laboratory findings indicated that the kit was able to detect all laboratory and clinical isolate strains of P. gingivalis at 5×10(4)/mL to 5×10(5)/mL. It was also able to detect the RgpA-Kgp complex and vesicles at 5×10(4) and 5×10(5) cell equivalent doses, respectively. Saliva and plaque were then collected from 50 subjects with moderate-severe chronic periodontitis and 50 age- and sex-matched subjects with healthy periodontium. Real-time PCR was utilised to analyse levels of P. gingivalis in both

  1. Lysine acetylation is a common post-translational modification of key metabolic pathway enzymes of the anaerobe Porphyromonas gingivalis.

    PubMed

    Butler, Catherine A; Veith, Paul D; Nieto, Matthew F; Dashper, Stuart G; Reynolds, Eric C

    2015-10-14

    Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy

  2. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. PMID:23075153

  3. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion.

  4. Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

    PubMed Central

    Pike, R N; Potempa, J; McGraw, W; Coetzer, T H; Travis, J

    1996-01-01

    Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces. PMID:8631676

  5. Purification and characterization of three types of proteases from culture supernatants of Porphyromonas gingivalis.

    PubMed Central

    Hinode, D; Hayashi, H; Nakamura, R

    1991-01-01

    Three types of caseinolytic proteases (Pase-A, Pase-B, and Pase-C) were isolated and purified from culture supernatants of Porphyromonas gingivalis 381 by the combined procedures of acetone precipitation, gel filtration, solubilization with octylthioglucoside followed by affinity chromatography on arginine-Sepharose 4B, high-performance liquid chromatography (HPLC) on Biofine IEC-DEAE, and HPLC on TSK-G4000SW. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pase-A and -B showed diffuse protein bands of 105 to 110 and 72 to 80 kDa, respectively, and Pase-C showed a clear band of about 44 kDa. Pase-B and -C hydrolyzed some synthetic substrates for trypsin, but Pase-B did not act on the carboxyl side of lysine in insulin chain B or on a synthetic substrate which trypsin and Pase-C acted on. Pase-A did not act on the synthetic substrates but cleaved the peptide bonds Glu-Ala and Ala-Leu of insulin. Leupeptin inhibition of the caseinolytic activity of both Pase-A and -B was similar to its inhibition of Pase-C. Phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate strongly inhibited Pase-A, but no significant effect on the other enzymes was observed, suggesting that only Pase-A is a serine protease. The inhibitory characteristics of Pase-B and -C were very similar. Pase-A was not thiol dependent for enzyme activity, but Pase-B was strongly dependent, i.e., even more so than Pase-C. Pase-A inactivated the inhibitory activity of plasma alpha-1-antitrypsin, but the other two did not. These results show that P. gingivalis produces different types of proteases other than the trypsinlike protease generally reported. Images PMID:1879930

  6. Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis.

    PubMed Central

    Sojar, H T; Hamada, N; Genco, R J

    1997-01-01

    Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain. PMID:9172351

  7. Expression Profiles of TGF-β and TLR Pathways in Porphyromonas gingivalis and Prevotella intermedia Challenged Osteoblasts

    PubMed Central

    Aydin, Kubra; Ekinci, Fatma Yesim; Korachi, May

    2015-01-01

    Background: The presence of certain oral pathogens at implant sites can hinder the osseointegration process. However, it is unclear how and by what microorganisms it happens. Objectives: This study investigated whether the presence of oral pathogens of Porphyromonas gingivalis and Prevotella intermedia individually, play a role in the failure of bone formation by determining the expression profiles of Transforming Growth Factor Beta (TGF-β/Bone Morphogenic Protein (BMP) and Toll-Like Receptor (TLR) pathways in challenged osteoblasts. Materials and Methods: Cell viability of P. gingivalis and P. intermedia challenged osteoblasts were determined by WST assay. Changes in osteoblast morphology and inhibition of mineralization were observed by Scanning Electron Microscopy (SEM) and Von Kossa staining, respectively. Expression of TGF-β and TLR pathway genes on challenged cells were identified by RT profiler array. Both P. gingivalis and P. intermedia challenges resulted in reduced viability and mineralization of osteoblasts. Results: Viability was reduced to 56.8% (P. gingivalis) and 52.75% (P. intermedia) at 1000 multiplicity. Amongst 48 genes examined, expressions of BMPER, SMAD1, IL8 and NFRKB were found to be highly upregulated by both bacterial challenges (Fold Change > 4). Conclusions: P. gingivalis and P. intermedia could play a role in implant failure by changing the expression profiles of genes related to bone formation and resorption. PMID:26034550

  8. Subcutaneous vaccination with Porphyromonas gingivalis ameliorates periodontitis by modulating Th17/Treg imbalance in a murine model.

    PubMed

    Wang, Linyuan; Guan, Ning; Jin, Ying; Lin, Xiaoping; Gao, Hong

    2015-03-01

    To date, Porphyromonas gingivalis (P. gingivalis) vaccination has been studied only in animals, and no effective prophylactic human periodontal vaccine has been developed, with the reason for the failure of prophylactic human periodontal vaccines unknown. T helper 17 cell (Th17)/regulatory T (Treg) cell responses play an important role in the development of periodontitis, and a Th17/Treg imbalance causes the pathogenesis of periodontitis. However, whether vaccination with P. gingivalis can prevent periodontitis through modulation of the Th17/Treg imbalance remains unknown. In this study, mice were subcutaneously vaccinated with formalin-killed P. gingivalis and then orally challenged with P. gingivalis. The vaccination protected the mice from alveolar bone resorption and inflammation. These protective effects might be ascribed to downregulation of Th17 cells and interleukin (IL)-17A production, upregulation of Treg and receptor activator of nuclear factor-kappa B ligand (RANKL)(+)CD4(+)T cells, and IL-10 and transforming growth factor-β1 production, and inhibition of lymphocyte proliferation. Our findings may provide a direction for the development of a vaccine or therapy against periodontitis by alteration of the Th17/Treg imbalance.

  9. Evaluation of the effect of andrographolide on atherosclerotic rabbits induced by Porphyromonas gingivalis.

    PubMed

    Al Batran, Rami; Al-Bayaty, Fouad; Al-Obaidi, Mazen M Jamil; Hussain, Saba F; Mulok, Tengku Z

    2014-01-01

    Epidemiologic evidence has demonstrated significant associations between atherosclerosis and Porphyromonas gingivalis (Pg). We had investigated the effect of andrographolide (AND) on atherosclerosis induced by Pg in rabbits. For experimental purpose, we separated thirty male white New Zealand rabbits into 5 groups. Group 1 received standard food pellets; Groups 2-5 were orally challenged with Pg; Group 3 received atorvastatin (AV, 5 mg/kg), and Groups 4-5 received 10 and 20 mg/kg of AND, respectively, over 12 weeks. Groups treated with AND showed significant decrease in TC, TG, and LDL levels (P<0.05) and significant increase in HDL level in the serum of rabbits. Furthermore, the treated groups (G3-G5) exhibited reductions in interleukins (IL-1β and IL-6) and C-reactive protein (CRP) as compared to atherogenicgroup (G2). The histological results showed that the thickening of atherosclerotic plaques were less significant in treated groups (G3-G5) compared with atherogenicgroup (G2). Also, alpha-smooth muscle actin (α-SMA) staining decreased within the plaques of atherogenicgroup (G2), while it was increased in treated groups (G3-G5). Lastly, groups treated with AV and AND (G3-G5) showed significant reduction of CD36 expression (P<0.05) compared to atherogenicgroup (G2). These results substantially proved that AND contain antiatherogenic activity.

  10. Gender-Specific Associations of Serum Antibody to Porphyromonas gingivalis and Inflammatory Markers

    PubMed Central

    Furuta, Michiko; Shimazaki, Yoshihiro; Tanaka, Shunichi; Takeuchi, Kenji; Shibata, Yukie; Takeshita, Toru; Nishimura, Fusanori; Yamashita, Yoshihisa

    2015-01-01

    It remains unclear whether serum antibody titer against Porphyromonas gingivalis (Pg) and inflammatory components lead to periodontal deterioration in each gender, as periodontal and systemic status is influenced by gender. The present study investigates the gender-specific probable effects of titer against Pg and inflammatory markers on periodontal health status in a longitudinal study. A retrospective study design was used. At two time points over an 8-year period (in 2003 and 2011), 411 individuals (295 males with a mean age of 57.6 ± 11.2 years and 116 females with a mean age of 59.2 ± 10.3 years) were surveyed. Periodontal status, serum antibody titer against Pg, and high-sensitive C-reactive protein (hsCRP) were evaluated. Poisson regression analyses revealed that the elevated titer against Pg and hsCRP significantly predicted the persistence of periodontal disease 8 years later in females with periodontal disease in 2003. Elevated hsCRP was significantly associated with the incidence of periodontal disease 8 years later in females who were periodontally healthy in 2003. Males had a weaker association among titer against Pg, inflammatory markers, and periodontal disease. These findings suggest that immune response to Pg infection in addition to inflammatory components affects periodontal deterioration in females. PMID:25756052

  11. Streptococcus salivarius promotes mucin putrefaction and malodor production by Porphyromonas gingivalis.

    PubMed

    Sterer, N; Rosenberg, M

    2006-10-01

    Although the contribution of the oral microbiota to oral malodor is well-documented, the potential role of Gram-positive micro-organisms is unclear. In the current study, we tested the hypothesis that Gram-positive micro-organisms contribute to malodor production by deglycosylating oral glycoproteins, rendering them susceptible to subsequent proteolysis. To this end, we examined the effect of Streptococcus salivarius on Porphyromonas gingivalis-mediated putrefaction of a model glycoprotein (pig gastric mucin). Malodor was scored by two odor judges, and volatile sulfides were determined with the use of a sulfide monitor. Mucin degradation was followed by electrophoresis on SDS-PAGE. Results showed that the addition of S. salivarius or beta-galactosidase promoted mucin degradation and concomitant malodor production. Addition of glycosidic inhibitors (p-APTG and glucose) inhibited this process. These results suggest that Gram-positive micro-organisms such as S. salivarius contribute to oral malodor production by deglycosylating salivary glycoproteins, thus exposing their protein core to further degradation by Gram-negative micro-organisms. PMID:16998130

  12. Evaluation of the Effect of Andrographolide on Atherosclerotic Rabbits Induced by Porphyromonas gingivalis

    PubMed Central

    Al-Bayaty, Fouad; Al-Obaidi, Mazen M. Jamil; Hussain, Saba F.; Mulok, Tengku Z.

    2014-01-01

    Epidemiologic evidence has demonstrated significant associations between atherosclerosis and Porphyromonas gingivalis (Pg). We had investigated the effect of andrographolide (AND) on atherosclerosis induced by Pg in rabbits. For experimental purpose, we separated thirty male white New Zealand rabbits into 5 groups. Group 1 received standard food pellets; Groups 2–5 were orally challenged with Pg; Group 3 received atorvastatin (AV, 5 mg/kg), and Groups 4-5 received 10 and 20 mg/kg of AND, respectively, over 12 weeks. Groups treated with AND showed significant decrease in TC, TG, and LDL levels (P < 0.05) and significant increase in HDL level in the serum of rabbits. Furthermore, the treated groups (G3–G5) exhibited reductions in interleukins (IL-1β and IL-6) and C-reactive protein (CRP) as compared to atherogenicgroup (G2). The histological results showed that the thickening of atherosclerotic plaques were less significant in treated groups (G3–G5) compared with atherogenicgroup (G2). Also, alpha-smooth muscle actin (α-SMA) staining decreased within the plaques of atherogenicgroup (G2), while it was increased in treated groups (G3–G5). Lastly, groups treated with AV and AND (G3–G5) showed significant reduction of CD36 expression (P < 0.05) compared to atherogenicgroup (G2). These results substantially proved that AND contain antiatherogenic activity. PMID:25215291

  13. Bortezomib Inhibits Osteoclastogenesis and Porphyromonas gingivalis Lipopolysaccharide-induced Alveolar Bone Resorption.

    PubMed

    Kim, Y-G; Kang, J H; Kim, H J; Kim, H J; Kim, H-H; Kim, J-Y; Lee, Y

    2015-09-01

    Healthy bone is maintained by the coordinated activities of osteoblast-mediated bone formation and osteoclast-dependent bone resorption. Pathologic conditions such as hormonal imbalance and inflammation cause increased osteoclastogenesis resulting in osteoporosis, rheumatoid arthritis, and periodontitis. Bortezomib is novel antimyeloma agent that has a direct beneficial effect on bone formation. However, the role of bortezomib in osteoclastogenesis and underlying mechanisms remains to be fully comprehended. In the present study, we show that bortezomib directly inhibited the receptor activator of nuclear factor κB ligand (RANKL)- and lipopolysaccharide-dependent osteoclast differentiation. Interestingly, the bortezomib-mediated inhibition of osteoclastogenesis was transient, since the removal of bortezomib from culture completely restored osteoclast differentiation. Bortezomib impeded the induction and nuclear localization of nuclear factor of activated T cells, cytoplasmic 1 and reduced both macrophage colony-stimulating factor- and RANKL-induced extracellular-signal-regulated kinase (ERK) phosphorylation. In a mouse model of periodontitis, bortezomib prevented alveolar bone erosion induced by Porphyromonas gingivalis lipopolysaccharide. These data not only suggest a previously unappreciated mechanism by which bortezomib regulates bone resorption but also propose novel applications of bortezomib beyond its use as an antimyeloma agent.

  14. Rosiglitazone impedes Porphyromonas gingivalis-accelerated atherosclerosis by downregulating the TLR/NF-κB signaling pathway in atherosclerotic mice.

    PubMed

    Pan, Shengbo; Lei, Lang; Chen, Shuai; Li, Houxuan; Yan, Fuhua

    2014-12-01

    Porphyromonas gingivalis,a predominant periodontal pathogen, is known to accelerate atherosclerosis in hyperlipidemic animals via aberrant inflammatory responses. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to exert anti-inflammatory effects in vitro. The purpose of the present study was to investigate the potential protective role of the PPARγ agonist rosiglitazone in pathogen accelerated atherosclerosis in an apolipoprotein E-deficient (ApoE-/-) mouse model. ApoE-/- mice were inoculated intravenously with live P. gingivalis (strain 33277) or the buffer vehicle and treated with rosiglitazone or saline over a 10-week period. Their atherosclerotic status in aortic artery was assessed through histomorphometric analysis, inflammatory agent and lipid profiles in blood was determined by ELISA, and levels of relevant cytokines and Toll-like receptors (TLRs) in aortic tissues were evaluated using immunohistochemistry and quantitative PCR. P. gingivalis inoculation was associated with increased atherosclerotic plaque formation in the aorta and higher levels of serum pro-inflammatory cytokines (tumor necrosis factor-α, monocyte chemotactic protein-1 and interleukin-1β), but the serum lipid profile was not affected by P. gingivalis infection. Levels of tumor necrosis factor-α, monocyte chemotactic protein-1 intercellular cell adhesion molecule-1 and TLRs were higher in the aortic tissues of mice exposed to P. gingivalis, and activation of nuclear factor-κB was also observed. In both P. gingivalis-treated and -untreated ApoE-/- mice, rosiglitazone treatment was associated with less atherosclerotic plaque formation; lower serum inflammatory cytokines, total cholesterol, and low density lipoprotein cholesterol; higher levels of PPARγ, lower amounts of TLR2/4 and downregulated nuclear factor-κB activity in aortic tissues. These findings suggest that rosiglitazone mitigates or prevents P. gingivalis-accelerated atherosclerosis by

  15. Xylitol, an Anticaries Agent, Exhibits Potent Inhibition of Inflammatory Responses in Human THP-1-Derived Macrophages Infected With Porphyromonas gingivalis

    PubMed Central

    Park, Eunjoo; Na, Hee Sam; Kim, Sheon Min; Wallet, Shannon; Cha, Seunghee; Chung, Jin

    2016-01-01

    Background Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. Methods Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis–induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. Results Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1β, in a multiplicity of infection– and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ–induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis– induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted anti-phagocytic activity against both Escherichia coli and P. gingivalis. Conclusion These findings suggest that xylitol acts as an antiinflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis. PMID:24592909

  16. Aging and contribution of MyD88 and TRIF in expression of TLR pathway associated genes to Porphyromonas gingivalis

    PubMed Central

    Shaik-Dasthagirisaheb, Yazdani B.; Huang, Nasi; Weinberg, Ellen O.; Shen, Steve S.; Genco, Caroline A.; Gibson, Frank C.

    2014-01-01

    BACKGROUND AND OBJECTIVE Periodontal disease is a highly complex chronic inflammatory disease of the oral cavity. Multiple factors influence periodontal disease including socioeconomic status, genetics, age, however, inflammation elicited by the presence of specific bacteria in the subgingival space is thought to drive the majority of soft and hard tissue destruction. Porphyromonas gingivalis is closely associated with periodontal disease. Toll-like receptors (TLRs) and their intracellular signaling pathways play roles in host responses to P. gingivalis. The focus of current study was to use microarray analysis to define the contributions that TLR adaptor molecules MyD88 and TRIF, and aging have on TLR pathway associated mRNA expression in response to P. gingivalis. MATERIALS AND METHODS Bone marrow derived macrophages (BMØ) from wild type (Wt), MyD88-KO and TrifLps2 mice at 2-months and 12-months of age were cultured with P. gingivalis. Expression of genes in BMØ cultured with P. gingivalis was determined in comparison to medium alone control. RESULTS Using a two-fold cut-off in mRNA expression criteria, differential expression of 32 genes was observed when Wt BMØ from 2-month old mice were cultured with P. gingivalis compared with medium alone control. When compared with 2-month old Wt, 21 and 12 genes were differentially expressed (P<0.05) as a result of MyD88 or TRIF mutations respectively. The expression of 5 genes was significantly (P<0.05) reduced in the 12-month group compared to the 2-month group in Wt BMØ following culture with P. gingivalis. Age also influenced expression of genes in MyD88-KO and TrifLps2 mice challenged with P. gingivalis. CONCLUSION Our results indicate that P. gingivalis induces differential expression of TLR pathway associated genes, and both MyD88, and TRIF play roles in the expression of these genes. Age also played a role in the expression of TLR-associated genes following stimulation of BMØ with P. gingivalis. PMID:24862405

  17. Assessing the Antimicrobial Effect of the Essential Oil of Myrtus communis on the Clinical Isolates of Porphyromonas gingivalis: An in vitro Study

    PubMed Central

    Hedayati, Azita; Khosropanah, Hengameh; Bazargani, Abdollah; Abed, Molud; Emami, Amir

    2013-01-01

    Background One of the major diseases affecting the oral health is periodontal disease. Various therapeutic methods have been introduced to eliminate the periodonto-pathic subgingival microflora. Among these, Porphyromonas gingivalis (P. gingivalis) has a major role in the pathogenesis of different forms of periodontal diseases. Objectives The present study investigated the antimicrobial effect of the essential oil of Myrtus communis on Porphyromonas gingivalis (P. gingivalis) as the most destructive periodontal pathogens. Materials and Methods The subjects included 27 male and 3 female patients with advanced chronic periodontitis. The mean age of the patients was 47.6 ± 2.0 years old. P. gingivalis was isolated from the samples and identified by various diagnostic tests, including Gram staining, Indol test, and fluorescent test. Minimum inhibitory concentration (MIC) of the essential oil against isolated P. gingivalis was determined by broth micro-dilution method. Results In this study, 0.12 - 64 μL/mL Myrtus communis essence were used for 30 P. gingivalis isolates and the MIC50 and MIC90 concentration of Myrtus communis essence against the isolates was equal to 1 and 8 μL/mL respectively. Conclusions The results showed that Myrtus communis has antimicrobial effects against P. gingivalis. Further studies are suggested to include this essence in therapeutic protocols of periodontal disease. PMID:24624208

  18. Quantification of Porphyromonas gingivalis in chronic periodontitis patients associated with diabetes mellitus using real-time polymerase chain reaction

    PubMed Central

    Padmalatha, GV; Bavle, Radhika M; Satyakiran, Gadavalli Vera Venkata; Paremala, K; Sudhakara, M; Makarla, Soumya

    2016-01-01

    Introduction: Periodontal diseases, if left untreated, can lead to tooth loss and affect at least one tooth in 80% of adults worldwide, with the main cause being a bacterial plaque. Among subgingival plaque bacterial species, Porphyromonas gingivalis has been implicated as a major etiological agent causing tooth loss. Diabetics and smokers are two patient groups at high risk for periodontal disease. The increase in the number of this organism with the coexistence of other pathogenic microbes leads to rapid destruction of the periodontium, premature loss of teeth and also because of its virulence has implications in systemic pathology. Our aim was to observe the involvement of P. gingivalis in diabetes mellitus (DM) patients associated with periodontitis with and without tobacco-associated habits and to compare them with periodontitis patients having no other systemic pathologies. Materials and Methods: Subgingival plaque samples from a total of seventy subjects were included in the study. DNA was isolated from the collected sample and was quantified using spectrophotometer for standardizing the polymerase chain reaction. The quantity of the isolated DNA was checked in a ultraviolet-visible spectrophotomer. Statistics: One-way ANOVA and Tukey's multiple post hoc procedures were carried out. Results: The maximum score of P. gingivalis was seen in periodontitis patients having DM, whereas the least score was seen in periodontitis patients having DM with tobacco smoking habit compared to the other groups. Conclusion: P. gingivalis count is significantly reduced in periodontitis patients having DM with smoking habit; it is concluded that P. gingivalis might not be a key causative organism responsible for the periodontal destruction in case of smokers despite the DM condition. The decrease in counts may be attributed to change in the local environment like chemical (tobacco nitrosamines) and physical changes preventing the growth of P. gingivalis. PMID:27721606

  19. Porphyromonas gingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility

    PubMed Central

    Nakayama, K

    2015-01-01

    Porphyromonas gingivalis is a gram-negative, non-motile, anaerobic bacterium implicated as a major pathogen in periodontal disease. P. gingivalis grows as black-pigmented colonies on blood agar, and many bacteriologists have shown interest in this property. Studies of colonial pigmentation have revealed a number of important findings, including an association with the highly active extracellular and surface proteinases called gingipains that are found in P. gingivalis. The Por secretion system, a novel type IX secretion system (T9SS), has been implicated in gingipain secretion in studies using non-pigmented mutants. In addition, many potent virulence proteins, including the metallocarboxypeptidase CPG70, 35 kDa hemin-binding protein HBP35, peptidylarginine deiminase PAD and Lys-specific serine endopeptidase PepK, are secreted through the T9SS. These findings have not been limited to P. gingivalis but have been extended to other bacteria belonging to the phylum Bacteroidetes. Many Bacteroidetes species possess the T9SS, which is associated with gliding motility for some of these bacteria. PMID:25546073

  20. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions

    PubMed Central

    Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2013-01-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces. PMID:23867843

  1. Punica granatum L. (Pomegranate) Extract: In Vivo Study of Antimicrobial Activity against Porphyromonas gingivalis in Galleria mellonella Model

    PubMed Central

    Aparecida Procópio Gomes, Livia; Alves Figueiredo, Lívia Mara; Corrêa Geraldo, Barbara Maria; Isler Castro, Kelly Cristine; Ruano de Oliveira Fugisaki, Luciana; Olavo Cardoso Jorge, Antônio; Dias de Oliveira, Luciane; Campos Junqueira, Juliana

    2016-01-01

    Due to the increase of bacterial resistance, medicinal alternatives are being explored. Punica granatum L. is an effective herbal extract with broad spectrum of action and bactericidal, antifungal, anthelmintic potential and being able to modulate the immune response. The aim was to evaluate the antimicrobial activity of pomegranate glycolic extract (PGE) against the periodontal pathogen Porphyromonas gingivalis by using Galleria mellonella as in vivo model. Fifteen larvae were used per group. Injection of high concentration (200, 100, and 25 mg/mL) of PGE showed a toxic effect, leading them to death. A suspension of P. gingivalis (106 cells/mL) was inoculated in the left last proleg and PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) were injected into the right proleg. The larvae were then kept at 37°C under the dark. Injection of PGE at any dose statistically improved larvae survival rates. The data were analysed (log-rank test, Mantel-Cox, P < 0.05) and showed that all concentrations of PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) presented higher larval survival rates, with significant statistical difference in relation to control group (P. gingivalis). In conclusion, the PGE had antimicrobial action against P. gingivalis in vivo model using G. mellonella. PMID:27668280

  2. Punica granatum L. (Pomegranate) Extract: In Vivo Study of Antimicrobial Activity against Porphyromonas gingivalis in Galleria mellonella Model

    PubMed Central

    Aparecida Procópio Gomes, Livia; Alves Figueiredo, Lívia Mara; Corrêa Geraldo, Barbara Maria; Isler Castro, Kelly Cristine; Ruano de Oliveira Fugisaki, Luciana; Olavo Cardoso Jorge, Antônio; Dias de Oliveira, Luciane; Campos Junqueira, Juliana

    2016-01-01

    Due to the increase of bacterial resistance, medicinal alternatives are being explored. Punica granatum L. is an effective herbal extract with broad spectrum of action and bactericidal, antifungal, anthelmintic potential and being able to modulate the immune response. The aim was to evaluate the antimicrobial activity of pomegranate glycolic extract (PGE) against the periodontal pathogen Porphyromonas gingivalis by using Galleria mellonella as in vivo model. Fifteen larvae were used per group. Injection of high concentration (200, 100, and 25 mg/mL) of PGE showed a toxic effect, leading them to death. A suspension of P. gingivalis (106 cells/mL) was inoculated in the left last proleg and PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) were injected into the right proleg. The larvae were then kept at 37°C under the dark. Injection of PGE at any dose statistically improved larvae survival rates. The data were analysed (log-rank test, Mantel-Cox, P < 0.05) and showed that all concentrations of PGE (12.5, 6.25, 3.1, and 2.5 mg/mL) presented higher larval survival rates, with significant statistical difference in relation to control group (P. gingivalis). In conclusion, the PGE had antimicrobial action against P. gingivalis in vivo model using G. mellonella.

  3. Porphyromonas gingivalis and Disease-Related Autoantibodies in Individuals at Increased Risk of Rheumatoid Arthritis

    PubMed Central

    Mikuls, Ted R.; Thiele, Geoffrey M.; Deane, Kevin D.; Payne, Jeffrey B.; O'Dell, James R.; Yu, Fang; Sayles, Harlan; Weisman, Michael H.; Gregersen, Peter K.; Buckner, Jane H.; Keating, Richard M.; Derber, Lezlie A.; Robinson, William H.; Holers, V. Michael; Norris, Jill M.

    2012-01-01

    Purpose To examine the relationship of Porphyromonas gingivalis (Pg) with the presence of autoantibodies in individuals at risk for rheumatoid arthritis (RA). Methods Participants included: 1) a cohort enriched with HLA-DR4 and 2) those at risk for RA by virtue of having a first-degree relative with RA. None satisfied 1987 ACR RA classification criteria. Autoantibodies measured included anti-citrullinated protein antibody (ACPA) and rheumatoid factor (RF; nephelometry, IgA, IgM, IgG). Individuals were considered autoantibody positive (n = 113) with ≥ 1 positive autoantibody with individuals further categorized as `high-risk' (n = 38; positive ACPA or ≥ 2 RF assays). Autoantibody negative individuals served as comparators (n = 171). Antibody to Pg, P. intermedia (Pi), and F. nucleatum (Fn) were measured. Associations of bacterial antibodies with group status were examined using logistic regression. Results Anti-Pg concentrations were higher in high-risk (p = 0.011) and autoantibody positive group (p = 0.010) than in the autoantibody negative group. There were no group differences in anti-Pi or anti-Fn concentrations. After multivariable adjustment, anti-Pg concentrations (but not anti-Pi or anti-Fn) were significantly associated with autoantibody positive and high-risk status (p < 0.05). Conclusion Immunity to Pg, but not Pi or Fn, is significantly associated with the presence of RA-related autoantibodies in individuals at risk for RA. These results support the hypothesis that infection with Pg may play a central role in the early loss of tolerance to self-antigens in RA pathogenesis. PMID:22736291

  4. Porphyromonas gingivalis within Placental Villous Mesenchyme and Umbilical Cord Stroma Is Associated with Adverse Pregnancy Outcome

    PubMed Central

    Vanterpool, Sizzle F.; Been, Jasper V.; Houben, Michiel L.; Nikkels, Peter G. J.; De Krijger, Ronald R.; Zimmermann, Luc J. I.; Kramer, Boris W.; Progulske-Fox, Ann; Reyes, Leticia

    2016-01-01

    Intrauterine presence of Porphyromonas gingivalis (Pg), a common oral pathobiont, is implicated in preterm birth. Our aim was to determine if the location of Pg within placental and/or umbilical cord sections was associated with a specific delivery diagnosis at preterm delivery (histologic chorioamnionitis, chorioamnionitis with funisitis, preeclampsia, and preeclampsia with HELLP-syndrome, small for gestational age). The prevalence and location of Pg within archived placental and umbilical cord specimens from preterm (25 to 32 weeks gestation) and term control cohorts were evaluated by immunofluorescent histology. Detection of Pg was performed blinded to pregnancy characteristics. Multivariate analyses were performed to evaluate independent effects of gestational age, being small for gestational age, specific preterm delivery diagnosis, antenatal steroids, and delivery mode, on the odds of having Pg in the preterm tissue. Within the preterm cohort, 49 of 97 (51%) placentas and 40 of 97 (41%) umbilical cord specimens were positive for Pg. Pg within the placenta was significantly associated with shorter gestation lengths (OR 0.63 (95%CI: 0.48–0.85; p = 0.002) per week) and delivery via caesarean section (OR 4.02 (95%CI: 1.15–14.04; p = 0.03), but not with histological chorioamnionitis or preeclampsia. However, the presence of Pg in the umbilical cord was significantly associated with preeclampsia: OR 6.73 (95%CI: 1.31–36.67; p = 0.02). In the term cohort, 2 of 35 (6%) placentas and no umbilical cord term specimens were positive for Pg. The location of Pg within the placenta was different between preterm and term groups in that Pg within the villous mesenchyme was only detected in the preterm cohort, whereas Pg associated with syncytiotrophoblasts was found in both preterm and term placentas. Taken together, our results suggest that the presence of Pg within the villous stroma or umbilical cord may be an important determinant in Pg-associated adverse pregnancy

  5. Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

    PubMed Central

    Zhang, Xinwen; Ni, Junjun; Yu, Weixian; Nakanishi, Hiroshi

    2013-01-01

    We report here that the leptomeningeal cells transduce inflammatory signals from peripheral macrophages to brain-resident microglia in response to Porphyromonas gingivalis (P.g.) LPS. The expression of Toll-like receptor 2 (TLR2), TLR4, TNF-α, and inducible NO synthase was mainly detected in the gingival macrophages of chronic periodontitis patients. In in vitro studies, P.g. LPS induced the secretion of TNF-α and IL-1β from THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. Surprisingly, the mean mRNA levels of TNF-α and IL-1β in leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated RAW264.7 macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean mRNA levels of TNF-α and IL-1β in microglia after treatment with the conditioned medium from P.g. LPS-stimulated leptomeningeal cells were significantly higher than those after P.g. LPS alone. These observations suggest that leptomeninges serve as an important route for transducing inflammatory signals from macrophages to microglia by secretion of proinflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1 β production by leptomeningeal cells through inhibiting the nuclear factor-κB signaling pathway. Together with the inhibitory effect on microglial activation, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis. PMID:24363500

  6. Serpine1 Mediates Porphyromonas gingivalis Induced Insulin Secretion in the Pancreatic Beta Cell Line MIN6

    PubMed Central

    Bhat, Uppoor G.; Watanabe, Keiko

    2015-01-01

    Periodontitis is an inflammatory disease resulting in destruction of gingiva and alveolar bone caused by an exuberant host immunological response to periodontal pathogens. Results from a number of epidemiological studies indicate a close association between diabetes and periodontitis. Results from cross-sectional studies indicate that subjects with periodontitis have a higher odds ratio of developing insulin resistance (IR). However, the mechanisms by which periodontitis influences the development of diabetes are not known. Results from our previous studies using an animal model of periodontitis suggest that periodontitis accelerates the onset of hyperinsulinemia and IR. In addition, LPS from a periodontal pathogen, Porphyromonas gingivalis (Pg), stimulates Serpine1 expression in the pancreatic beta cell line MIN6. Based on these observations, we hypothesized that a periodontal pathogen induces hyperinsulinemia and Serpine1 may be involved in this process. To test this hypothesis, we co-incubated Pg with the pancreatic beta cell line MIN6 and measured the effect on insulin secretion by MIN6 cells. We further determined the involvement of Serpine1 in insulin secretion by downregulating Serpine1 expression. Our results indicated that Pg stimulated insulin secretion by approximately 3.0 fold under normoglycemic conditions. In a hyperglycemic state, Pg increased insulin secretion by 1.5 fold. Pg significantly upregulated expression of the Serpine1 gene and this was associated with increased secretion of insulin by MIN6 cells. However, cells with downregulated Serpine1 expression were resistant to Pg stimulated insulin secretion under normoglycemic conditions. We conclude that the periodontal pathogen, Pg, induced insulin secretion by MIN6 cells and this induction was, in part, Serpine1 dependent. Thus, Serpine1 may play a pivotal role in insulin secretion during the accelerated development of hyperinsulinemia and the resulting IR in the setting of periodontitis. PMID

  7. Involvement of PG2212 Zinc Finger Protein in the Regulation of Oxidative Stress Resistance in Porphyromonas gingivalis W83

    PubMed Central

    Dou, Yuetan; Aruni, Wilson; Luo, Tianlong; Roy, Francis; Wang, Charles

    2014-01-01

    The adaptation of Porphyromonas gingivalis to H2O2-induced stress while inducible is modulated by an unknown OxyR-independent mechanism. Previously, we reported that the PG_2212 gene was highly upregulated in P. gingivalis under conditions of prolonged oxidative stress. Because this gene may have regulatory properties, its function in response to H2O2 was further characterized. PG2212, annotated as a hypothetical protein of unknown function, is a 10.3-kDa protein with a cysteine 2-histidine 2 (Cys2His2) zinc finger domain. The isogenic mutant P. gingivalis FLL366 (ΔPG_2212) showed increased sensitivity to H2O2 and decreased gingipain activity compared to the parent strain. Transcriptome analysis of P. gingivalis FLL366 revealed that approximately 11% of the genome displayed altered expression (130 downregulated genes and 120 upregulated genes) in response to prolonged H2O2-induced stress. The majority of the modulated genes were hypothetical or of unknown function, although some are known to participate in oxidative stress resistance. The promoter region of several of the most highly modulated genes contained conserved motifs. In electrophoretic mobility shift assays, the purified rPG2212 protein did not bind its own promoter region but bound a similar region in several of the genes modulated in the PG_2212-deficient mutant. A metabolome analysis revealed that PG2212 can regulate a number of genes coding for proteins involved in metabolic pathways critical for its survival under the conditions of oxidative stress. Collectively, our data suggest that PG2212 is a transcriptional regulator that plays an important role in oxidative stress resistance and virulence regulation in P. gingivalis. PMID:25225267

  8. The hemagglutinin gene A (hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, direct repeats.

    PubMed Central

    Han, N; Whitlock, J; Progulske-Fox, A

    1996-01-01

    Porphyromonas gingivalis is a gram-negative anaerobic bacterial species strongly associated with adult periodontitis. One of its distinguishing characteristics and putative virulence properties is the ability to agglutinate erythrocytes. We have previously reported the cloning of multiple hemagglutinin genes from P. gingivalis 381. Subsequent sequencing of clone ST 2 revealed that the cloned fragment contained only an internal portion of the gene which lacked both start and stop codons. We here report the cloning and sequencing of the entire gene, designated hagA, as well as its relationship to other genes of this species. By use of inverse PCR technology and the construction of several additional genomic libraries, the complete open reading frame of hagA was found to be 7,887 bp in length, encoding a protein of 2,628 amino acids with a molecular mass of 283.3 kDa, which is among the largest genes ever cloned from a prokaryote to date. Within its open reading frame, four large, contiguous, direct repeats (varying from 1,318 to 1,368 bp) were identified. The repeat unit (HArep), which is assumed to contain the hemagglutinin domain, is also present in other recently reported protease and hemagglutinin genes in P. gingivalis. Thus, we propose that hagA and the other genes which share the HArep sequence form a multigene family with hagA as a central member. PMID:8926061

  9. Inhibitory effect of gels loaded with a low concentration of antibiotics against biofilm formation by Enterococcus faecalis and Porphyromonas gingivalis.

    PubMed

    A Algarni, Amnah; H Yassen, Ghaeth; L Gregory, Richard

    2015-09-01

    We explored longitudinally the inhibitory effect of gels loaded with 1 mg/mL modified triple antibiotic paste (MTAP) or double antibiotic paste (DAP) against biofilm formation by Enterococcus faecalis and Porphyromonas gingivalis. Methylcellulose-based antibiotic gels of MTAP (ciprofloxacin, metronidazole and clindamycin) and DAP (ciprofloxacin and metronidazole) were prepared at a concentration of 1 mg/mL. Individually cultured E. faecalis and P. gingivalis bacterial suspensions were treated with MTAP, DAP, or placebo (vehicle only) gels at different dilutions and allowed to grow in 96-well microtiter plates. Untreated bacterial suspensions served as a negative control. Crystal violet assays were used to evaluate biofilm formation after 48 h. The ability of the gels to inhibit biofilm formation was determined immediately, and at 1 month and 3 months after the gels had been prepared. Data were analyzed using a mixed-model ANOVA. The MTAP and DAP gels significantly reduced biofilm formation by both bacterial species at all time points, regardless of the tested dilution. No-significant differences in biofilm-inhibitory effects between MTAP and DAP gels were observed at the majority of the tested dilutions through various time points. Gels loaded with 1 mg/mL MTAP and DAP demonstrated a significant antibiofilm effect against E.faecalis and P. gingivalis. PMID:26369485

  10. Inhibitory Effect of Dodonaea viscosa var. angustifolia on the Virulence Properties of the Oral Pathogens Streptococcus mutans and Porphyromonas gingivalis

    PubMed Central

    Owotade, Foluso John

    2013-01-01

    Aim. This study investigated the effect of Dodonaea viscosa var. angustifolia (DVA) on the virulence properties of cariogenic Streptococcus mutans and Porphyromonas gingivalis implicated in periodontal diseases. Methods. S. mutans was cultured in tryptone broth containing a crude leaf extract of DVA for 16 hours, and the pH was measured after 10, 12, 14, and 16 h. Biofilms of S. mutans were grown on glass slides for 48 hours and exposed to plant extract for 30 minutes; the adherent cells were reincubated and the pH was measured at various time intervals. Minimum bactericidal concentration of the extracts against the four periodontal pathogens was determined. The effect of the subinhibitory concentration of plant extract on the production of proteinases by P. gingivalis was also evaluated. Results. DVA had no effect on acid production by S. mutans biofilms; however, it significantly inhibited acid production in planktonic cells. Periodontal pathogens were completely eliminated at low concentrations ranging from 0.09 to 0.02 mg/mL of crude plant extracts. At subinhibitory concentrations, DVA significantly reduced Arg-gingipain (24%) and Lys-gingipain (53%) production by P. gingivalis (P ≤ 0.01). Conclusions. These results suggest that DVA has the potential to be used to control oral infections including dental caries and periodontal diseases. PMID:24223061

  11. Porphyromonas gingivalis Lipopolysaccharide Upregulates Insulin Secretion From Pancreatic β Cell Line MIN6

    PubMed Central

    Bhat, Uppoor G.; Ilievski, Vladimir; Unterman, Terry G.; Watanabe, Keiko

    2015-01-01

    Background A close association between periodontitis and diabetes has been demonstrated in human cross-sectional studies, but an exact relationship between periodontitis and prediabetes has not been established. Previous studies using animal model systems consistently have shown that hyperinsulinemia occurs in animals with periodontitis compared to animals with healthy periodontium (while maintaining normoglycemia). Because bacterial lipopolysaccharide (LPS) plays an important role in the pathogenesis of periodontitis, we hypothesized that LPS may stimulate insulin secretion through a direct effect on β cell function. To test this hypothesis, pancreatic β cell line MIN6 cells were used to determine the effect of Porphyromonas gingivalis (Pg) LPS on insulin secretion. Furthermore, expression of genes altered by Pg LPS in innate immunity and insulin-signaling pathways was determined. Methods MIN6 cells were grown in medium with glucose concentration of normoglycemia (5.5 mM). Pg LPS was added to each well at final concentrations of 50, 200, and 500 ng/mL. Insulin secretion was measured using enzyme-linked immunosorbent assay. Gene expression levels altered by Pg LPS were determined by polymerase chain reaction (PCR) array for mouse innate and adaptive immunity response and mouse insulin-signaling pathways, and results were confirmed for specific genes of interest by quantitative PCR. Results Pg LPS stimulated insulin secretion in the normoglycemic condition by ≈1.5- to 3.0-fold depending on the concentration of LPS. Pg LPS treatment altered the expression of several genes involved in innate and adaptive immune response and insulin-signaling pathway. Pg LPS upregulated the expression of the immune response–related genes cluster of differentiation 8a (Cd8a), Cd14, and intercellular adhesion molecule-1 (Icam1) by about two-fold. LPS also increased the expression of two insulin signaling–related genes, glucose-6-phosphatase catalytic subunit (G6pc) and insulin

  12. Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2

    PubMed Central

    Hasegawa, Yoshiaki; Iwami, Jun; Sato, Keiko; Park, Yoonsuk; Nishikawa, Kiyoshi; Atsumi, Tatsuo; Moriguchi, Keiichi; Murakami, Yukitaka; Lamont, Richard J.; Nakamura, Hiroshi; Ohno, Norikazu; Yoshimura, Fuminobu

    2009-01-01

    Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components. PMID:19589838

  13. Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

    NASA Astrophysics Data System (ADS)

    Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P.; Ren, Ling; Yang, Ke

    2016-07-01

    Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility.

  14. Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis

    PubMed Central

    Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P.; Ren, Ling; Yang, Ke

    2016-01-01

    Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility. PMID:27457788

  15. Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis.

    PubMed

    Liu, Rui; Memarzadeh, Kaveh; Chang, Bei; Zhang, Yumei; Ma, Zheng; Allaker, Robert P; Ren, Ling; Yang, Ke

    2016-01-01

    Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility. PMID:27457788

  16. An Overview of the Carbonic Anhydrases from Two Pathogens of the Oral Cavity: Streptococcus mutans and Porphyromonas gingivalis.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2016-01-01

    Among the crowd of bacteria provoking disease of the oral cavity during the weakened of immune system, Streptococcus mutans and Porphyromonas gingivalis are the main microorganisms implicated in caries formation and periodontitis, respectively. The life cycle of the pathogens, such as protozoa, fungi and bacteria, is influenced by a superfamily of enzymes, called carbonic anhydrases (CAs, EC 4.2.1.1). These metalloenzymes, being crucial for the survival of the pathogen, have been considered as novel anti-infective targets. In fact, bicarbonate and protons, produced by the CA catalyzed carbon dioxide as substrate, are two fundamental ions implicated in the pH regulation, biosynthetic reactions, and adaptation of the pathogen to the host or in the possibility of the pathogen to avoid the host immune system. Bacteria genome encodes for the α-, β- and γ-CAs. Recently, our groups using the recombinant DNA technology prepared and characterized the CAs belonging to the β- and γ-classes encoded by the genome of the two oral cavity pathogens S. mutans and P. gingivalis. An extensive inhibition study was carried out using typical anion/sulfonamide inhibitors of these classes of CAs. We discovered numerous inhibitors, which had in vitro an effective inhibitory activity against the bacterial CAs considered, here, as alternative anti-infective targets.

  17. Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model.

    PubMed

    Jäsberg, Heli; Söderling, Eva; Endo, Akihito; Beighton, David; Haukioja, Anna

    2016-06-01

    There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar-overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited. PMID:27061393

  18. Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts

    PubMed Central

    Herath, Thanuja D. K.; Darveau, Richard P.; Seneviratne, Chaminda J.; Wang, Cun-Yu; Wang, Yu; Jin, Lijian

    2016-01-01

    Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis. PMID:27538450

  19. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

    PubMed Central

    Quirke, Anne-Marie; Lugli, Elena Birgitta; Wegner, Natalia; Hamilton, Bart C; Charles, Peter; Chowdhury, Muslima; Ytterberg, A Jimmy; Zubarev, Roman A; Potempa, Jan; Culshaw, Shauna; Guo, Yonghua; Fisher, Benjamin A; Thiele, Geoffrey; Mikuls, Ted R; Venables, Patrick JW

    2014-01-01

    Background Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. Objectives To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. Methods PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). Results Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. Conclusions The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy. PMID:23463691

  20. Fur homolog regulates Porphyromonas gingivalis virulence under low-iron/heme conditions through a complex regulatory network.

    PubMed

    Ciuraszkiewicz, J; Smiga, M; Mackiewicz, P; Gmiterek, A; Bielecki, M; Olczak, M; Olczak, T

    2014-12-01

    Porphyromonas gingivalis is a key pathogen responsible for initiation and progression of chronic periodontitis. Little is known about the regulatory mechanisms of iron and heme uptake that allow P. gingivalis to express virulence factors and survive in the hostile environment of the oral cavity, so we initiated characterization of a P. gingivalis Fur homolog (PgFur). Many Fur paralogs found in microbial genomes, including Bacteroidetes, confirm that Fur proteins have a tendency to be subjected to a sub- or even neofunctionalization process. PgFur revealed extremely high sequence divergence, which could be associated with its functional dissimilarity in comparison with other Fur homologs. A fur mutant strain constructed by insertional inactivation exhibited retarded growth during the early growth phase and a significantly lower tendency to form a homotypic biofilm on abiotic surfaces. The mutant also showed significantly weaker adherence and invasion to epithelial cells and macrophages. Transcripts of many differentially regulated genes identified in the fur mutant strain were annotated as hypothetical proteins, suggesting that PgFur can play a novel role in the regulation of gene expression. Inactivation of the fur gene resulted in decreased hmuY gene expression, increased expression of other hmu components and changes in the expression of genes encoding hemagglutinins and proteases (mainly gingipains), HtrA, some extracytoplasmic sigma factors and two-component systems. Our data suggest that PgFur can influence in vivo growth and virulence, at least in part by affecting iron/heme acquisition, allowing efficient infection through a complex regulatory network.

  1. Blocking Pro-Inflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas Gingivalis

    PubMed Central

    Berker, Ezel; Kantarci, Alpdogan; Hasturk, Hatice; Van Dyke, Thomas E.

    2013-01-01

    Background Chronic periodontitis is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti-inflammatory cytokines (IL-4 and IL-10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the pro- and anti-inflammatory mediators. We have tested the hypothesis that there is cellular cross-talk mediated by pro- and anti-inflammatory cytokines and that blocking pro-inflammatory cytokine (TNF-α and IL-1) production will enhance anti-inflammatory cytokine (IL-4 and IL-10) production from peripheral blood mononuclear cells (PBMC) in response to P. gingivalis. Methods PBMC were isolated from individuals diagnosed with chronic periodontitis or healthy individuals and cultured for 24 hours. Concanavalin-A (ConA) was used as an activator of lymphocyte function. Live and heat-killed P .gingivalis or lipopolysaccharide from P. gingivalis was used as the bacterial stimulants. TNF-α and IL-1 production was neutralized by specific antibodies against TNF-α and IL-1α or β. Culture supernatants were evaluated by ELISA for TNF-α, IL-1β, IL-4, and IL-10 production. Results Live P. gingivalis did not result in any significant IL-10 or IL-4 release while heat-killed P. gingivalis led to a significant increase in IL-10 levels compared to unstimulated or live P. gingivalis-stimulated cells from both healthy and periodontitis individuals. Overall, PBMC from patients with chronic periodontitis produced significantly lower IL-10 in response to ConA and P. gingivalis suggesting chronic suppression of the anti-inflammatory cytokine production. Blocking the pro-inflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response to live P. gingivalis. Blocking the pro-inflammatory cytokine response restored IL-10 production by cells from chronic periodontitis in response to P. gingivalis LPS. Conclusion These findings suggest that PBMC from patients with chronic

  2. Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin.

    PubMed

    Love, R M; McMillan, M D; Park, Y; Jenkinson, H F

    2000-03-01

    Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology

  3. Effects of the antimicrobial peptide cathelicidin (LL-37) on immortalized gingival fibroblasts infected with Porphyromonas gingivalis and irradiated with 625-nm LED light.

    PubMed

    Kim, JiSun; Kim, SangWoo; Lim, WonBong; Choi, HongRan; Kim, OkJoon

    2015-11-01

    Porphyromonas gingivalis causes chronic inflammatory diseases (periodontal diseases) that destroy the periodontal ligament and alveolar bone. Antimicrobial peptides are crucial components of the host defense response required to maintain cellular homeostasis during microbial invasion. Because light-emitting diode (LED) irradiation influences the host defense response against bacterial infections, we investigated its effect on immortalized gingival fibroblasts (IGFs) infected with P. gingivalis. IGFs were incubated with P. gingivalis following LED irradiation at 425, 525, and 625 nm. The dark 1 group comprised noninfected, nonirradiated IGFs, and the dark 2 group comprised nonirradiated IGFs infected with P. gingivalis. These groups served as controls. Infected cells and controls were assayed for reactive oxygen species (ROS) and were subjected to RT-PCR and Western blotting analyses to determine the levels of expression of antimicrobial peptides. LED irradiation enhanced the bactericidal effects of the antimicrobial peptide LL-37 in cells infected with P. gingivalis. Irradiation at 625 nm decreased inflammatory responses involving the release of prostaglandin E2 induced by ROS in P. gingivalis-infected IGFs. LED irradiation at 625 nm induces an anti-inflammatory response that elicits the production of antimicrobial peptides, providing an efficacious method of treatment for periodontal diseases.

  4. Detection of Porphyromonas gingivalis fimA Type I Genotype in Gingivitis by Real-Time PCR–A Pilot Study

    PubMed Central

    Krishnan, Mahalakshmi; Chandrasekaran, S.C.

    2016-01-01

    Introduction Published literature till date reveals a high prevalence of Porphyromonas gingivalis fimA type I genotype among healthy subjects. Quite a few studies have reported its prevalence also in periodontitis patients. Nevertheless incidence of this genotype in gingivitis is lacking in adult population. Aim The present study was chosen to detect P. gingivalis fimA type I genotype among chronic gingivitis patients. Materials and Methods A total of 46 subgingival plaque samples collected from chronic marginal gingivitis (n=23) and chronic periodontitis subjects (control group) (n=23) were subjected to Real-Time Polymerase Chain Reaction to detect the P. gingivalis fimA type I gene. Statistical analysis was performed using chi-square test. Results Prevalence of P. gingivalis fimA type I gene among chronic periodontitis and chronic gingivitis patients were 8.7% and 30.4% respectively. P. gingivalis fimA type I genotype prevalence was found to be statistically insignificant between the two study groups (p=0.135). Conclusion The avirulent P. gingivalis fimA type I genotype, occurred in high prevalence among chronic gingivitis patients, while its presence was low in chronic periodontitis patients. Presence of this avirulent genotype in chronic marginal gingivitis signifies its reversible condition. PMID:27504406

  5. Dual Action of Myricetin on Porphyromonas gingivalis and the Inflammatory Response of Host Cells: A Promising Therapeutic Molecule for Periodontal Diseases

    PubMed Central

    Grenier, Daniel; Chen, Huangqin; Ben Lagha, Amel; Fournier-Larente, Jade; Morin, Marie-Pierre

    2015-01-01

    Periodontitis that affects the underlying structures of the periodontium, including the alveolar bone, is a multifactorial disease, whose etiology involves interactions between specific bacterial species of the subgingival biofilm and the host immune components. In the present study, we investigated the effects of myricetin, a flavonol largely distributed in fruits and vegetables, on growth and virulence properties of Porphyromonas gingivalis as well as on the P. gingivalis-induced inflammatory response in host cells. Minimal inhibitory concentration values of myricetin against P. gingivalis were in the range of 62.5 to 125 μg/ml. The iron-chelating activity of myricetin may contribute to the antibacterial activity of this flavonol. Myricetin was found to attenuate the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including proteinases (rgpA, rgpB, and kgp) and adhesins (fimA, hagA, and hagB). Myricetin dose-dependently prevented NF-κB activation in a monocyte model. Moreover, it inhibited the secretion of IL-6, IL-8 and MMP-3 by P. gingivalis-stimulated gingival fibroblasts. In conclusion, our study brought clear evidence that the flavonol myricetin exhibits a dual action on the periodontopathogenic bacterium P. gingivalis and the inflammatory response of host cells. Therefore, myricetin holds promise as a therapeutic agent for the treatment/prevention of periodontitis. PMID:26121135

  6. A study of the uptake of toluidine blue O by Porphyromonas gingivalis and the mechanism of lethal photosensitization.

    PubMed

    Bhatti, M; MacRobert, A; Meghji, S; Henderson, B; Wilson, M

    1998-09-01

    The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (3H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a heliumneon (HeNe) laser and TBO was investigated by using deuterium oxide (D2O), which prolongs the lifetime of singlet oxygen, and the free radical and signlet oxygen scavenger L-tryptophan. There were 9.0 log10 and 2 log10 reductions in the presence of D2O and H2O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm2), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/cm2) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggregation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.

  7. Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts*

    PubMed Central

    Kassem, Ali; Henning, Petra; Lundberg, Pernilla; Souza, Pedro P. C.; Lindholm, Catharina; Lerner, Ulf H.

    2015-01-01

    Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease. PMID:26085099

  8. Unique Structure and Stability of HmuY, a Novel Heme-Binding Protein of Porphyromonas gingivalis

    PubMed Central

    Wójtowicz, Halina; Guevara, Tibisay; Tallant, Cynthia; Olczak, Mariusz; Sroka, Aneta; Potempa, Jan; Solà, Maria; Olczak, Teresa; Gomis-Rüth, F. Xavier

    2009-01-01

    Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection. PMID:19424422

  9. Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

    PubMed Central

    Taguchi, Yuko; Sato, Keiko; Yukitake, Hideharu; Inoue, Tetsuyoshi; Nakayama, Masaaki; Naito, Mariko; Kondo, Yoshio; Kano, Konami; Hoshino, Tomonori; Nakayama, Koji; Takashiba, Shogo

    2015-01-01

    The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence. PMID:26502912

  10. Structure and Mechanism of Cysteine Peptidase Gingipain K (Kgp), a Major Virulence Factor of Porphyromonas gingivalis in Periodontitis*

    PubMed Central

    de Diego, Iñaki; Veillard, Florian; Sztukowska, Maryta N.; Guevara, Tibisay; Potempa, Barbara; Pomowski, Anja; Huntington, James A.; Potempa, Jan; Gomis-Rüth, F. Xavier

    2014-01-01

    Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys477-His444-Asp388, rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates. PMID:25266723

  11. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  12. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis.

    PubMed

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  13. In-vivo effect of andrographolide on alveolar bone resorption induced by Porphyromonas gingivalis and its relation with antioxidant enzymes.

    PubMed

    Al Batran, Rami; Al-Bayaty, Fouad H; Al-Obaidi, Mazen M Jamil

    2013-01-01

    Alveolar bone resorption is one of the most important facts in denture construction. Porphyromonas gingivalis (Pg) causes alveolar bone resorption, and morphologic measurements are the most frequent methods to identify bone resorption in periodontal studies. This study has aimed at evaluating the effect of Andrographolide (AND) on alveolar bone resorption in rats induced by Pg. 24 healthy male Sprague Dawley rats were divided into four groups as follows: normal control group and three experimental groups challenged orally with Pg ATCC 33277 five times a week supplemented with 20 mg/kg and 10 mg/kg of AND for twelve weeks. Alveolar bones of the left and right sides of the mandible were assessed by a morphometric method. The bone level, that is, the distance from the alveolar bone crest to cementumenamel junction (CEJ), was measured using 6.1 : 1 zoom stereomicroscope and software. AND reduced the effect of Pg on alveolar bone resorption and decreased the serum levels of Hexanoyl-Lysine (HEL); furthermore the reduced glutathione/oxidised glutathione (GSH/GSSG) ratio in AND treated groups (10 and 20 mg/kg) significantly increased when compared with the Pg group (P < 0.05). We can conclude that AND suppresses alveolar bone resorption caused by Pg in rats.

  14. Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats

    PubMed Central

    Delbosc, Sandrine; Alsac, Jean-Marc; Journe, Clement; Louedec, Liliane; Castier, Yves; Bonnaure-Mallet, Martine; Ruimy, Raymond; Rossignol, Patrick; Bouchard, Philippe; Michel, Jean-Baptiste; Meilhac, Olivier

    2011-01-01

    Background Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. Methods and Findings Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. Conclusions Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression. PMID:21533243

  15. Effects of Porphyromonas gingivalis lipopolysaccharide on the expression of key genes involved in cholesterol metabolism in macrophages

    PubMed Central

    Liu, Fen; Wang, Yi; Xu, Jing; Liu, Fangqiang

    2016-01-01

    Introduction Cardiovascular diseases are positively correlated with periodontal disease. However, the molecular mechanisms linking atherosclerosis and periodontal infection are not clear. This study aimed to determine whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) altered the expression of genes regulating cholesterol metabolism in macrophages in the presence of low-density lipoprotein (LDL). Material and methods THP-1-derived macrophages were exposed to different concentrations (0.1, 1, 10 µg/ml) of LPS in the presence of 50 µg/ml native LDL. Macrophages were also incubated with 1 µg/ml LPS for varying times (0, 24, 48, or 72 h) in the presence of native LDL. Foam cell formation was determined by oil red O staining and cholesterol content quantification. CD36, lectin-like oxidized LDL receptor-1 (LOX-1), ATP-binding cassette G1 (ABCG1), and acetyl CoA acyltransferase 1 (ACAT1) expression levels were measured by western blot and qRT-PCR. Results Foam cell formation was induced in a time- and concentration-dependent manner as assessed by both morphological and biochemical criteria. Pg-LPS caused downregulation of CD36 and ABCG1 but upregulation of ACAT1, while LOX-1 expression was not affected (p = 0.137). Conclusions Pg-LPS appears to be an important link in the development of atherosclerosis by mechanisms targeting cholesterol homeostasis, namely, excess cholesterol ester formation via ACAT1 and reduced cellular cholesterol efflux via ABCG1.

  16. Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

    PubMed Central

    Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada

    2015-01-01

    The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589

  17. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling

    PubMed Central

    ZHANG, YI; WANG, XI-CHAO; BAO, XING-FU; HU, MIN; YU, WEI-XIAN

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites. PMID:24348768

  18. Effects of Porphyromonas gingivalis lipopolysaccharide on osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling.

    PubMed

    Zhang, Yi; Wang, Xi-Chao; Bao, Xing-Fu; Hu, Min; Yu, Wei-Xian

    2014-01-01

    In bone remodeling, the Eph family is involved in regulating the process of osteoclast and osteoblast coordination in order to maintain bone homeostasis. In this study, the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the osteoblast-osteoclast bidirectional EphB4-EphrinB2 signaling were investigated. An osteoblast-osteoclast co-culture system was achieved successfully. Hence, direct contact and communication between osteoblasts and osteoclasts was permitted. Regarding the protein expression and gene expression of EphB4 and EphrinB2, it was shown that Pg-LPS increased the expression of EphB4 while inhibiting the expression of EphrinB2. Therefore, the results indicate that, when treated with Pg-LPS, the EphB4 receptor on osteoblasts and the EphrinB2 ligand on osteoclasts may generate bidirectional anti-osteoclastogenic and pro-osteoblastogenic signaling into respective cells and potentially facilitate the transition from bone resorption to bone formation. This study may contribute to the control of osteoblast differentiation and bone formation at remodeling, and possibly also modeling, sites.

  19. Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses

    NASA Astrophysics Data System (ADS)

    Borgwardt, Derek S.; Martin, Aaron D.; van Hemert, Jonathan R.; Yang, Jianyi; Fischer, Carol L.; Recker, Erica N.; Nair, Prashant R.; Vidva, Robinson; Chandrashekaraiah, Shwetha; Progulske-Fox, Ann; Drake, David; Cavanaugh, Joseph E.; Vali, Shireen; Zhang, Yang; Brogden, Kim A.

    2014-01-01

    Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 μM and one site had a weaker affinity with a KD2 of 60.0 μM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 μM histatin 5 attenuated (p < 0.05) 0.02 μM HagB-induced CCL3/MIP-1α, CCL4/MIP-1β, and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

  20. Gingipains from the Periodontal Pathogen Porphyromonas gingivalis Play a Significant Role in Regulation of Angiopoietin 1 and Angiopoietin 2 in Human Aortic Smooth Muscle Cells

    PubMed Central

    Khalaf, Hazem; Sirsjö, Allan; Bengtsson, Torbjörn

    2015-01-01

    Angiopoietin 1 (Angpt1) and angiopoietin 2 (Angpt2) are the ligands of tyrosine kinase (Tie) receptors, and they play important roles in vessel formation and the development of inflammatory diseases, such as atherosclerosis. Porphyromonas gingivalis is a Gram-negative periodontal bacterium that is thought to contribute to the progression of cardiovascular disease. The aim of this study was to investigate the role of P. gingivalis infection in the modulation of Angpt1 and Angpt2 in human aortic smooth muscle cells (AoSMCs). We exposed AoSMCs to wild-type (W50 and 381), gingipain mutant (E8 and K1A), and fimbrial mutant (DPG-3 and KRX-178) P. gingivalis strains and to different concentrations of tumor necrosis factor (TNF). The atherosclerosis risk factor TNF was used as a positive control in this study. We found that P. gingivalis (wild type, K1A, DPG3, and KRX178) and TNF upregulated the expression of Angpt2 and its transcription factor ETS1, respectively, in AoSMCs. In contrast, Angpt1 was inhibited by P. gingivalis and TNF. However, the RgpAB mutant E8 had no effect on the expression of Angpt1, Angpt2, or ETS1 in AoSMCs. The results also showed that ETS1 is critical for P. gingivalis induction of Angpt2. Exposure to Angpt2 protein enhanced the migration of AoSMCs but had no effect on proliferation. This study demonstrates that gingipains are crucial to the ability of P. gingivalis to markedly increase the expressed Angpt2/Angpt1 ratio in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. These findings further support the association between periodontitis and cardiovascular disease. PMID:26283334

  1. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively-parallel sequencing of transposon libraries (Tn-seq)

    PubMed Central

    Klein, Brian A.; Duncan, Margaret J.; Hu, Linden T.

    2016-01-01

    Summary Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify non-essential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures. PMID:25636611

  2. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system.

    PubMed

    Stathopulos, Julien; Cambillau, Christian; Cascales, Eric; Roussel, Alain; Leone, Philippe

    2015-01-01

    PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P4(3)2(1)2. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation. PMID:25615973

  3. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

    PubMed Central

    Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

    2016-01-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  4. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  5. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  6. The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

    PubMed

    Jayaprakash, K; Demirel, I; Khalaf, H; Bengtsson, T

    2015-10-01

    Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.

  7. Inhibition of Sprouty2 polarizes macrophages toward an M2 phenotype by stimulation with interferon γ and Porphyromonas gingivalis lipopolysaccharide.

    PubMed

    Atomura, Ryo; Sanui, Terukazu; Fukuda, Takao; Tanaka, Urara; Toyoda, Kyosuke; Taketomi, Takaharu; Yamamichi, Kensuke; Akiyama, Hajime; Nishimura, Fusanori

    2016-03-01

    Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small-interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)-10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3-kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage-based anti-inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.

  8. Biochemical characterization of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor.

    PubMed Central

    Rangarajan, M; Smith, S J; U, S; Curtis, M A

    1997-01-01

    Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an alpha chain of approximately 54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked alpha and beta chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70-80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg-Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nalpha-benzoyl-L-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 degrees C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 degrees C of 6. 5+/-0.05 and 9.7+/-0.05, and DeltaH values of approximately 29 kJ/mol and approximately 24 kJ/mol in the enzyme-substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [L-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the alpha/beta structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by

  9. Inhibition of in vitro adhesion and virulence of Porphyromonas gingivalis by aqueous extract and polysaccharides from Rhododendron ferrugineum L. A new way for prophylaxis of periodontitis?

    PubMed

    Löhr, G; Beikler, T; Hensel, A

    2015-12-01

    The effect of an aqueous extract from the leaves of Rhododendron ferrugineum (RF) was investigated for its capacity of inhibiting the adhesion of Porphyromonas gingivalis cells to epithelial buccal KB cells. RF was characterized by HPLC (12.1% taxifolin-3-O-β-l-arabinopyranoside, 1.6% hyperoside, 0.9% isoquercitrin, 1.6% chlorogenic acid and a tannin content of 8.7%). Additionally raw polysaccharides (RPS) were obtained from the leaves of R. ferrugineum by aqueous extraction. RF and RPS interacted in a dose-dependent manner (max. 25% reduction at 1mg/ml each) with the adhesion of P. gingivalis by influencing bacterial outer membrane proteins. On protein level a time- and concentration-dependent inhibition of Arg-gingipain activity by RF was observed, while the Lys-gingipain activity remained unaltered. In addition, RF and RPS inhibited the bacterial hemagglutinin. RF affected the P. gingivalis adhesion also by interacting with KB cells in pre-incubation assays of the eukaryotic host cells, leading to reduced bacterial adhesion of about 75%. Gene expression analysis by RT-PCR indicated significant downregulation for arginine-specific gingipain rgpA by RF, while lysin-specific gingipain kgp and fimbrillinA fimA were strongly upregulated. Moreover, pre-incubation with RF abolished the P. gingivalis induced expression of IL-1β, IL-6, IL-8 and TNFα in KB cells. Results of this study indicate that an aqueous extract from R. ferrugineum combines cytoprotective and antimicrobial effects by both downregulating the expression of pro-inflammatory genes and inhibiting the adhesion of P. gingivalis. Thus RF may be potential candidate for the development of an adjunctive antimicrobial approach in the prevention of periodontal diseases.

  10. Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii.

    PubMed

    Valappil, Sabeel P; Coombes, Marc; Wright, Lucy; Owens, Gareth J; Lynch, Richard J M; Hope, Christopher K; Higham, Susan M

    2012-05-01

    Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity.

  11. Synergic phototoxic effect of visible light or Gallium-Arsenide laser in the presence of different photo-sensitizers on Porphyromonas gingivalis and Fusobacterium nucleatum

    PubMed Central

    Ghanbari, Habibollah; Mousavi, Seyed Amir; Forouzanfar, Ali; Zakeri, Mahdi; Shafaee, Hooman; Shahnaseri, Shirin

    2015-01-01

    Background: According to the development of resistant strains of pathogenic bacteria following treatment with antimicrobial chemotherapeutic agents, alternative approaches such as lethal photosensitization are being used. The aim of this study was to evaluate the effect of visible light and laser beam radiation in conjugation with three different photosensitizers on the survival of two main periodontopathogenic bacteria including Porphyromonas gingivalis and Fusobacterium nucleatum in different exposure periods. Materials and Methods: In this in vitro prospective study, strains of P. gingivalis and F. nucleatum. were exposed to visible light at wavelengths of 440 nm and diode laser light, Gallium-Arsenide, at wavelength of 830 nm in the presence of a photosensitizer (erythrosine, curcuma, or hydrogen peroxide). They were exposed 1-5 min to each light. Each experiment was repeated 3 times for each strain of bacteria. Data were analyzed by two-ways ANOVA and least significant difference post-hoc tests. P < 0.05 was considered as significant. After 4 days the colonies were counted. Results: Viability of P. gingivalis was reduced 10% and 20% subsequent to exposure to visible light and diode laser, respectively. The values were 65% and 75% for F. nucleatum in a period of 5-min, respectively. Exposure to visible light or laser beam in conjugation with the photosensitizers suspension caused significant reduction in the number of P. gingivalis in duration of 5-min, suggesting a synergic phototoxic effect. However, the survival rate of F. nucleatum following the exposure to laser with hydrogen peroxide, erythrosine and rhizome of Curcuma longa (curcumin) after 5-min was 10%, 20% and 90% respectively. Conclusion: Within the limitations of this study, the synergic phototoxic effect of visible light in combination with each of the photosensitizers on P. gingivalis and F. nucleatum. However, the synergic phototoxic effect of laser exposure and hydrogen peroxide and curcumin as

  12. Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii.

    PubMed

    Valappil, Sabeel P; Coombes, Marc; Wright, Lucy; Owens, Gareth J; Lynch, Richard J M; Hope, Christopher K; Higham, Susan M

    2012-05-01

    Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity. PMID:22314314

  13. Enhancing Specific-Antibody Production to the ragB Vaccine with GITRL That Expand Tfh, IFN-γ+ T Cells and Attenuates Porphyromonas gingivalis Infection in Mice

    PubMed Central

    Su, Zhaoliang; Kong, Fanzhi; Shi, Xiaoju; Tong, Jia; Shen, Pei; Peng, Tianqing; Wang, Shengjun; Xu, Huaxi

    2013-01-01

    The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ+ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ+ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ+ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ+ T cells and antibody production to P. gingivalis. PMID:23560053

  14. Anti-Inflammatory Effect of Heme Oxygenase-1 Toward Porphyromonas gingivalis Lipopolysaccharide in Macrophages Exposed to Gomisins A, G, and J

    PubMed Central

    Ryu, Eun Yeon; Park, Sun Young; Kim, Sun Gun; Park, Da Jung; Kang, Jum Soon; Kim, Young Hun; Seetharaman, Rajaseker

    2011-01-01

    Abstract Periodontitis, a chronic inflammatory periodontal disease that develops from gingivitis, is caused by periodontal pathogenic bacteria such as Porphyromonas gingivalis. Recent studies have focused on the antioxidant, anti–human immunodeficiency virus, anticarcinogenic, and anti-inflammatory properties of gomisins. However, the anti-inflammatory activities of gomisin plants through heme oxygenase-1 (HO-1) signals remain poorly defined. We found that gomisins' anti-inflammatory activity occurs via the induction of HO-1 expression. Gomisins G and J inhibit the production of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 and also block nuclear factor-κB activation in Raw264.7 cells stimulated with P. gingivalis lipopolysaccharide. Furthermore, pro-inflammatory cytokine production is inhibited through the induction of HO-1 expression. HO-1 expression is induced by all gomisins, but their anti-inflammatory activity via HO-1 signaling is observed with gomisins G and J, and not A. We found that gomisins G and J extracted from Schisandria chinensis can inhibit the P. gingivalis lipopolysaccharide induced-inflammatory responses in Raw264.7 cells. PMID:22145771

  15. Porphyromonas gingivalis Differentially Modulates Cell Death Profile in Ox-LDL and TNF-α Pre-Treated Endothelial Cells

    PubMed Central

    Bugueno, Isaac Maximiliano; Khelif, Yacine; Seelam, Narendra; Morand, David-Nicolas; Tenenbaum, Henri; Davideau, Jean-Luc; Huck, Olivier

    2016-01-01

    Objective Clinical studies demonstrated a potential link between atherosclerosis and periodontitis. Porphyromonas gingivalis (Pg), one of the main periodontal pathogen, has been associated to atheromatous plaque worsening. However, synergism between infection and other endothelial stressors such as oxidized-LDL or TNF-α especially on endothelial cell (EC) death has not been investigated. This study aims to assess the role of Pg on EC death in an inflammatory context and to determine potential molecular pathways involved. Methods Human umbilical vein ECs (HUVECs) were infected with Pg (MOI 100) or stimulated by its lipopolysaccharide (Pg-LPS) (1μg/ml) for 24 to 48 hours. Cell viability was measured with AlamarBlue test, type of cell death induced was assessed using Annexin V/propidium iodide staining. mRNA expression regarding caspase-1, -3, -9, Bcl-2, Bax-1 and Apaf-1 has been evaluated with RT-qPCR. Caspases enzymatic activity and concentration of APAF-1 protein were evaluated to confirm mRNA results. Results Pg infection and Pg-LPS stimulation induced EC death. A cumulative effect has been observed in Ox-LDL pre-treated ECs infected or stimulated. This effect was not observed in TNF-α pre-treated cells. Pg infection promotes EC necrosis, however, in infected Ox-LDL pre-treated ECs, apoptosis was promoted. This effect was not observed in TNF-α pre-treated cells highlighting specificity of molecular pathways activated. Regarding mRNA expression, Pg increased expression of pro-apoptotic genes including caspases-1,-3,-9, Bax-1 and decreased expression of anti-apoptotic Bcl-2. In Ox-LDL pre-treated ECs, Pg increased significantly the expression of Apaf-1. These results were confirmed at the protein level. Conclusion This study contributes to demonstrate that Pg and its Pg-LPS could exacerbate Ox-LDL and TNF-α induced endothelial injury through increase of EC death. Interestingly, molecular pathways are differentially modulated by the infection in function of the

  16. Morbidly obese patient with non-alcoholic steatohepatitis-related cirrhosis who died from sepsis caused by dental infection of Porphyromonas gingivalis: A case report.

    PubMed

    Omura, Yuno; Kitamoto, Mikiya; Hyogo, Hideyuki; Yamanoue, Takao; Tada, Yoshihiro; Boku, Noriko; Nishisaka, Takashi; Miyauchi, Mutsumi; Takata, Takashi; Chayama, Kazuaki

    2016-03-01

    Non-alcoholic steatohepatitis (NASH) is associated with increased risks of developing lifestyle-related diseases including type 2 diabetes, cardiovascular disease and cerebral vessel disease. While the two-hit hypothesis and, recently, multiple parallel hits hypothesis of NASH pathogenesis were proposed, further details have not emerged. Recently, dental infection of Porphyromonas gingivalis (P. gingivalis) has been reported as a critical risk factor for NASH progression, which acts as multiple parallel hits to induce inflammation and fibrogenic responses in steatosis. We describe here a 54-year-old woman who died from sepsis and was diagnosed with NASH. Briefly, her body mass index (BMI) at the age of 35 years old had been 25.6 kg/m(2) , but she became obese after withdrawing into her home at the age of 45 years. Severe obesity continued over 19 years without diabetes mellitus. She was admitted to our hospital due to a sudden disturbance of consciousness. On admission, her BMI was 48.5 kg/m(2) . Computed tomography revealed cirrhotic liver with massive ascites, and laboratory data indicated increased inflammatory responses, renal failure and C grade Child-Pugh classification, suggesting the diagnosis of sepsis. Also, severe periodontal disease was present, because the patient's front teeth fell out easily during intubation. Although the focus of infection was not specified, the oral flora Parvimonas micra, a periodontal pathogen, was detected in venous blood. In spite of intensive care including artificial respiration management and continuous hemodiafiltration, she died on the 43rd day after admission. Surprisingly, P. gingivalis was detected in her hepatocytes. This case may represent the significance of P. gingivalis in the progress to cirrhosis in NASH patients. PMID:25943712

  17. Identification of ragAB as a temperature-regulated operon of Porphyromonas gingivalis W50 using differential display of randomly primed RNA.

    PubMed

    Bonass, W A; Marsh, P D; Percival, R S; Aduse-Opoku, J; Hanley, S A; Devine, D A; Curtis, M A

    2000-07-01

    Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalis has the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalis by changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalis grown to steady state at temperatures of 37, 39, and 41 degrees C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragAB locus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragAB may be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

  18. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by upregulating toll-like receptor 4 expression.

    PubMed

    Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Lin, Yi-Wen; Shih, Chun-Che; Chiang, Kuang-Hsing; Shyue, Song-Kun; Chang, Yu-Jia; Hsieh, Chi-Kun; Lin, Feng-Yen

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is a bacterial species that causes periodontitis. GroEL from P. gingivalis may possess biological activity and may be involved in the destruction of periodontal tissues. However, it is unclear whether P. gingivalis GroEL enhances the appearance of atherogenic phenomena in endothelial cells and vessels. Here, we constructed recombinant GroEL from P. gingivalis to investigate its effects in human coronary artery endothelial cells (HCAECs) in vitro and on aortas of high-cholesterol (HC)-fed B57BL/6 and B57BL/6-Tlr4(lps-del) mice in vivo. The results showed that GroEL impaired tube-formation capacity under non-cytotoxic conditions in HCAECs. GroEL increased THP-1 cell/HCAEC adhesion by increasing the expression of intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in endothelial cells. Additionally, GroEL increased DiI-oxidized low density lipoprotein (oxLDL) uptake, which may be mediated by elevated lectin-like oxLDL receptor (LOX)-1 but not scavenger receptor expressed by endothelial cells (SREC) and scavenger receptor class B1 (SR-B1) expression. Furthermore, GroEL interacts with toll-like receptor 4 (TLR4) and plays a causal role in atherogenesis in HCAECs. Human antigen R (HuR), an RNA-binding protein with a high affinity for the 3' untranslated region (3'UTR) of TLR4 mRNA, contributes to the up-regulation of TLR4 induced by GroEL in HCAECs. In a GroEL animal administration study, GroEL elevated ICAM-1, VCAM-1, LOX-1 and TLR4 expression in the aortas of HC diet-fed wild C57BL/6 but not C57BL/6-Tlr4(lps-del) mice. Taken together, our findings suggest that P. gingivalis GroEL may contribute to cardiovascular disorders by affecting TLR4 expression. PMID:27158334

  19. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by upregulating toll-like receptor 4 expression

    PubMed Central

    Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Lin, Yi-Wen; Shih, Chun-Che; Chiang, Kuang-Hsing; Shyue, Song-Kun; Chang, Yu-Jia; Hsieh, Chi-Kun; Lin, Feng-Yen

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is a bacterial species that causes periodontitis. GroEL from P. gingivalis may possess biological activity and may be involved in the destruction of periodontal tissues. However, it is unclear whether P. gingivalis GroEL enhances the appearance of atherogenic phenomena in endothelial cells and vessels. Here, we constructed recombinant GroEL from P. gingivalis to investigate its effects in human coronary artery endothelial cells (HCAECs) in vitro and on aortas of high-cholesterol (HC)-fed B57BL/6 and B57BL/6-Tlr4lps-del mice in vivo. The results showed that GroEL impaired tube-formation capacity under non-cytotoxic conditions in HCAECs. GroEL increased THP-1 cell/HCAEC adhesion by increasing the expression of intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in endothelial cells. Additionally, GroEL increased DiI-oxidized low density lipoprotein (oxLDL) uptake, which may be mediated by elevated lectin-like oxLDL receptor (LOX)-1 but not scavenger receptor expressed by endothelial cells (SREC) and scavenger receptor class B1 (SR-B1) expression. Furthermore, GroEL interacts with toll-like receptor 4 (TLR4) and plays a causal role in atherogenesis in HCAECs. Human antigen R (HuR), an RNA-binding protein with a high affinity for the 3’ untranslated region (3’UTR) of TLR4 mRNA, contributes to the up-regulation of TLR4 induced by GroEL in HCAECs. In a GroEL animal administration study, GroEL elevated ICAM-1, VCAM-1, LOX-1 and TLR4 expression in the aortas of HC diet-fed wild C57BL/6 but not C57BL/6-Tlr4lps-del mice. Taken together, our findings suggest that P. gingivalis GroEL may contribute to cardiovascular disorders by affecting TLR4 expression. PMID:27158334

  20. Myxomavirus anti-inflammatory chemokine binding protein reduces the increased plaque growth induced by chronic Porphyromonas gingivalis oral infection after balloon angioplasty aortic injury in mice.

    PubMed

    Lucas, Alexandra R; Verma, Raj K; Dai, Erbin; Liu, Liying; Chen, Hao; Kesavalu, Sheela; Rivera, Mercedes; Velsko, Irina; Ambadapadi, Sriram; Chukkapalli, Sasanka; Kesavalu, Lakshmyya

    2014-01-01

    Thrombotic occlusion of inflammatory plaque in coronary arteries causes myocardial infarction. Treatment with emergent balloon angioplasty (BA) and stent implant improves survival, but restenosis (regrowth) can occur. Periodontal bacteremia is closely associated with inflammation and native arterial atherosclerosis, with potential to increase restenosis. Two virus-derived anti-inflammatory proteins, M-T7 and Serp-1, reduce inflammation and plaque growth after BA and transplant in animal models through separate pathways. M-T7 is a broad spectrum C, CC and CXC chemokine-binding protein. Serp-1 is a serine protease inhibitor (serpin) inhibiting thrombotic and thrombolytic pathways. Serp-1 also reduces arterial inflammation and improves survival in a mouse herpes virus (MHV68) model of lethal vasculitis. In addition, Serp-1 demonstrated safety and efficacy in patients with unstable coronary disease and stent implant, reducing markers of myocardial damage. We investigate here the effects of Porphyromonas gingivalis, a periodontal pathogen, on restenosis after BA and the effects of blocking chemokine and protease pathways with M-T7 and Serp-1. ApoE-/- mice had aortic BA and oral P. gingivalis infection. Arterial plaque growth was examined at 24 weeks with and without anti-inflammatory protein treatment. Dental plaques from mice infected with P. gingivalis tested positive for infection. Neither Serp-1 nor M-T7 treatment reduced infection, but IgG antibody levels in mice treated with Serp-1 and M-T7 were reduced. P. gingivalis significantly increased monocyte invasion and arterial plaque growth after BA (P<0.025). Monocyte invasion and plaque growth were blocked by M-T7 treatment (P<0.023), whereas Serp-1 produced only a trend toward reductions. Both proteins modified expression of TLR4 and MyD88. In conclusion, aortic plaque growth in ApoE-/- mice increased after angioplasty in mice with chronic oral P. gingivalis infection. Blockade of chemokines, but not serine

  1. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

    PubMed Central

    de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2016-01-01

    In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

  2. A Porphyromonas gingivalis Mutant Defective in a Putative Glycosyltransferase Exhibits Defective Biosynthesis of the Polysaccharide Portions of Lipopolysaccharide, Decreased Gingipain Activities, Strong Autoaggregation, and Increased Biofilm Formation▿ †

    PubMed Central

    Yamaguchi, Mikiyo; Sato, Keiko; Yukitake, Hideharu; Noiri, Yuichiro; Ebisu, Shigeyuki; Nakayama, Koji

    2010-01-01

    The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. The gtfB mutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation of P. gingivalis. PMID:20624909

  3. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism.

    PubMed

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  4. Structure of the fimbrial protein Mfa4 from Porphyromonas gingivalis in its precursor form: implications for a donor-strand complementation mechanism

    PubMed Central

    Kloppsteck, Patrik; Hall, Michael; Hasegawa, Yoshiaki; Persson, Karina

    2016-01-01

    Gingivitis and periodontitis are chronic inflammatory diseases that can lead to tooth loss. One of the causes of these diseases is the Gram-negative Porphyromonas gingivalis. This periodontal pathogen is dependent on two fimbriae, FimA and Mfa1, for binding to dental biofilm, salivary proteins, and host cells. These fimbriae are composed of five proteins each, but the fimbriae assembly mechanism and ligands are unknown. Here we reveal the crystal structure of the precursor form of Mfa4, one of the accessory proteins of the Mfa1 fimbria. Mfa4 consists of two β-sandwich domains and the first part of the structure forms two well-defined β-strands that run over both domains. This N-terminal region is cleaved by gingipains, a family of proteolytic enzymes that encompass arginine- and lysine-specific proteases. Cleavage of the N-terminal region generates the mature form of the protein. Our structural data allow us to propose that the new N-terminus of the mature protein may function as a donor strand in the polymerization of P. gingivalis fimbriae. PMID:26972441

  5. Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

    NASA Astrophysics Data System (ADS)

    Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

    2007-01-01

    Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

  6. Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway.

    PubMed Central

    Imamura, T; Pike, R N; Potempa, J; Travis, J

    1994-01-01

    To elucidate the mechanism of production of an inflammatory exudate, gingival crevicular fluid (GCF), from periodontal pockets in periodontitis, we examined the vascular permeability enhancement (VPE) activity induced by an arginine-specific cysteine proteinase, Arg-gingipain-1 (RGP-1), produced by a major periopathogenic bacterium, Porphyromonas gingivalis. Intradermal injections into guinea pigs of RGP-1 (> 10(-8) M), or human plasma incubated with RGP-1 (> 10(-9) M), induced VPE in a dose- and activity-dependent manner but with different time courses for the two routes of production. VPE activity induced by RGP-1 was augmented by kininase inhibitors, inhibited by a kallikrein inhibitor and unaffected by an antihistamine drug. The VPE activity in human plasma incubated with RGP-1 also correlated closely with generation of bradykinin (BK). RGP-1 induced 30-40% less VPE activity in Hageman factor-deficient plasma and no VPE in plasma deficient in either prekallikrein (PK) or high molecular weight kininogen (HMWK). After incubation with RGP-1, plasma deficient in PK or HMWK, reconstituted with each missing protein, caused VPE, as did a mixture of purified PK and HMWK, but RGP-1 induced no VPE from HMWK. The VPE of extracts of clinically isolated P. gingivalis were reduced to about 10% by anti-RGP-1-IgG, leupeptin, or tosyl-L-lysine chloromethyl ketone, which paralleled effects observed with RGP-1. These results indicate that RGP-1 is the major VPE factor of P. gingivalis, inducing this activity through PK activation and subsequent BK release, resulting in GCF production at sites of periodontitis caused by infection with this organism. Images PMID:8040277

  7. Aerosolized clindamycin is superior to aerosolized dexamethasone or clindamycin-dexamethasone combination in the treatment of severe Porphyromonas gingivalis aspiration pneumonia in an experimental murine model.

    PubMed

    Nemec, Ana; Pavlica, Zlatko; Nemec-Svete, Alenka; Eržen, Damijan; Milutinović, Aleksandra; Petelin, Milan

    2012-02-01

    Adjunctive corticosteroid treatment to reduce excessive local inflammatory response in pneumonia is controversial. To study the effects of an early local adjunct dexamethasone treatment on the course of pneumonia and inflammatory/cytokine response, mice were intratracheally inoculated with live Porphyromonas gingivalis and treated with either clindamycin (C), dexamethasone (D), C+D combination, or were not treated (Pg). Six mice from each group were euthanized at 6, 24, 72, and 168 hours after inoculation. Levels of tumor necrosis factor (TNF)-α, soluble TNF-α receptors (sTNFR1 and sTNFR2), interleukin (IL)-1β, and IL-6 in the serum and lung-homogenate supernatant were determined. Lung samples were histopathologically assessed and all findings compared to those found in 24 sham-inoculated mice (phosphate-buffered saline [PBS]). Severe P. gingivalis-induced bronchopneumonia progressed from 24 hours, peaked at 72 hours, and resolved after 168 hours with changes in local and systemic cytokine levels. Clindamycin-treated mice developed only mild bronchopneumonia that resolved fast (72 hours) with an early (6-24 hours) normalization of local and systemic cytokine levels. Similar course of pneumonia and cytokine level changes were observed in mice treated with C+D, but later. Early (6-24 hours) local elevation of sTNFRs was observed in C and C+D groups of mice, whereas nontreated (Pg) mice had increased systemic sTNFRs. Severe bronchopneumonia with delayed resolution was observed in D-group mice, with an early local and systemic decrease in sTNFR1 and persistent elevation of local TNF-α. Clindamycin or a clindamycin-dexamethasone combination treatment significantly improves the course of P. gingivalis-aspiration pneumonia, but more so if clindamycin alone is used. A favorable course of pneumonia seems to be associated with an early elevation of sTNFRs and normalization of TNF-α.

  8. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis.

    PubMed

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35-76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants' health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0-0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1-57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0-88.2) and body mass index (OR = 3.0; 95%CI 1.7-5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is suggested. PMID

  9. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    PubMed

    Śmiga, Michał; Bielecki, Marcin; Olczak, Mariusz; Smalley, John W; Olczak, Teresa

    2015-01-01

    Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. PMID:25658942

  10. The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities.

    PubMed

    Fournier-Larente, Jade; Azelmat, Jabrane; Yoshioka, Masami; Hinode, Daisuke; Grenier, Daniel

    2016-01-01

    Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest. PMID:26859747

  11. Social stress enhances IL-1beta and TNF-alpha production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells.

    PubMed

    Bailey, Michael T; Kinsey, Steven G; Padgett, David A; Sheridan, John F; Leblebicioglu, Binnaz

    2009-09-01

    Psychological stress is associated with an increased expression of markers of peripheral inflammation, and there is a growing literature describing a link between periodontal pathogens and systemic inflammation. The hypothesis of the present work is that exposing mice to the social stressor, called social disruption (SDR), would enhance the inflammatory response to lipopolysaccharide (LPS) derived from the oral pathogen, Porphyromonas gingivalis. Mice were exposed to SDR for 2h per day on 6 consecutive days. On the morning following the last cycle of SDR, mice were tested for anxiety-like behavior in the open field test and novel object test. The mice were sacrificed the following day and their spleens harvested. Spleen cells were stimulated with LPS derived from P. gingivalis in the absence or presence of increasing doses of corticosterone. Social disruption resulted in anxiety-like behavior, and the production of IL-1beta and TNF-alpha was significantly higher in spleen cells from mice exposed to SDR in comparison to levels from non-stressed control mice. In addition, the viability of spleen cells from mice exposed to SDR was significantly greater than the viability of cells from non-stressed control mice, even in the presence of high doses of corticosterone. The use of cultures enriched for CD11b+ cells indicated that the stressor was affecting the activity of splenic myeloid cells. This study demonstrates that social stress enhances the inflammatory response to an oral pathogen and could provide a critical clue in the reported associations between stress, inflammation, and oral pathogens.

  12. Porphyromonas gingivalis-specific serum IgG and IgA antibodies originate from immunoglobulin-secreting cells in inflamed gingiva.

    PubMed Central

    Ogawa, T; Kono, Y; McGhee, M L; McGhee, J R; Roberts, J E; Hamada, S; Kiyono, H

    1991-01-01

    Patients with adult periodontitis (AP) exhibit elevated serum antibody levels to Porphyromonas (Bacteroides) gingivalis; however, it is not known whether these antibodies originate from plasma cells in the local disease site or from peripheral lymphoid tissues. We studied the isotype and subclass levels and origin of antibodies to P. gingivalis fimbriae, since elevated serum anti-fimbriae responses were seen when compared with sera of healthy controls. IgG anti-fibriae titres were dominant and the subclass response was IgG3 much greater than IgG1 greater than IgG2 much greater than IgG4; however, some IgA anti-fimbriae antibodies were also seen. The IgA subclass fimbriae-specific response was mainly IgA1; however, significant IgA2 anti-fimbrae antibodies were seen. We also assessed numbers of anti-fimbriae antibody producing cells from peripheral blood mononuclear cells (PMBC) and from either healthy or inflamed gingiva of AP subjects. Gingival mononuclear cells (GMC) of AP patients exhibited high numbers of immunoglobulin-producing (spot-forming) cells (SFC) including fimbriae-specific antibody secreting cells in a pattern of IgG greater than IgA greater than greater than greater than IgM. However, low numbers of SFC were seen in GMC from healthy gingiva; further, no anti-fimbriae SFC responses were noted in healthy GMC. Although no fimbriae-specific immunoglobulin-producing cells were seen in PBMC, low numbers of antigen-specific SFC were found in pokeweed mitogen-triggered PBMC from AP subjects. Treatment of AP patients for plaque and surgical removal of inflamed gingiva resulted in significant reductions in serum anti-fimbriae responses. These studies show that AP patients exhibit brisk serum IgG and IgA subclass anti-fimbriae antibodies, whose origin appear to be the plasma cells present in the localized inflamed tissues. PMID:1671564

  13. Effects of immunization with Porphyromonas gingivalis and Prevotella intermedia on progression of ligature-induced periodontitis in the nonhuman primate Macaca fascicularis.

    PubMed Central

    Ebersole, J L; Brunsvold, M; Steffensen, B; Wood, R; Holt, S C

    1991-01-01

    The nonhuman primate (Nhp) has proven to be a useful model of human periodontitis. This study describes the immunological characteristics of this model and the ability of active immunization to interfere with ecological changes in the microbiota and its associated disease symptoms. Nhps were parenterally immunized with whole-cell antigens of Porphyromonas gingivalis and Prevotella intermedia. The immunization elicited an approximate 2-log increase in serum immunoglobulin G (IgG), IgM, and IgA isotype antibody that was highly specific for these immunogens. Postimmunization and postligation, there was minimal change in the levels of specific antibody. P. gingivalis immunization significantly inhibited the emergence of this species during disease progression. In contrast, induction of anti-P. intermedia antibody had a minimal effect on this species within the subgingival plaque. Plaque indices showed few changes that could be attributed to active immunization. Both bleeding on probing and loss of attachment were higher in ligated sites of immunized animals than in the placebo-treated group. A significant increase in bone density loss was observed in the ligated teeth from immunized versus control animals. These findings indicate that active immunization of Nhps can elicit a substantial systemic immune response; however, while this response may effect the emergence of an individual microorganism, it appears that other ecological considerations are critical in disease progression. It is also possible that the induction of a broad-based immune response to multiple bacterial antigens can result in increased disease, potentially associated with hypersensitivity reactions to the bacteria in the subgingival plaque. PMID:1894349

  14. Genome of the pathogen Porphyromonas gingivalis recovered from a biofilm in a hospital sink using a high-throughput single-cell genomics platform

    PubMed Central

    McLean, Jeffrey S.; Lombardo, Mary-Jane; Ziegler, Michael G.; Novotny, Mark; Yee-Greenbaum, Joyclyn; Badger, Jonathan H.; Tesler, Glenn; Nurk, Sergey; Lesin, Valery; Brami, Daniel; Hall, Adam P.; Edlund, Anna; Allen, Lisa Z.; Durkin, Scott; Reed, Sharon; Torriani, Francesca; Nealson, Kenneth H.; Pevzner, Pavel A.; Friedman, Robert; Venter, J. Craig; Lasken, Roger S.

    2013-01-01

    Although biofilms have been shown to be reservoirs of pathogens, our knowledge of the microbial diversity in biofilms within critical areas, such as health care facilities, is limited. Available methods for pathogen identification and strain typing have some inherent restrictions. In particular, culturing will yield only a fraction of the species present, PCR of virulence or marker genes is mainly focused on a handful of known species, and shotgun metagenomics is limited in the ability to detect strain variations. In this study, we present a single-cell genome sequencing approach to address these limitations and demonstrate it by specifically targeting bacterial cells within a complex biofilm from a hospital bathroom sink drain. A newly developed, automated platform was used to generate genomic DNA by the multiple displacement amplification (MDA) technique from hundreds of single cells in parallel. MDA reactions were screened and classified by 16S rRNA gene PCR sequence, which revealed a broad range of bacteria covering 25 different genera representing environmental species, human commensals, and opportunistic human pathogens. Here we focus on the recovery of a nearly complete genome representing a novel strain of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis JCVI SC001) using the single-cell assembly tool SPAdes. Single-cell genomics is becoming an accepted method to capture novel genomes, primarily in the marine and soil environments. Here we show for the first time that it also enables comparative genomic analysis of strain variation in a pathogen captured from complex biofilm samples in a healthcare facility. PMID:23564253

  15. The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities

    PubMed Central

    Fournier-Larente, Jade; Azelmat, Jabrane; Yoshioka, Masami; Hinode, Daisuke; Grenier, Daniel

    2016-01-01

    Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest. PMID:26859747

  16. Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

    PubMed

    Morozova, E A; Kulikova, V V; Yashin, D V; Anufrieva, N V; Anisimova, N Y; Revtovich, S V; Kotlov, M I; Belyi, Y F; Pokrovsky, V S; Demidkina, T V

    2013-07-01

    The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

  17. CCL3 and CXCL12 production in vitro by dental pulp fibroblasts from permanent and deciduous teeth stimulated by Porphyromonas gingivalis LPS

    PubMed Central

    SIPERT, Carla Renata; MORANDINI, Ana Carolina de Faria; MODENA, Karin Cristina da Silva; DIONÍSIO, Thiago José; MACHADO, Maria Aparecida Andrade Moreira; de OLIVEIRA, Sandra Helena Penha; CAMPANELLI, Ana Paula; SANTOS, Carlos Ferreira

    2013-01-01

    Objective: The aim of this study was to compare the production of the chemokines CCL3 and CXCL12 by cultured dental pulp fibroblasts from permanent (PDPF) and deciduous (DDPF) teeth under stimulation by Porphyromonas gingivalis LPS (PgLPS). Material and Methods: Primary culture of fibroblasts from permanent (n=3) and deciduous (n=2) teeth were established using an explant technique. After the fourth passage, fibroblasts were stimulated by increasing concentrations of PgLPS (0 - 10 µg/mL) at 1, 6 and 24 h. The cells were tested for viability through MTT assay, and production of the chemokines CCL3 and CXCL12 was determined through ELISA. Comparisons among samples were performed using One-way ANOVA for MTT assay and Two-way ANOVA for ELISA results. Results: Cell viability was not affected by the antigen after 24 h of stimulation. PgLPS induced the production of CCL3 by dental pulp fibroblasts at similar levels for both permanent and deciduous pulp fibroblasts. Production of CXCL12, however, was significantly higher for PDPF than DDPF at 1 and 6 h. PgLPS, in turn, downregulated the production of CXCL12 by PDPF but not by DDPF. Conclusion: These data suggest that dental pulp fibroblasts from permanent and deciduous teeth may present a differential behavior under PgLPS stimulation. PMID:23739851

  18. Structural Significance of the β1K396 Residue Found in the Porphyromonas gingivalis Sialidase β-Propeller Domain: A Computational Study with Implications for Novel Therapeutics Against Periodontal Disease

    PubMed Central

    Kamio, Noriaki; Imai, Kenichi; Ohya, Manabu; Tamura, Muneaki

    2014-01-01

    Abstract Porphyromonas gingivalis sialidase activity is associated with virulence and initiated by sialic acid (SA) binding to the β-propeller domain (BPD). Sialidase BPD is structurally conserved in various bacterial species and the protein binding interfaces have the tendency to form salt bridges, whereas uncommitted charged residues may affect binding and protein structure. However, it is not clear whether the sialidase BPD of varying strains of the same bacterial species differ, particularly with regards to salt bridge formation. Here, we determined the P. gingivalis ATCC 33277 and W50 sialidase homology models and sialidase activities, while the putative salt bridge residues found in the sialidase BPDs were compared. We established that both ATCC 33277 and W50 have different sialidase homology models and activities, whereas, the BPD (β1–6) is structurally conserved with most salt bridge-forming residues following a common orientation. Moreover, β2D444–β6K338 distance measurement in ATCC 33277 (5.99 Å) and W50 (3.09 Å) differ, while β1K396A substitution alters the β2D444–β6K338 distance measurements in ATCC 33277 (3.09 Å) and W50 (3.01 Å) consequentially affecting each model. P. gingivalis plays a major role in periodontitis induction and its virulence is greatly influenced by the sialidase enzyme wherein the sialidase BPD is highly conserved. Our results suggest that alterations in the salt bridge formation within the BPD interface may affect the P. gingivalis sialidase structure. This would imply that disrupting the salt bridge formation within the P. gingivalis sialidase BPD could serve as a potential therapeutic strategy for the treatment of P. gingivalis-related periodontitis. PMID:25000206

  19. Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus.

    PubMed Central

    Loesche, W J; Lopatin, D E; Giordano, J; Alcoforado, G; Hujoel, P

    1992-01-01

    Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed. PMID:1311335

  20. CD36/SR-B2-TLR2 Dependent Pathways Enhance Porphyromonas gingivalis Mediated Atherosclerosis in the Ldlr KO Mouse Model.

    PubMed

    Brown, Paul M; Kennedy, David J; Morton, Richard E; Febbraio, Maria

    2015-01-01

    There is strong epidemiological association between periodontal disease and cardiovascular disease but underlying mechanisms remain ill-defined. Because the human periodontal disease pathogen, Porphyromonas gingivalis (Pg), interacts with innate immune receptors Toll-like Receptor (TLR) 2 and CD36/scavenger receptor-B2 (SR-B2), we studied how CD36/SR-B2 and TLR pathways promote Pg-mediated atherosclerosis. Western diet fed low density lipoprotein receptor knockout (Ldlr°) mice infected orally with Pg had a significant increase in lesion burden compared with uninfected controls.This increase was entirely CD36/SR-B2-dependent, as there was no significant change in lesion burden between infected and uninfected Cd36o/Ldlro mice [corrected]. Western diet feeding promoted enhanced CD36/SR-B2-dependent IL1β generation and foam cell formation as a result of Pg lipopolysaccharide (PgLPS) exposure. CD36/SR-B2 and TLR2 were necessary for inflammasome activation and optimal IL1ß generation, but also resulted in LPS induced lethality (pyroptosis). Modified forms of LDL inhibited Pg-mediated IL1ß generation in a CD36/SR-B2-dependent manner and prevented pyroptosis, but promoted foam cell formation. Our data show that Pg infection in the oral cavity can lead to significant TLR2-CD36/SR-B2 dependent IL1ß release. In the vessel wall, macrophages encountering systemic release of IL1ß, PgLPS and modified LDL have increased lipid uptake, foam cell formation, and release of IL1ß, but because pyroptosis is inhibited, this enables macrophage survival and promotes increased plaque development. These studies may explain increased lesion burden as a result of periodontal disease, and suggest strategies for development of therapeutics.

  1. Serum Immunoglobulin G Levels to Porphyromonas gingivalis Peptidylarginine Deiminase Affect Clinical Response to Biological Disease-Modifying Antirheumatic Drug in Rheumatoid Arthritis

    PubMed Central

    Kobayashi, Tetsuo; Ito, Satoshi; Kobayashi, Daisuke; Shimada, Atsushi; Narita, Ichiei; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

    2016-01-01

    Objectives To determine whether serum immunity to Porphyromonas gingivalis peptidylarginine deiminase (PPAD) affects the clinical response to biological disease-modifying antirheumatic drug (bDMARD) in patients with rheumatoid arthritis (RA). Methods In a retrospective study, rheumatologic and periodontal conditions of 60 patients with RA who had been treated with conventional synthetic DMARD were evaluated before (baseline) and after 3 and 6 months of bDMARD therapy. After serum levels of anti-PPAD immunoglobulin G (IgG) were determined at baseline, the patients were respectively divided into two groups for high and low anti-PPAD IgG titers according to the median measurements. Genotypes at 8 functional single nucleotide polymorphisms (SNPs) related to RA were also determined. Results After 3 and 6 months of therapy, patients with low anti-PPAD IgG titers showed a significantly greater decrease in changes in the Disease Activity Score including 28 joints using C-reactive protein (DAS28-CRP) (P = 0.04 for both) and anti-cyclic citrullinated peptide (CCP) IgG levels (P = 0.03 and P = 0.04) than patients with high anti-PPAD IgG titers, although these parameter values were comparable at baseline. The anti-PPAD IgG titers were significantly positively correlated with changes in the DAS28-CRP (P = 0.01 for both) and the anti-CCP IgG levels (P = 0.02 for both) from baseline to 3 and 6 months later. A multiple regression analysis revealed a significantly positive association between the anti-PPAD IgG titers and changes in the DAS28-CRP after 6 months of bDMARD therapy (P = 0.006), after adjusting for age, gender, smoking, periodontal condition, and RA-related SNPs. Conclusion The serum IgG levels to PPAD affect the clinical response to bDMARD in patients with RA. PMID:27111223

  2. Revised sequence of the Porphyromonas gingivalis prtT cysteine protease/hemagglutinin gene: homology with streptococcal pyrogenic exotoxin B/streptococcal proteinase.

    PubMed Central

    Madden, T E; Clark, V L; Kuramitsu, H K

    1995-01-01

    The prtT gene from Porphyromonas gingivalis ATCC 53977 was previously isolated from an Escherichia coli clone possessing trypsinlike protease activity upstream of a region encoding hemagglutinin activity (J. Otogoto and H. Kuramitsu, Infect. Immun. 61;117-123, 1993). Subsequent molecular analysis of this gene has revealed that the PrtT protein is larger than originally reported, encompassing the hemagglutination region. Results of primer extension experiments indicate that the translation start site was originally misidentified. An alternate open reading frame of nearly 2.7 kb, which encodes a protein in the size range of 96 to 99 kDa, was identified. In vitro transcription-translation experiments confirm this size, and Northern (RNA) blot experiments indicate that the protease is translated from a 3.3-kb mRNA. Searching the EMBL protein database revealed that the amino acid sequence of the revised PrtT is similar to sequences of two related proteins from Streptococcus pyogenes. PrtT is 31% identical and 73% similar over 401 amino acids to streptococcal pyrogenic exotoxin B. In addition, it is 36% identical and 74% similar over 244 amino acids with streptococcal proteinase, which is closely related to streptococcal pyrogenic exotoxin B. The similarity is particularly high at the putative active site of streptococcal proteinase, which is similar to the active sites of the family of cysteine proteases. Thus, we conclude that PrtT is a 96- to 99-kDa cysteine protease and hemagglutinin with significant similarity to streptococcal enzymes. PMID:7806362

  3. Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis-induced salivary gland acinar cell apoptosis.

    PubMed

    Slomiany, B L; Slomiany, A

    2010-06-01

    Recent advances in identifying the salivary constituents capable of influencing the oral mucosal inflammatory responses have brought to focus the importance of a peptide hormone, ghrelin. Here, we report on the involvement of ghrelin in controlling the apoptotic processes induced in sublingual salivary gland acinar cells by the lipopolysaccharide (LPS) of a periodontopathic bacterium, Porphyromonas gingivalis. We show that the countering effect of ghrelin on the LPS-induced acinar cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (iNOS). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, but not the iNOS inhibitor, 1400W. The effect of ghrelin on the LPS-induced changes in cNOS activity, moreover, was reflected in the increased cNOS phosphorylation that was sensitive to PP2 as well as SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by SH-5 and L-NAME. The findings point to the involvement of ghrelin in Src/Akt kinase-mediated cNOS activation and the apoptogenic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

  4. Periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum promote tumor progression in an oral-specific chemical carcinogenesis model.

    PubMed

    Binder Gallimidi, Adi; Fischman, Stuart; Revach, Brurya; Bulvik, Raanan; Maliutina, Alina; Rubinstein, Ariel M; Nussbaum, Gabriel; Elkin, Michael

    2015-09-01

    Oral squamous cell carcinoma (OSCC) is a lethal disease whose incidence is increasing. Epidemiologic studies demonstrate an association between periodontitis and oral cancer, and periodontal pathogens are implicated in the pathogenesis of numerous disorders, including rheumatoid arthritis, cardiovascular diseases, diabetes and gastrointestinal malignancies. Nevertheless, a causal role for periodontal pathogens in OSCC has not been shown, partly due to the lack of an appropriate animal model. Here, utilizing a newly-established murine model of periodontitis-associated oral tumorigenesis, we report that chronic bacterial infection promotes OSCC, and that augmented signaling along the IL-6-STAT3 axis underlies this effect. Our results indicate that periodontal pathogens P. gingivalis and F. nucleatum stimulate tumorigenesis via direct interaction with oral epithelial cells through Toll-like receptors. Furthermore, oral pathogens stimulate human OSCC proliferation and induce expression of key molecules implicated in tumorigenesis. To the best of our knowledge, these findings represent the first demonstration of a mechanistic role for oral bacteria in chemically induced OSCC tumorigenesis. These results are highly relevant for the design of effective prevention and treatment strategies for OSCC.

  5. A novel approach for purification and selective capture of membrane vesicles of the periodontopathic bacterium, Porphyromonas gingivalis: membrane vesicles bind to magnetic beads coated with epoxy groups in a noncovalent, species-specific manner.

    PubMed

    Nakao, Ryoma; Kikushima, Kenji; Higuchi, Hideo; Obana, Nozomu; Nomura, Nobuhiko; Bai, Dongying; Ohnishi, Makoto; Senpuku, Hidenobu

    2014-01-01

    Membrane vesicles (MVs) of Porphyromonas gingivalis are regarded as an offensive weapon of the bacterium, leading to tissue deterioration in periodontal disease. Therefore, isolation of highly purified MVs is indispensable to better understand the pathophysiological role of MVs in the progression of periodontitis. MVs are generally isolated by a conventional method based on ultracentrifugation of the bacterial culture supernatant. However, the resulting MVs are often contaminated with co-precipitating bacterial appendages sheared from the live bacteria. Here, we report an intriguing property of P. gingivalis MVs--their ability to bind superparamagnetic beads coated with epoxy groups (SB-Epoxy). Analysis of fractions collected during the purification revealed that all MVs of five tested P. gingivalis stains bound to SB-Epoxy. In contrast, free fimbriae in the crude MV preparation did not bind to the SB-Epoxy. The SB-Epoxy-bound MVs were easily dissociated from the SB-Epoxy using a mild denaturation buffer. These results suggest that the surface chemistry conferred by epoxy on the beads is responsible for the binding, which is mediated by noncovalent bonds. Both the structural integrity and purity of the isolated MVs were confirmed by electron microscopy. The isolated MVs also caused cell detachment from culture dishes at a physiologically relevant concentration. Assays of competitive binding between the SB-Epoxy and mixtures of MVs from five bacterial species demonstrated that only P. gingivalis MVs could be selectively eliminated from the mixtures. We suggest that this novel approach enables efficient purification and selective elimination of P. gingivalis MVs. PMID:24830438

  6. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis

    PubMed Central

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35–76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants’ health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0–0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1–57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0–88.2) and body mass index (OR = 3.0; 95%CI 1.7–5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is

  7. The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

    PubMed Central

    Aduse-Opoku, J; Slaney, J M; Rangarajan, M; Muir, J; Young, K A; Curtis, M A

    1997-01-01

    The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a

  8. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  9. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  10. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  11. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  12. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells.

    PubMed

    Slomiany, Bronislaw L; Slomiany, Amalia

    2010-01-01

    Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS). We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  13. A combination of both arginine- and lysine-specific gingipain activity of Porphyromonas gingivalis is necessary for the generation of the micro-oxo bishaem-containing pigment from haemoglobin.

    PubMed Central

    Smalley, John W; Thomas, Michael F; Birss, Andrew J; Withnall, Robert; Silver, Jack

    2004-01-01

    The black pigment of Porphyromonas gingivalis is composed of the mu-oxo bishaem complex of Fe(III) protoporphyrin IX (mu-oxo oligomer, dimeric haem), namely [Fe(III)PPIX]2O. P. gingivalis W50 and Rgp (Arg-gingipain)- and Kgp (Lys-gingipain)-deficient mutants K1A, D7, E8 and W501 [Aduse-Opoku, Davies, Gallagher, Hashim, Evans, Rangarajan, Slaney and Curtis (2000) Microbiology 146, 1933-1940] were grown on horse blood/agar for 14 days and examined for the production of mu-oxo bishaem. Mu-oxo Bishaem was detected by UV-visible, Mössbauer and Raman spectroscopies in wild-type W50 and in the black-pigmented RgpA- and RgpB-deficient mutants (W501 and D7 respectively), whereas no haem species were detected in the straw-coloured colonies of Kgp-deficient strain K1A. The dark brown pigment of the double RgpA/RgpB knockout mutant (E8) was not composed of mu-oxo bishaem, but of a high-spin monomeric Fe(III) protoporphyrin IX species (possibly a haem-albumin complex). In vitro incubation of oxyhaemoglobin with cells of the W50 strain and the RgpA- and RgpB-deficient mutants (W501 and D7) resulted in the formation of mu-oxo bishaem via methaemoglobin as an intermediate. Although the Kgp-deficient strain K1A converted oxyhaemoglobin into methaemoglobin, this was not further degraded into mu-oxo bishaem. The double RgpA/RgpB knockout was also not capable of producing mu-oxo bishaem from oxyhaemoglobin, but instead generated a haemoglobin haemichrome. Inhibition of Arg-X protease activity of W50, W501, D7 and K1A with leupeptin, under conditions where Lys-X protease activity was unaffected, prevented the production of mu-oxo bishaem from oxyhaemoglobin, but resulted in the formation of a haemoglobin haemichrome. These results show that one or both of RgpA and RgpB gingipains, in addition to the lysine-specific gingipain, is necessary for the production of mu-oxo bishaem from haemoglobin by whole cells of P. gingivalis. PMID:14741050

  14. Sulfonamide inhibition study of the carbonic anhydrases from the bacterial pathogen Porphyromonas gingivalis: the β-class (PgiCAb) versus the γ-class (PgiCA) enzymes.

    PubMed

    Prete, Sonia Del; Vullo, Daniela; Osman, Sameh M; Scozzafava, Andrea; AlOthman, Zeid; Capasso, Clemente; Supuran, Claudiu T

    2014-09-01

    The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrases (CAs, EC 4.2.1.1) one belonging to the γ-class (PgiCA) and another one to the β-class (PgiCAb). This last enzyme has been cloned and characterized here for its inhibition profile with the main class of CA inhibitors, the sulfonamides. Many of the clinically used sulfonamides as well as simple aromatic/heterocyclic sulfonamides were ineffective as PgiCAb inhibitors whereas better inhibition was observed with simple derivatives such as sulfanilamide, metanilamide, 4-aminoalkylbenzenesulfonamides (KIs of 364-475nM). The halogenosulfanilamides incorporating heavy halogens, 4-hydroxy- and 4-hydroxyalkyl-benzenesulfonamides, were also micromolar, ineffective PgiCAb inhibitors. The best inhibitors of the β-class enzyme were acetazolamide and ethoxzolamide, with KIs of 214-280nM. Interestingly, the γ-class enzyme was much more sensitive to sulfonamide inhibitors compared to the β-class one, PgiCAb. Identification of potent and possibly selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available. PMID:25129169

  15. Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

    PubMed Central

    White, P A; Wilson, M; Nair, S P; Kirby, A C; Reddi, K; Henderson, B

    1995-01-01

    The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP. PMID:7790076

  16. Extract from Rumex acetosa L. for Prophylaxis of Periodontitis: Inhibition of Bacterial In Vitro Adhesion and of Gingipains of Porphyromonas gingivalis by Epicatechin-3-O-(4β→8)-Epicatechin-3-O-Gallate (Procyanidin-B2-Di-Gallate)

    PubMed Central

    Schmuch, Jana; Beckert, Sabine; Brandt, Simone; Löhr, Gesine; Hermann, Fabian; Schmidt, Thomas J.; Beikler, Thomas; Hensel, Andreas

    2015-01-01

    Background The aerial parts of Rumex acetosa L. have been used in traditional European medicine for inflammatory diseases of the mouth epithelial tissue. The following study aimed to investigate the influence of a proanthocyanidin-enriched extract from R. acetosa extract against the adhesion of Porphyromonas gingivalis (P. gingivalis), a pathogen strongly involved in chronic and aggressive periodontitis. A further goal was to define the bioactive lead structures responsible for a potential antiadhesive activity and to characterize the underlying molecular mechanisms of the antiadhesive effects. Methodology An extract of R. acetosa (RA1) with a defined mixture of flavan-3-ols, oligomeric proanthocyanidins and flavonoids, was used. Its impact on P. gingivalis adhesion to KB cells was studied by flow cytometry, confocal laser scanning microscopy and in situ adhesion assay using murine buccal tissue. RA1 and its compounds 1 to 15 were further investigated for additional effects on gingipain activity, hemagglutination and gene expression by RT-PCR. Principal Findings RA1 (5 to 15 μg/mL) reduced P. gingivalis adhesion in a dose-dependent manner to about 90%. Galloylated proanthocyanidins were confirmed to be responsible for this antiadhesive effect with epicatechin-3-O-gallate-(4β,8)-epicatechin-3’-O-gallate (syn. procyanidin B2-di-gallate) being the lead compound. Ungalloylated flavan-3-ols and oligomeric proanthocyanidins were inactive. RA1 and the galloylated proanthocyanidins strongly interact with the bacterial virulence factor Arg-gingipain, while the corresponding Lys-gingipain was hardly influenced. RA1 inhibited also hemagglutination. In silico docking studies indicated that epicatechin-3-O-gallate-(4β,8)-epicatechin-3’-O-gallate interacts with the active side of Arg-gingipain and hemaglutinin from P. gingivalis; the galloylation of the molecule seems to be responsible for fixation of the ligand to the protein. In conclusion, the proanthocyanidin

  17. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  18. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections.

  19. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  20. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections.

    PubMed

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S; Somannavar, Pradeep D; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion . The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  1. Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen

    SciTech Connect

    Lantz, M.S.; Allen, R.D.; Bounelis, P.; Switalski, L.M.; Hook, M. )

    1990-02-01

    Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains.

  2. Bacteroides gingivalis and Bacteroides intermedius recognize different sites on human fibrinogen.

    PubMed Central

    Lantz, M S; Allen, R D; Bounelis, P; Switalski, L M; Hook, M

    1990-01-01

    Bacteroides (Porphyromonas) gingivalis and Bacteroides (Porphyromonas) intermedius have been implicated in the etiology of human periodontal diseases. These organisms are able to bind and degrade human fibrinogen, and these interactions may play a role in the pathogenesis of periodontal disease. In attempts to map the bacterial binding sites along the fibrinogen molecule, we have found that strains of B. gingivalis and B. intermedius, respectively, recognize spatially distant and distinct sites on the fibrinogen molecule. Isolated reduced and alkylated alpha-, beta-, and gamma-fibrinogen chains inhibited binding of 125I-fibrinogen to both Bacteroides species in a concentration-dependent manner. Plasmin fragments D and to some extent fragment E, however, produced a concentration-dependent inhibition of 125I-fibrinogen binding to B. intermedius strains but did not affect binding of 125I-fibrinogen to B. gingivalis strains. Radiolabeled fibrinogen chains and fragments were compared with 125I-fibrinogen with respect to specificity and reversibility of binding to bacteria. According to these criteria, gamma chain most closely resembled the native fibrinogen molecule in behavior toward B. gingivalis strains and fragments D most closely resembled fibrinogen in behavior toward B. intermedius strains. The ability of anti-human fibrinogen immunoglobulin G (IgG) to inhibit binding of 125I-fibrinogen to B. intermedius strains was greatly reduced by absorbing the IgG with fragments D. Absorbing the IgG with fragments D had no effect on the ability of the antibody to inhibit binding of 125I-fibrinogen to B. gingivalis strains. A purified staphylococcal fibrinogen-binding protein blocked binding of 125I-fibrinogen to B. intermedius strains but not to B. gingivalis strains. PMID:2404954

  3. Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis.

    PubMed

    O'Flynn, Ciaran; Deusch, Oliver; Darling, Aaron E; Eisen, Jonathan A; Wallis, Corrin; Davis, Ian J; Harris, Stephen J

    2015-12-01

    Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity. PMID:26568374

  4. Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis

    PubMed Central

    O’Flynn, Ciaran; Deusch, Oliver; Darling, Aaron E.; Eisen, Jonathan A.; Wallis, Corrin; Davis, Ian J.; Harris, Stephen J.

    2015-01-01

    Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity. PMID:26568374

  5. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria.

    PubMed

    Paster, B J; Dewhirst, F E; Olsen, I; Fraser, G J

    1994-02-01

    The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis. Approximately 95% of the 16S rRNA sequence was determined for 36 representative strains of species of Prevotella, Bacteroides, and Porphyromonas and related species by a modified Sanger sequencing method. A phylogenetic tree was constructed from a corrected distance matrix by the neighbor-joining method, and the reliability of tree branching was established by bootstrap analysis. The bacteroides subgroup was divided primarily into three major phylogenetic clusters which contained most of the species examined. The first cluster, termed the prevotella cluster, was composed of 16 species of Prevotella, including P. melaninogenica, P. intermedia, P. nigrescens, and the ruminal species P. ruminicola. Two oral species, P. zoogleoformans and P. heparinolytica, which had been recently placed in the genus Prevotella, did not fall within the prevotella cluster. These two species and six species of Bacteroides, including the type species B. fragilis, formed the second cluster, termed the bacteroides cluster. The third cluster, termed the porphyromonas cluster, was divided into two subclusters. The first contained Porphyromonas gingivalis, P. endodontalis, P. asaccharolytica, P. circumdentaria, P. salivosa, [Bacteroides] levii (the brackets around genus are used to indicate that the species does not belong to the genus by the sensu stricto definition), and [Bacteroides] macacae, and the second subcluster contained [Bacteroides] forsythus and [Bacteroides] distasonis. [Bacteroides] splanchnicus fell just outside the three major clusters but still belonged within the bacteroides subgroup. With few exceptions, the 16 S rRNA data were in overall agreement with previously proposed reclassifications of species of Bacteroides, Prevotella, and Porphyromonas. Suggestions are made to accommodate those species which do not

  6. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

    PubMed Central

    Kalmar, J R; Arnold, R R

    1988-01-01

    Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen. PMID:3417349

  7. The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

    PubMed

    Hall, E R; Falkler, W A; Martin, S A; Suzuki, J B

    1991-12-01

    The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients. PMID:1765941

  8. Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Shenker, B J; Kushner, M E; Tsai, C C

    1982-01-01

    We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis. PMID:7152684

  9. P. gingivalis in Periodontal Disease and Atherosclerosis – Scenes of Action for Antimicrobial Peptides and Complement

    PubMed Central

    Hussain, Mehak; Stover, Cordula M.; Dupont, Aline

    2015-01-01

    According to the NHS, it is estimated that over 50% of the adult population are, to some extent, affected by gum disease and approximately 15% of UK population have been diagnosed with severe periodontitis. Periodontitis, a chronic polymicrobial disease of the gums, causes inflammation in its milder form, whereas in its severe form affects the surrounding tissues and can result in tooth loss. During periodontitis, plaque accumulates and sits between the junctional epithelium and the tooth itself, resulting in inflammation and the formation of a periodontal pocket. An interface is formed directly between the subgingival bacteria and the junctional epithelial cells. Bacterial pathogens commonly associated with periodontal disease are, among others, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, together known as the “red complex.” This review will mostly concentrate on the role of P. gingivalis, a Gram-negative anaerobic bacterium and one of the major and most studied contributors of this disease. Because periodontal disease is associated with the development of atherosclerosis, it is important to understand the local immune response to P. gingivalis. Innate immune players, in particular, complement and antimicrobial peptides and their effects with regard to P. gingivalis during periodontitis and in the development of atherosclerosis will be presented. PMID:25713575

  10. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    PubMed Central

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  11. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  12. Soluble CD14 Enhances the Response of Periodontal Ligament Stem Cells to P. gingivalis Lipopolysaccharide

    PubMed Central

    Andrukhov, Oleh; Andrukhova, Olena; Özdemir, Burcu; Haririan, Hady; Müller-Kern, Michael; Moritz, Andreas; Rausch-Fan, Xiaohui

    2016-01-01

    Periodontal ligament stem cells (PDLSCs) are lacking membrane CD14, which is an important component of lipopolysaccharide (LPS) signaling through toll-like receptor (TLR) 4. In the present study we investigated the effect of soluble CD14 on the response of human PDLSCs to LPS of Porphyromonas (P.) gingivalis. Human PDLSCs (hPDLSCs) were stimulated with P. gingivalis LPS in the presence or in the absence of soluble CD14 (sCD14) and the production of interleukin (IL)-6, chemokine C-X-C motif ligand 8 (CXCL8), and chemokine C-C motif ligand 2 (CCL2) was measured. The response to P. gingivalis LPS was compared with that to TLR4 agonist Escherichia coli LPS and TLR2-agonist Pam3CSK4. The response of hPDLSCs to both P. gingivalis LPS and E. coli LPS was significantly enhanced by sCD14. In the absence of sCD14, no significant difference in the hPDLSCs response to two kinds of LPS was observed. These responses were significantly lower compared to that to Pam3CSK4. In the presence of sCD14, the response of hPdLSCs to P. gingivalis LPS was markedly higher than that to E. coli LPS and comparable with that to Pam3CSK4. The response of hPdLSCs to bacterial LPS is strongly augmented by sCD14. Local levels of sCD14 could be an important factor for modulation of the host response against periodontal pathogens. PMID:27504628

  13. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  14. Identification of Fur-regulated genes in Actinobacillus actinomycetemcomitans.

    PubMed

    Haraszthy, Violet I; Jordan, Shawn F; Zambon, Joseph J

    2006-03-01

    Actinobacillus actinomycetemcomitans is an oral pathogen that causes aggressive periodontitis as well as sometimes life-threatening, extra-oral infections. Iron regulation is thought to be important in the pathogenesis of A. actinomycetemcomitans infections and, consistent with this hypothesis, the fur gene has recently been identified and characterized in A. actinomycetemcomitans. In this study, 14 putatively Fur-regulated genes were identified by Fur titration assay (Furta) in A. actinomycetemcomitans, including afuA, dgt, eno, hemA, tbpA, recO and yfe - some of which are known to be Fur regulated in other species. A fur mutant A. actinomycetemcomitans strain was created by selecting for manganese resistance in order to study the Fur regulon. Comparisons between the fur gene sequences revealed that nucleotide 66 changed from C in the wild-type to T in the mutant strain, changing leucine to isoleucine. The fur mutant strain expressed a nonfunctional Fur protein as determined by Escherichia coli-based ferric uptake assays and Western blotting. It was also more sensitive to acid stress and expressed higher levels of minC than the wild-type strain. minC, which inhibits cell division in other bacterial species and whose regulation by iron has not been previously described, was found to be Fur regulated in A. actinomycetemcomitans by Furta, by gel shift assays, and by RT-qPCR assays for gene expression. PMID:16514158

  15. Entamoeba gingivalis in sputum smears.

    PubMed

    Dao, A H

    1985-01-01

    Entamoeba gingivalis is a common parasite of the human buccal cavity whose rare appearance in Papanicolaou-stained sputum smears may be missed. Two such cases are described, including the morphologic features of this ameba. The trophozoites were seen to phagocytize leukocytes as well as red blood cells, in distinction to E. histiolytica, which phagocytizes only red blood cells and also can cause pulmonary abscesses. The concomitant finding of Actinomyces sp. organisms in one patient reinforces the possible symbiotic relationship between the two organisms, as has been suggested for their appearance in other extraoral sites, such as the female genital tract. PMID:3861055

  16. Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans.

    PubMed

    Diaz, Roger; Ghofaily, Lourdes Al; Patel, Jigna; Balashova, Nataliya V; Freitas, Anna C; Labib, Irene; Kachlany, Scott C

    2006-02-01

    Actinobacillus actinomycetemcomitans is a Gram negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP), a rapidly progressing and severe disease of the oral cavity that affects predominantly adolescents. A. actinomycetemcomitans is also found in extraoral infections including infective endocarditis. As one of its many virulence determinants, A. actinomycetemcomitans produces the RTX (repeats in toxin) exotoxin, leukotoxin (LtxA). LtxA specifically kills leukocytes of humans and Old World Monkeys. All of our current knowledge of A. actinomycetemcomitans LtxA is based on the protein from strain JP2, a nonadherent laboratory isolate. Because laboratory isolates can lose virulence properties, we wished to examine LtxA from a clinical isolate, NJ4500. We show that localization patterns of LtxA do not differ between the strains. Subcellular localization studies with NJ4500 revealed that LtxA localizes to the outer membrane and that the interaction between LtxA and the surface of cells is specific. Surface localized LtxA was not removed with NaCl treatment and protease protection experiments revealed that approximately 10 kDa of LtxA is exposed. We purified secreted LtxA from NJ4500 and found that the specific activity of this toxin was greater than that of secreted LtxA from JP2. For other RTX toxins, fatty acid modification affects toxin activity, and A. actinomycetemcomitans LtxA is predicted to be modified. We show by two-dimensional gel electrophoresis that NJ4500 LtxA is more highly modified than JP2 LtxA, suggesting that the difference in activities could be due to differential modification. Studies of A. actinomycetemcomitans pathogenesis should therefore consider LtxA from clinical isolates.

  17. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    SciTech Connect

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M. )

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37{degree}C. A functional fibrinogen-binding component (M{sub r}, 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with {sup 125}I-fibrinogen. Fibrinogen degradation did not occur at 4{degree}C but did occur at 22 and 37{degree}C. When bacteria and iodinated fibrinogen were incubated at 37{degree}C, two major fibrinogen fragments (M{sub r}, 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M{sub r}, 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M{sub r}-120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-{alpha}-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

  18. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen.

    PubMed Central

    Lantz, M S; Allen, R D; Vail, T A; Switalski, L M; Hook, M

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate. Images PMID:1987144

  19. Laser antisepsis of Phorphyromonas gingivalis in vitro with dental lasers

    NASA Astrophysics Data System (ADS)

    Harris, David M.

    2004-05-01

    It has been shown that both pulsed Nd:YAG (1064nm) and continuous diode (810nm) dental lasers kill pathogenic bacteria (laser antisepsis), but a quantitative method for determining clinical dosimetry does not exist. The purpose of this study was to develop a method to quantify the efficacy of ablation of Porphyromonas gingivalis (Pg) in vitro for two different lasers. The ablation thresholds for the two lasers were compared in the following manner. The energy density was measured as a function of distance from the output of the fiber-optic delivery system. Pg cultures were grown on blood agar plates under standard anaerobic conditions. Blood agar provides an approximation of gingival tissue for the wavelengths tested in having hemoglobin as a primary absorber. Single pulses (Nd:YAG: 100- Œs diode: 100-msec) of laser energy were delivered to Pg colonies and the energy density was increased until the appearance of a small plume was observed coincident with a laser pulse. The energy density at this point defines the ablation threshold. Ablation thresholds to a single pulse were determined for both Pg and for blood agar alone. The large difference in ablation thresholds between the pigmented pathogen and the host matrix for pulsed-Nd:YAG represented a significant therapeutic ratio and Pg was ablated without visible effect on the blood agar. Near threshold the 810-nm diode laser destroyed both the pathogen and the gel. Clinically, the pulsed Nd:YAG may selectively destroy pigmented pathogens leaving the surrounding tissue intact. The 810-nm diode laser may not demonstrate this selectivity due to its longer pulse length and greater absorption by hemoglobin.

  20. Stability of the JP2 clone of Aggregatibacter actinomycetemcomitans.

    PubMed

    Haubek, D; Ennibi, O-K; Vaeth, M; Poulsen, S; Poulsen, K

    2009-09-01

    The JP2 clone of Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis. To obtain information about colonization dynamics of the JP2 clone, we used PCR to examine its presence in 365 Moroccan juveniles from whom periodontal plaque samples were collected at baseline and after one and two years. Periodontal attachment loss was measured at baseline and at the two-year follow-up. At baseline, 43 (12%) carriers of the JP2 clone were found. Nearly half (44 %) of these were persistently colonized with the clone. The relative risk for the development of aggressive periodontitis, adjusted for the concomitant presence of other genotypes of A. actinomycetemcomitans, was highest for individuals continuously infected by the JP2 clone (RR = 13.9; 95% CI, 9.0 to 21.4), indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis.

  1. An in-vitro evaluation of the efficacy of garlic extract as an antimicrobial agent on periodontal pathogens: A microbiological study

    PubMed Central

    Shetty, Sunaina; Thomas, Biju; Shetty, Veena; Bhandary, Rahul; Shetty, Raghavendra M.

    2013-01-01

    With the rise in bacterial resistance to antibiotics, there is considerable interest in the development of other classes of antimicrobials for the control of infection. Garlic (Allium sativum Linn.) has been used as medicine since ancient times and has long been known to have antibacterial, antifungal, and antiviral properties. This study was undertaken to assess the inhibitory effect of garlic on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, to assess the time-kill curve of P. gingivalis and A. actinomycetemcomitans, and to determine the antiproteolytic activity of garlic on P. gingivalis. Ethanolic garlic extract (EGE) and aqueous garlic extract (AGE) were prepared and the inhibitory effects of these extracts for two periodontal pathogens (P. gingivalis and A. actinomycetemcomitans) were tested. Antiproteolytic activity on protease of P. gingivalis was determined. 25 microliter (μl), 50 μl, and 75 μl of AGE showed 16 mm, 20 mm, and 25 mm zone of inhibition, respectively, on P. gingivalis. The AGE showed greater bacteriostatic activity against the P. gingivalis with minimum inhibitory concentration determined at 16.6 μl/ml. The time-kill assay of AGE and EGE were compared for P. gingivalis and A. actinomycetemcomitans. AGE showed better antiproteolytic activity on total protease of P. gingivalis compared to the EGE. Thus, the study concludes the antimicrobial activity of garlic extract against periodontal pathogens, P. gingivalis, A. actinomycetemcomitans. Its action against P. gingivalis includes inhibition of total protease activity, and this raises the possibility that garlic may have therapeutic use for periodontitis and possibly other oral infections. PMID:24695825

  2. Occurrence of periodontal pathogens among patients with chronic periodontitis

    PubMed Central

    Farias, B.C.; Souza, P.R.E.; Ferreira, B.; Melo, R.S.A.; Machado, F.B.; Gusmão, E.S.; Cimões, R.

    2012-01-01

    The aim of the present study was to evaluate the presence of the periodontal pathogens that form the red complex (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) and Aggregatibacter actinomycetemcomitans in patients with chronic periodontitis. The sample consisted of 29 patients with a clinical and radiographic diagnosis of chronic periodontitis based on the criteria of the American Academy of Periodontology (3). Samples for microbiological analysis were collected from the four sites of greatest probing depth in each patient, totaling 116 samples. These samples were processed using conventional polymerase chain reaction, which achieved the following positive results: 46.6% for P. gingivalis, 41.4% for T. forsythia, 33.6% for T. denticola and 27.6% for A. actinomycetemcomitans. P. gingivalis and T. forsythia were more prevalent (p < 0.05) in periodontal pockets ≥ 8 mm. The combinations T. forsythia + P. gingivalis (23.2%) and T. forsythia + P. gingivalis + T. denticola (20.0%) were more frequent in sites with a probing depth ≥ 8 mm. Associations with the simultaneous presence of A. actinomycetemcomitans + P. gingivalis, A. actinomycetemcomitans + T. forsythia, P. gingivalis + T. forsythia and T. forsythia + T. denticola were statistically significant (p < 0.05). It was concluded that the red complex pathogens are related to chronic periodontitis, presenting a higher occurrence in deep periodontal pockets. Moreover, the simultaneous presence of these bacteria in deep sites suggests a symbiotic relationship between these virulent species, favoring, in this way, a further progression of periodontal disease. PMID:24031906

  3. Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Serotype g Strain NUM4039 (JCM 30399)

    PubMed Central

    Saito, Masanori; Hirasawa, Masaaki; Kuwahara, Noriko; Okada, Tamami; Umezawa, Koji; Kobayashi, Taira; Okamoto, Masaaki; Naito, Mariko; Hirasawa, Masatomo

    2016-01-01

    Aggregatibacter actinomycetemcomitans is considered to be a major etiological agent of aggressive periodontitis and includes serotype a to g strains. We herein report the first complete genome sequence of A. actinomycetemcomitans serotype g strain NUM4039. The genome is 2,382,853 bp in length with a G+C content of 44.34%. PMID:26988057

  4. Mucosal Langerhans Cells Promote Differentiation of Th17 Cells in a Murine Model of Periodontitis but Are Not Required for Porphyromonas gingivalis–Driven Alveolar Bone Destruction

    PubMed Central

    Bittner-Eddy, Peter D.; Fischer, Lori A.; Kaplan, Daniel H.; Thieu, Kathleen

    2016-01-01

    Periodontitis is a chronic oral inflammatory disease affecting one in five individuals that can lead to tooth loss. CD4+ Th cells activated by a microbial biofilm are thought to contribute to the destruction of alveolar bone surrounding teeth by influencing osteoclastogenesis through IL-17A and receptor activator for NF-κB ligand effects. The relative roles of mucosal Ag presentation cells in directing Th cell immune responses against oral pathogens and their contribution to destruction of alveolar bone remain unknown. We tested the contribution of mucosal Langerhans cells (LCs) to alveolar bone homeostasis in mice following oral colonization with a well-characterized human periodontal pathogen, Porphyromonas gingivalis. We found that oral mucosal LCs did not protect from or exacerbate crestal alveolar bone destruction but were responsible for promoting differentiation of Th17 cells specific to P. gingivalis. In mice lacking LCs the Th17 response was suppressed and a Th1 response predominated. Bypassing LCs with systemic immunization of P. gingivalis resulted in a predominantly P. gingivalis–specific Th1 response regardless of whether LCs were present. Interestingly, we find that in vivo clonal expansion of P. gingivalis–specific Th cells and induced regulatory T cells does not depend on mucosal LCs. Furthermore, destruction of crestal alveolar bone induced by P. gingivalis colonization occurred regardless of the presence of mucosal LCs or P. gingivalis–specific Th17 cells. Our data indicate that both LCs and Th17 cells are redundant in contributing to alveolar bone destruction in a murine model of periodontitis. PMID:27402698

  5. Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.

    PubMed Central

    Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J

    1983-01-01

    The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be

  6. Oral Administration of P. gingivalis Induces Dysbiosis of Gut Microbiota and Impaired Barrier Function Leading to Dissemination of Enterobacteria to the Liver

    PubMed Central

    Nakajima, Mayuka; Arimatsu, Kei; Kato, Tamotsu; Matsuda, Yumi; Minagawa, Takayoshi; Takahashi, Naoki; Ohno, Hiroshi; Yamazaki, Kazuhisa

    2015-01-01

    Although periodontitis has been implicated as a risk factor for various systemic diseases, the precise mechanisms by which periodontitis induces systemic disease remain to be elucidated. We have previously revealed that repeated oral administration of Porphyromonas gingivalis elicits endotoxemia via changes in the gut microbiota of the ileum, and thereby induces systemic inflammation and insulin resistance. However, it is not clear to what extent a single administration of P. gingivalis could affect gut microbiota composition, gut barrier function, and subsequent influx of gut microbiota into the liver. Therefore, in the present study, C57BL/6 mice were orally administered P. gingivalis (strain W83) once and compared to sham-inoculated mice. The phylogenetic structure and diversity of microbial communities in the gut and liver were analyzed by pyrosequencing the 16S ribosomal RNA genes. Serum endotoxin activity was determined by a Limulus amebocyte lysate test. Gene expression in the intestine and expression of 16S rRNA genes in the blood and liver were examined by quantitative polymerase chain reaction. Administration of P. gingivalis significantly altered gut microbiota, with an increased proportion of phylum Bacteroidetes, a decreased proportion of phylum Firmicutes, and increased serum endotoxin levels. In the intestinal tissues, gene expression of tjp-1 and occludin, which are involved in intestinal permeability, were downregulated. Higher amounts of bacterial DNA were detected in the liver of infected mice. Importantly, changes in gut microbiota preceded systemic inflammatory changes. These results further support the idea that disturbance of the gut microbiota composition by orally derived periodontopathic bacteria may be a causal mechanism linking periodontitis and systemic disease. PMID:26218067

  7. Oral Administration of P. gingivalis Induces Dysbiosis of Gut Microbiota and Impaired Barrier Function Leading to Dissemination of Enterobacteria to the Liver.

    PubMed

    Nakajima, Mayuka; Arimatsu, Kei; Kato, Tamotsu; Matsuda, Yumi; Minagawa, Takayoshi; Takahashi, Naoki; Ohno, Hiroshi; Yamazaki, Kazuhisa

    2015-01-01

    Although periodontitis has been implicated as a risk factor for various systemic diseases, the precise mechanisms by which periodontitis induces systemic disease remain to be elucidated. We have previously revealed that repeated oral administration of Porphyromonas gingivalis elicits endotoxemia via changes in the gut microbiota of the ileum, and thereby induces systemic inflammation and insulin resistance. However, it is not clear to what extent a single administration of P. gingivalis could affect gut microbiota composition, gut barrier function, and subsequent influx of gut microbiota into the liver. Therefore, in the present study, C57BL/6 mice were orally administered P. gingivalis (strain W83) once and compared to sham-inoculated mice. The phylogenetic structure and diversity of microbial communities in the gut and liver were analyzed by pyrosequencing the 16S ribosomal RNA genes. Serum endotoxin activity was determined by a Limulus amebocyte lysate test. Gene expression in the intestine and expression of 16S rRNA genes in the blood and liver were examined by quantitative polymerase chain reaction. Administration of P. gingivalis significantly altered gut microbiota, with an increased proportion of phylum Bacteroidetes, a decreased proportion of phylum Firmicutes, and increased serum endotoxin levels. In the intestinal tissues, gene expression of tjp-1 and occludin, which are involved in intestinal permeability, were downregulated. Higher amounts of bacterial DNA were detected in the liver of infected mice. Importantly, changes in gut microbiota preceded systemic inflammatory changes. These results further support the idea that disturbance of the gut microbiota composition by orally derived periodontopathic bacteria may be a causal mechanism linking periodontitis and systemic disease. PMID:26218067

  8. In vitro activity of azithromycin compared with that of erythromycin against Actinobacillus actinomycetemcomitans.

    PubMed Central

    Pajukanta, R; Asikainen, S; Saarela, M; Alaluusua, S; Jousimies-Somer, H

    1992-01-01

    The in vitro susceptibility of Actinobacillus actinomycetemcomitans to azithromycin, a new macrolide antibiotic of a new class known as azalides, was compared with that of erythromycin by the agar dilution method on Mueller-Hinton Haemophilus test medium. Eighty-two A. actinomycetemcomitans strains, 79 recent clinical isolates obtained from 40 periodontally healthy or diseased subjects, and 3 type strains were included in the study. Erythromycin showed poor in vitro activity against A. actinomycetemcomitans. Azithromycin, however, was highly effective against A. actinomycetemcomitans: all strains were inhibited at 2.0 micrograms/ml. Azithromycin exhibited the best in vitro activity against the serotype a subpopulation of A. actinomycetemcomitans: 100% of the strains were inhibited at 1.0 micrograms/ml. The lowest MICs were, however, recorded by serotype b strains. Since azithromycin has favorable pharmacokinetic properties, including excellent distribution into tissues, it could be expected to pass into gingival crevicular fluid at levels sufficient to inhibit A. actinomycetemcomitans in vivo. Therefore, it is a good candidate for future clinical trials in A. actinomycetemcomitans-associated periodontitis. PMID:1329617

  9. Aggregatibacter actinomycetemcomitans accelerates atherosclerosis with an increase in atherogenic factors in spontaneously hyperlipidemic mice.

    PubMed

    Zhang, Tao; Kurita-Ochiai, Tomoko; Hashizume, Tomomi; Du, Yuan; Oguchi, Sumito; Yamamoto, Masafumi

    2010-07-01

    Cariogenic and periodontal pathogens are thought to be etiological factors in the development of cardiovascular disease. We assessed the involvement of the periodontal pathogen Aggregatibacter actinomycetemcomitans and cariogenic pathogen Streptococcus mutans in the development of atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. The mice were treated intravenously with A. actinomycetemcomitans HK1651, S. mutans GS-5, or phosphate-buffered saline three times a week for 3 weeks and killed at 15 weeks of age. The areas of the aortic sinus that were covered with atherosclerotic plaque were significantly larger in Apoe(shl) mice challenged with A. actinomycetemcomitans compared with S. mutans- or vehicle-challenged mice. Aggregatibacter actinomycetemcomitans challenge increased serum high-sensitive C-reactive protein and lipopolysaccharide levels. Bacterial DNA was detected in the blood, heart, and spleen, but not in the liver. Furthermore, serum interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, and MCP-1 levels and Toll-like receptor (TLR)2, TLR4, ICAM-1, E-selectin, P-selectin, LOX-1, HSP60, CCL19, CCL21, CCR7, and MCP-1 expressions in the aorta were significantly increased in mice challenged with A. actinomycetemcomitans. These results suggest that systemic infection with A. actinomycetemcomitans accelerates atherosclerosis in Apoe(shl) mice by exposing the whole microorganisms or their products, followed by initiating inflammation. Increases in proatherogenic factors may explain the aggravation of atherosclerosis by A. actinomycetemcomitans infection. PMID:20482627

  10. LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

    PubMed

    Sol, Asaf; Ginesin, Ofir; Chaushu, Stella; Karra, Laila; Coppenhagen-Glazer, Shunit; Ginsburg, Isaac; Bachrach, Gilad

    2013-10-01

    Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

  11. Mature Biofilm Degradation by Potential Probiotics: Aggregatibacter actinomycetemcomitans versus Lactobacillus spp.

    PubMed Central

    Mizuno, Kouhei; Okinaga, Toshinori

    2016-01-01

    The biofilm degradation of Aggregatibacter actinomycetemcomitans is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as Lactobacillus spp. for the biofilm of several serotypes of A. actinomycetemcomitans strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in A. actinomycetemcomitans Y4 (serotype b) as well as four of the seven species for the biofilm of A. actinomycetemcomitans OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of A. actinomycetemcomitans SUNY 75 (serotype a) biofilm. The dispersed A. actinomycetemcomitans Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of A. actinomycetemcomitans Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of A. actinomycetemcomitans strains. On the other hand, the biofilm of A. actinomycetemcomitans SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics. PMID:27438340

  12. Immunosuppressive factor from Actinobacillus actinomycetemcomitans down regulates cytokine production.

    PubMed Central

    Kurita-Ochiai, T; Ochiai, K

    1996-01-01

    A cytoplasmic soluble fraction of Actinobacillus actinomycetemcomitans Y4 was isolated and characterized as suppressing mitogen-stimulated proliferation of and cytokine production by C3H/HeN mouse splenic T cells. This factor, designated suppressive factor 1 (SF1), was isolated from the supernatant of sonicated whole bacteria and purified by Q-Sepharose Fast Flow column chromatography, DEAE-Sepharose Fast Flow column chromatography, hydroxyapatite high-pressure liquid chromatography (HPLC), and Protein Pack 300 & 125 gel filtration HPLC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified SF1 migrated as a single band corresponding to a molecular mass of 14 kDa. This molecule was protease labile, heat resistant, and noncytotoxic. N'-terminal sequence analysis revealed no homology with any known peptides of periodontopathic bacteria or with any host-derived growth factors. Purified SF1 suppressed the proliferation of mouse splenic T cells which had been stimulated with concanavalin A, as well as suppressing the production of interleukin-2 (IL-2), gamma interferon, IL-4, and IL-5 from CD4+ T cells as 0.1 microgram/ml or more. These data suggest that SF1 produced by the periodontal pathogen A. actinomycetemcomitans functions as a virulence factor by down regulating T-cell proliferation and cytokine production at local defense sites. PMID:8557373

  13. A longitudinal microbiological investigation of Actinobacillus actinomycetemcomitans and Eikenella corrodens in juvenile periodontitis.

    PubMed Central

    Mandell, R L

    1984-01-01

    Longitudinal clinical and microbiological monitoring of subjects with localized juvenile periodontitis indicated that Actinobacillus actinomycetemcomitans and Eikenella corrodens were significantly associated (P less than 0.05) with active tissue destruction. PMID:6381313

  14. Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Miyasaki, K T; Zambon, J J; Jones, C A; Wilson, M E

    1987-01-01

    The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans. PMID:3032796

  15. Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

    PubMed

    Park, Se-Ra; Kim, Dong-Jae; Han, Seung-Hyun; Kang, Min-Jung; Lee, Jun-Young; Jeong, Yu-Jin; Lee, Sang-Jin; Kim, Tae-Hyoun; Ahn, Sang-Gun; Yoon, Jung-Hoon; Park, Jong-Hwan

    2014-05-01

    Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.

  16. Conjugal transfer of broad-host-range incompatibility group P and Q plasmids from Escherichia coli to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Goncharoff, P; Yip, J K; Wang, H; Schreiner, H C; Pai, J A; Furgang, D; Stevens, R H; Figurski, D H; Fine, D H

    1993-01-01

    The first example of conjugal transfer of DNA from Escherichia coli to the periodontal pathogen Actinobacillus actinomycetemcomitans is presented. Derivatives of the incompatibility group P (IncP) plasmid RK2 successfully transferred from an E. coli donor to an A. actinomycetemcomitans recipient. The resulting A. actinomycetemcomitans transconjugants transferred the plasmids back to E. coli recipients. The IncP transfer functions were also used in trans to mobilize the IncQ plasmid pBK1 from E. coli to A. actinomycetemcomitans. The IncP and IncQ plasmids both transferred into A. actinomycetemcomitans at high frequencies (0.3 to 0.5 transconjugants per donor) and showed no gross deletions, insertions, or rearrangements. Determinations of MICs of various antibiotics for the A. actinomycetemcomitans transconjugant strains demonstrated the expression of ampicillin, chloramphenicol, and kanamycin resistance determinants. Images PMID:8335386

  17. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed

    Braunthal, S D; Holt, S C; Tanner, A C; Socransky, S S

    1980-06-01

    Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content. PMID:7430333

  18. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed Central

    Braunthal, S D; Holt, S C; Tanner, A C; Socransky, S S

    1980-01-01

    Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content. PMID:7430333

  19. Actinobacillus actinomycetemcomitans contamination of toothbrushes from patients harbouring the organism.

    PubMed

    Müller, H P; Lange, D E; Müller, R F

    1989-07-01

    The main ecological niche of Actinobacillus actinomycetemcomitans (A.a.) seems to be the periodontal pocket, but it can also be isolated from supragingival plaque, buccal and tongue mucosa, or saliva. We examined toothbrushes from 21 patients, all identified as harbouring moderate to large numbers of A.a. in subgingival plaque, for contamination with this organism. 29% of the toothbrushes presented by our patients yielded detectable numbers of A.a. Immediately after toothbrushing this figure rose to 62%, but dropped to 50% after 1 h. Numbers of isolated A.a. on toothbrushes were weakly correlated with the degree of periodontal destruction, and significantly more numbers of A.a. on toothbrushes could be detected if the organism was found on mucous membranes or in saliva. There was no association with gingival inflammation, supragingival plaque nor mean numbers of isolated subgingival A.a. PMID:2760252

  20. Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis

    PubMed Central

    Kadkhoda, Zeinab; Amarlu, Zeinab; Eshraghi, Saeed; Samiei, Nazanin

    2016-01-01

    Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion. Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites.

  1. Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis

    PubMed Central

    Gaetti-Jardim Jr., Elerson; Wahasugui, Thais Cristiane; Tomazinho, Paulo Henrique; Marques, Márcia Martins; Nakano, Viviane; Avila-Campos, Mario Julio

    2008-01-01

    Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases. PMID:24031284

  2. Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis.

    PubMed

    Kadkhoda, Zeinab; Amarlu, Zeinab; Eshraghi, Saeed; Samiei, Nazanin

    2016-01-01

    Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion . Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites. PMID:27651884

  3. Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis

    PubMed Central

    Kadkhoda, Zeinab; Amarlu, Zeinab; Eshraghi, Saeed; Samiei, Nazanin

    2016-01-01

    Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion. Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites. PMID:27651884

  4. Frequency of Entamoeba gingivalis in human gingival scrapings.

    PubMed

    Dao, A H; Robinson, D P; Wong, S W

    1983-09-01

    A survey was made of gingival scrapings stained by the Papanicolaou method to assess the occurrence of Entamoeba gingivalis, a nonpathogenic-oral amoeba. Positive findings were recorded in 59% of 113 dental patients, and 32% of 96 healthy controls. These figures showed no significant changes during the last 20 years when compared with data published in 1960 and 1963. The existence of E. gingivalis and its rare appearance in the sputum should be known to cytologists because of the morphologic resemblance to Entamoeba histolytica, a pathogenic amoeba. Morphologic features are described to differentiate E. gingivalis from similar structures found in sputum. PMID:6881102

  5. Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans

    PubMed Central

    Tan, Kai Soo; Ong, Grace; Song, Keang Peng

    2005-01-01

    In eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria. Actinobacillus actinomycetemcomitans is a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within the cdt gene of A. actinomycetemcomitans. By use of reverse transcription-PCR, cdt transcripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carrying cdt was cloned into Escherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of the cdt RNA was located within the cdtC gene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in both cis and trans configurations. PMID:15629928

  6. Investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a “Red complex” by a multiplex PCR method

    PubMed Central

    Sanghavi, Tulsi Hasmukhrai; Shah, Nimisha; Shah, Ruchi Rani; Sanghavi, Akta

    2014-01-01

    Aim: The aim of this study was to investigate the correlation between endodontic clinical signs and symptoms and the presence of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia or their association by Multiplex polymerase chain reaction assay. Materials and Methods: Microbial samples were taken from 30 cases with necrotic pulp tissues in primary infections. DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens by using species-specific primers. Results: P. gingivalis, T. denticola, T. forsythia, and Red Complex were present in 11, 17, 4, and 2 canals, respectively. Clinical and statistically significant relationships were found between T. forsythia and mobility and between T. denticola and swelling. (P < 0.05). Presence of other Red complex bacteria shows clinical association with presence of signs and symptoms but no statistically significant relationship. Conclusion: The high prevalence of P. gingivalis, T. denticola, and T. forsythia in the examined samples suggests that these bacteria are related to the etiology of symptomatic periradicular diseases. PMID:25506144

  7. First Human Case of Fatal Halicephalobus gingivalis Meningoencephalitis in Australia

    PubMed Central

    Crawford, April; Moore, Casey V.; Gasser, Robin B.; Nelson, Renjy; Koehler, Anson V.; Bradbury, Richard S.; Speare, Rick; Dhatrak, Deepak; Weldhagen, Gerhard F.

    2015-01-01

    Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool. PMID:25694532

  8. Periodontal pathogens in erupting third molars of periodontally healthy subjects.

    PubMed

    Rajasuo, A; Sihvonen, O J; Peltola, M; Meurman, J H

    2007-09-01

    The presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythensis in bacteriologic samples of 5-7-mm deep mandibular third-molar pericoronal pockets was analysed by polymerase chain reaction, to test the hypothesis that these sites would harbour the bacteria. The patients were periodontally healthy 20-year-old Finnish male conscripts. Sixteen had acute pericoronitis, 28 chronic pericoronitis, and 15 were symptom-free controls. A. actinomycetemcomitans was detected in only 7% of the samples from chronic pericoronitis cases, whereas P. gingivalis was positive in 20% of the symptom-free versus 69% (P = 0.018) of the acute and 57% (P = 0.044) of the chronic cases. The percentages for P. intermedia were 93, 94 and 93%, and for T. forsythensis 47, 63 and 57%, respectively. These results confirm that, apart from A. actinomycetemcomitans, periodontopathogens are common in third-molar sites in periodontally healthy individuals.

  9. Expression cloning of a periodontitis-associated apoptotic effector, cagE homologue, in Actinobacillus actinomycetemcomitans.

    PubMed

    Teng, Yen-Tung A; Hu, Wenqi

    2003-04-18

    To study anti-bacterial immunity and to identify critical bacterial antigens associated with specific periodontal infection, we screened the genomic library of Actinobacillus actinomycetemcomitans, a major Gram(-) anaerobe causing human periodontitis, by expression cloning using disease-associated periodontal CD4(+)T cells derived from HuPBL-engrafted NOD/SCID mice. Here, we report one of the novel genes identified and designated, cagE homologue (in short: cagE) of A. actinomycetemcomitans, which encodes a putative bacterial type IV secretion system with significant homology to Helicobacter pylori CagE and Agrobacterium tumefaciens VirB4. All serum samples from A. actinomycetemcomitans-infected periodontitis patients, but not from the healthy controls, readily recognized CagE by ELISA and Western blot, suggesting its biological and clinical significance. The CagE protein, upon secretion, elicited significant apoptosis on primary human epithelia, endothelia, osteoblasts, and T cells by 4-12h in vitro. Importantly, both cagE(-) mutant strain and N-terminus truncated CagE protein drastically reduced (p<0.001) the induction of apoptosis on human epithelia in vitro. These data strongly suggest that a novel effector protein, CagE in A. actinomycetemcomitans, induces apoptosis of human cells and destructive immunity, thereby it may play an important role in the pathogenesis of A. actinomycetemcomitans-mediated infections. PMID:12684047

  10. Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

    PubMed Central

    Brown, Angela C.; Boesze-Battaglia, Kathleen; Du, Yurong; Stefano, Frank P.; Kieba, Irene R.; Epand, Raquel F.; Kakalis, Lazaros; Yeagle, Philip L.; Epand, Richard M.; Lally, Edward T.

    2012-01-01

    Summary The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization. PMID:22309134

  11. A molecular survey of S. mutans and P. gingivalis oral microbial burden in human saliva using Relative Endpoint Polymerase Chain Reaction (RE-PCR) within the population of a Nevada dental school revealed disparities among minorities

    PubMed Central

    2012-01-01

    Background The University of Nevada, Las Vegas School of Dental Medicine recently opened an orthodontic treatment clinic to address the needs of the racially and ethnically diverse population of Southern Nevada, primarily focusing on the treatment and care of low-income and minority patients. Although orthodontic treatment and therapy has been shown to induce changes in the oral cavity, much of this evidence was collected from traditional White, teenage orthodontic clinic populations. The primary goal of this study was to describe the microbial burden of the cariogenic and periodontal pathogens, Streptococcus mutans and Porphyromonas gingivalis within the UNLV-SDM patient population. Methods Representative saliva samples were collected from healthy adult patients for DNA isolation. Relative endpoint polymerase chain reaction (RE-PCR) was performed to ascertain the presence and relative microbial burden of these oral pathogens. Results Nearly one quarter (13/56) or 23.3% of these patients had elevated levels of S. mutans, while (10/56) and 17.8% of these samples were found to have elevated levels of P. gingivalis, - with (90%) of P. gingivalis-positive samples from minority patients (X2 = 17.921, d.f. = 1; p < 0.0001). Conclusions These findings of elevated P. gingivalis levels, primarily among minority patients, may suggest underlying oral health practices contributing to adverse oral health conditions within this population. Oral health knowledge and practices among minority patients may be strongly influenced by other factors, including education and socioeconomic status, suggesting additional research may be needed to accurately determine the most appropriate standards for care and oral health education within this patient population. PMID:22925755

  12. A Consortium of Aggregatibacter actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis Is Present in Sites Prior to Bone Loss in a Longitudinal Study of Localized Aggressive Periodontitis

    PubMed Central

    Markowitz, Kenneth; Fairlie, Karen; Tischio-Bereski, Debbie; Ferrendiz, Javier; Furgang, David; Paster, Bruce J.; Dewhirst, Floyd E.

    2013-01-01

    Aggregatibacter actinomycetemcomitans-induced localized aggressive periodontitis (LAP) in African-American adolescents has been documented but is poorly understood. Two thousand fifty-eight adolescents aged 11 to 17 years were screened for their periodontal status and the presence of A. actinomycetemcomitans in their oral cavity. Seventy-one A. actinomycetemcomitans-negative and 63 A. actinomycetemcomitans-positive periodontally healthy subjects were enrolled, sampled, examined, and radiographed yearly for 3 years. Gingival and periodontal pocket depth and attachment levels were recorded. Disease presentation was characterized by bone loss (BL). Subgingival sites were sampled every 6 months to assess (i) the role of A. actinomycetemcomitans in BL and (ii) the association of A. actinomycetemcomitans and other microbes in their relationships to BL. Sixteen of 63 subjects with A. actinomycetemcomitans developed BL (the other 47 subjects with A. actinomycetemcomitans had no BL). No A. actinomycetemcomitans-negative subjects developed BL. Human oral microbe identification microarray (HOMIM) was used for subgingival microbial assessment. On a subject level, pooled data from A. actinomycetemcomitans-positive subjects who remained healthy had higher prevalences of Streptococcus and Actinomyces species, while A. actinomycetemcomitans-positive subjects with BL had higher prevalences of Parvimonas micra, Filifactor alocis, A. actinomycetemcomitans, and Peptostreptococcus sp. human oral taxon 113 (HOT-113). At vulnerable sites, A. actinomycetemcomitans, Streptococcus parasanguinis, and F. alocis levels were elevated prior to BL. In cases where the three-organism consortium (versus A. actinomycetemcomitans alone) was detected, the specificity for detecting sites of future BL increased from 62% to 99%, with a sensitivity of 89%. We conclude that detecting the presence of A. actinomycetemcomitans, S. parasanguinis, and F. alocis together indicates sites of future BL in LAP. A

  13. Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β

    PubMed Central

    Paino, Annamari; Tuominen, Heidi; Jääskeläinen, Mari; Alanko, Jonna; Nuutila, Jari; Asikainen, Sirkka E.; Pelliniemi, Lauri J.; Pöllänen, Marja T.; Chen, Casey; Ihalin, Riikka

    2011-01-01

    Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation. PMID:21533109

  14. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  15. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  16. An outbreak of bovine meningoencephalomyelitis with identification of Halicephalobus gingivalis.

    PubMed

    Enemark, Heidi Larsen; Hansen, Mette Sif; Jensen, Tim Kåre; Larsen, Gitte; Al-Sabi, Mohammad Nafi Solaiman

    2016-03-15

    Halicephalobus gingivalis is an opportunistic parasite which is known to cause fatal meningoencephalomyelitis primarily in equines but sporadically also in humans. In April 2014, laboratory examination of the head of a young dairy calf, euthanized due to severe central nervous system symptoms, revealed the presence of granulomatous to necrotizing encephalitis and myriads of nematodes in the brain lesion. Morphologically the parasites were identified as H. gingivalis. The diagnosis was confirmed by molecular analysis of the large subunit (LSU) rRNA and the small subunit (SSU) rRNA genes, revealing genetic variations of 0.5-4.4% and 0.7-8.6%, respectively, between the H. gingivalis isolated from the Danish calf and published isolates, collected worldwide from free-living and parasitic stages of the nematode. Clinical symptoms and histological changes indicated infection with H. gingivalis from another three calves in the herd. This is the first scientific publication of H. gingivalis induced meningoencephalomyelitis in ruminants. As ante mortem diagnosis is a major challenge, the infection may easily remain undiagnosed in cattle. PMID:26872932

  17. Aggregatibacter actinomycetemcomitans induces Th17 cells in atherosclerotic lesions.

    PubMed

    Jia, Ru; Hashizume-Takizawa, Tomomi; Du, Yuan; Yamamoto, Masafumi; Kurita-Ochiai, Tomoko

    2015-04-01

    Th17 cells have been linked to the pathogenesis of several chronic inflammatory and autoimmune diseases. However, the role of Th17 cells and IL-17 in atherosclerosis remains poorly understood. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) bacteremia accelerated atherosclerosis accompanied by inflammation in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. In this study, we investigated whether Aa promotes the Th17 inducing pathway in Aa-challenged Apoe(shl) mice. Mice were intravenously injected with live Aa HK1651 or vehicles. Time-course analysis of splenic IL-17(+)CD4(+) cell frequencies, the proximal aorta lesion area, serum IL-17, IL-6, TGF-β and IL-1β levels, the mRNA expression of Th17-related molecules such as IL-1β, IL-6, IL17RA, STAT3, IL-21, IL-23, TGF-β and RORγt, Th17-related microRNA levels and the levels of AIM-2, Mincle and NLRP3 were examined. Challenge with Aa time dependently induced tropism of Th17 cells in the spleen and increase in atheromatous lesions in the aortic sinus of Apoe(shl) mice. Serum IL-17, IL-6, TGF-β and IL-1β levels were significantly enhanced by Aa. The gene expression of IL-1β, IL-6, IL-17RA, IL-21, IL-23, TGF-β, STAT3, RORγt, AIM-2, Mincle and NLRP3 was also time dependently stimulated in the aorta of Aa-challenged mice. Furthermore, Aa challenge significantly increased the expression of miR-146b and miR-155 in the aorta. Based on the results, it seems that Aa stimulates Th17 induction that affects the progression of Aa-accelerated atherosclerosis. PMID:25743474

  18. Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

    PubMed

    Stevens, Roy H; de Moura Martins Lobo Dos Santos, Caroline; Zuanazzi, David; de Accioly Mattos, Marcelo Barbosas; Ferreira, Davis Fernandes; Kachlany, Scott C; Tinoco, Eduardo M B

    2013-01-01

    Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens. PMID:23022667

  19. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss. PMID:26817919

  20. Virulence of Aggregatibacter actinomycetemcomitans serotypes and DGGE subtypes isolated from chronic adult periodontitis in Thailand.

    PubMed

    Pahumunto, Nuntiya; Ruangsri, Praphansri; Wongsuwanlert, Mutita; Piwat, Supatcharin; Dahlen, Gunnar; Teanpaisan, Rawee

    2015-12-01

    A high proportion of non-serotypeable isolates of Aggregatibacter actinomycetemcomitans among Thai periodontitis cases has been previously reported. The aim of this study was to investigate the expression of leukotoxin and toxicity, cytolethal distending toxin (Cdts), and internalization and the killing effect on fibroblasts by A. actinomycetemcomitans subtypes from Thai chronic periodontitis cases. A total of 96 A. actinomycetemcomitans strains from 37 periodontitis cases, previously serotyped with PCR and subtyped with DGGE, were examined for the presence of the ltx gene and cdt genes (cdtBC), and tested for leukotoxin expression, leukotoxicity, internalization, and apoptosis of fibroblast cells. The ltx gene was present in all isolates, while 84.4% showed the cdtBC gene. Two strains with a JP2-like ltx gene with a deletion of 530 bp in the promoter region, serotyped as c, showed virulence of similar magnitude to the JP2 strain. Furthermore, a higher virulence was found in the two non-serotypeable DGGE subtypes, NS1 and NS2, compared with the serotypeable strains (serotype a-f, serotype b and d were absent). Generally, the virulence of strains obtained from deep periodontal pockets was higher than those isolated from shallow non-bleeding pockets. A. actinomycetemcomitans subtypes isolated from adult Thais with chronic periodontitis showed a highly variable virulence, leukotoxin expression, leukotoxicity, internalization and apoptosis of fibroblast, and are regulated both genetically and environmentally. PMID:26529053

  1. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss.

  2. Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis.

    PubMed Central

    Han, N; Hoover, C I; Winkler, J R; Ng, C Y; Armitage, G C

    1991-01-01

    To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies. Images PMID:1761677

  3. Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis.

    PubMed

    Ohguchi, M; Ishisaki, A; Okahashi, N; Koide, M; Koseki, T; Yamato, K; Noguchi, T; Nishihara, T

    1998-12-01

    We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue. PMID:9826381

  4. Aggregatibacter actinomycetemcomitans arcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite

    PubMed Central

    Longo, PL; Ota-Tsuzuki, C; Nunes, ACR; Fernandes, BL; Mintz, K; Fives-Taylor, P; Mayer, MPA

    2009-01-01

    The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces. PMID:24031399

  5. Nonspecific Adherence by Actinobacillus actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea

    PubMed Central

    Kachlany, Scott C.; Planet, Paul J.; Bhattacharjee, Mrinal K.; Kollia, Evyenia; DeSalle, Rob; Fine, Daniel H.; Figurski, David H.

    2000-01-01

    The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903φkan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad− mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad

  6. Azithromycin Enhances Phagocytic Killing of Aggregatibacter actinomycetemcomitans Y4 by Human Neutrophils

    PubMed Central

    Lai, Pin-Chuang; Schibler, Mark R.; Walters, John D.

    2015-01-01

    Background Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. Methods Killing assays were conducted in the presence of either 2 μg/mL AZM or 16 μg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. Results Neutrophil incubation with 2 μg/mL AZM yielded an intracellular concentration of 10 μg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60- and 90-minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60- and 90-minute incubations (P <0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. Conclusions Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria. PMID:25186779

  7. Iron-Chelating Activity of Tetracyclines and Its Impact on the Susceptibility of Actinobacillus actinomycetemcomitans to These Antibiotics

    PubMed Central

    Grenier, Daniel; Huot, Marie-Pierre; Mayrand, Denis

    2000-01-01

    Three tetracyclines (tetracycline, doxycycline, and minocycline) were found to possess iron-chelating activity in a colorimetric siderophore assay. Determination of MICs indicated that the activity of doxycycline against the periodontopathogen Actinobacillus actinomycetemcomitans was only slightly influenced by the presence of an excess of iron that likely saturates the antibiotic. On the other hand, the MICs of doxycycline and minocycline were significantly lower for A. actinomycetemcomitans cultivated under iron-poor conditions than under iron-rich conditions. PMID:10681353

  8. C-Terminal Domain Residues Important for Secretion and Attachment of RgpB in Porphyromonas gingivalis▿

    PubMed Central

    Slakeski, Nada; Seers, Christine A.; Ng, Kaiting; Moore, Caroline; Cleal, Steven M.; Veith, Paul D.; Lo, Alvin W.; Reynolds, Eric C.

    2011-01-01

    Porphyromonas gingivalis, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a P. gingivalis host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur. PMID:20971915

  9. [Microbiological approach to a possible infective endocarditis case caused by Aggregatibacter actinomycetemcomitans].

    PubMed

    Gürcan, Şaban; Ünlü, Selahattin; Kuloğlu, Figen; Karadenizli, Aynur; Kuşkucu, Mert Ahmet

    2016-04-01

    Aggregatibacter (Actinobacillus) actinomycetemcomitans, a small, gram-negative coccobacillus that grows slow and fastidious, is generally colonized in the oral cavity. It is a rarely seen bacterium because of the difficulty of isolation but it can be a causative agent for dental infections and infective endocarditis (IE) particularly in the persons having prosthetic heart valves. In this report, a possible IE case caused by A.actinomycetemcomitans in a patient with aortic valve replacement has been presented. A 36-year-old man has admitted to Trakya University Hospital, Health Center for Medical Research and Practice, with the complaints of chills, malaise, intermittent fever, severe arthralgia and weight loss (20 kg). During his follow-up period, the blood cultures that were obtained three week intervals yielded the identical gram-negative coccobacilli morphology. The patient was then diagnosed as possible IE on the basis of having one major (growth of the typical microorganisms that may cause IE in two different blood cultures) and two minor (presence of prosthetic valve and high fever) criterias. The isolate could not be identified with conventional methods, while it was identified as Francisella tularensis with VITEK 2 (bioMerieux, France) system. Hence this identification was not confirmed by real-time Taqman polymerase chain reaction, so MALDI-TOF mass spectrometry was used to identify this bacteria. In the first run of the study, the isolate was named as Shigella dysenteriae initially, however when it was retested the next day it was identified as A.actinomycetemcomitans. In order to enlighten these conflicting results, 16S and 23S ribosomal DNA sequence analysis was performed, and consequently the bacterium was identified as A.actinomycetemcomitans. Doxycycline (2 x 100 mg po, 20 days) and streptomycin (2 x 10 mg/kg im, 10 days) therapy were initiated, considering the initial suspicious identification (F.tularensis), and on the fifth day of therapy the

  10. Detection and identification of Entamoeba gingivalis by specific amplification of rRNA gene.

    PubMed

    Kikuta, N; Yamamoto, A; Goto, N

    1996-12-01

    A pair of oligonucleotide primers were designed from the nucleotide sequence of the gene encoding the small subunit ribosomal RNA (SrRNA) of the oral protozoan parasite Entamoeba gingivalis. The primers amplified a 1.4-kb DNA fragment by polymerase chain reaction and were specific for Entamoeba gingivalis but not for other protozoa, oral protists and bacteria, or human leukocytes. With this method, the DNA from as few as 30 cells of Entamoeba gingivalis could be detected. These results suggest that this approach is applicable to the detection and identification of Entamoeba gingivalis in the human oral cavity.

  11. Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms.

    PubMed Central

    Calhoon, D A; Mayberry, W R; Slots, J

    1981-01-01

    The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles. PMID:7287893

  12. Microbial ecology of Actinobacillus actinomycetemcomitans, Eikenella corrodens and Capnocytophaga spp. in adult periodontitis.

    PubMed

    Müller, H P; Heinecke, A; Borneff, M; Knopf, A; Kiencke, C; Pohl, S

    1997-08-01

    Information on intraoral distribution of putative periodontal pathogens might be essential for controlling different forms of periodontal disease. Colonization may be either promoted or impeded by other bacteria competing in the subgingival ecosystem. In recent investigations microbial associations between dental organisms have been determined in a multitude of subgingival plaque samples within multiple patients and described by odds ratios, in most circumstances without taking into account the correlated structure of the observations within a single individual. The present investigation had 3 major objectives: (i) to describe the intraoral distribution of some facultatively anaerobic, Gram-negative rods, i.e. Actinobacillus actinomycetemcomitans, Eikenella corrodens-like organisms and Capnocytophaga spp., in a multitude of subgingival and extracrevicular samples of 10 adult subjects with A. actinomycetemcomitans-associated periodontitis; (ii) to analyse possible inconsistencies of microbial associations between these periodontal organisms; and (iii) to determine factors increasing the likelihood of isolating these bacteria in a given subgingival site by employing Generalized Estimation Equation (GEE) methods. Clinical examinations were carried out at 6 sites of every tooth present. In each subject, 13 extracrevicular (2 cheek mucosa, 3 tongue, 4 gingival, 2 tonsillar samples, 1 palatinal, 1 saliva sample) and between 22 and 44 subgingival samples from deepest sites of every tooth present (n = 296) were selectively cultivated for A. actinomycetemcomitans, E. corrodens and Capnocytophaga spp. In extracrevicular material, A. actinomycetemcomitans, Capnocytophaga spp. and E. corrodens were isolated in 9, 10 and 6 patients, and from 65, 82 and 15% samples, respectively. The organisms were recovered from 51, 62 and 27% subgingival plaque samples, respectively. Heterogeneity tests did not reveal significant inconsistencies of microbial associations between bacteria in

  13. In vitro activity of antibiotics alone and in combination against Actinobacillus actinomycetemcomitans.

    PubMed

    Yogev, R; Shulman, D; Shulman, S T; Glogowski, W G

    1986-01-01

    The MICs for 90% of the organisms tested (MIC90S) of 11 antibiotics against 24 clinical isolates of Actinobacillus actinomycetemcomitans were determined by the MIC 2000 system. The lowest MIC90S (16 micrograms/ml) were observed with ceftriaxone and rifampin. The next lowest MIC90S were found with cephapirin, tetracycline, and chloramphenicol (3.12 micrograms/ml). The MIC90S of penicillin, ampicillin, ticarcillin, piperacillin, and amikacin were each greater than or equal to 12.5 micrograms/ml. Antibiotic synergy was studied by the killing curve method and was defined as a greater than or equal to 2 log10 reduction in CFU when two antibiotics were used in combination at one-fourth the MBC for each compared with the effect of each antibiotic alone at one-half the MBC. Synergism between rifampin and penicillin, cephapirin, or ceftriaxone was tested for with 12 A. actinomycetemcomitans strains. In 7 of 37 instances, synergism was demonstrated for the combinations rifampin plus ceftriaxone (n = 3) or rifampin plus penicillin (n = 4); in 9 instances, an additive effect was noted, and impaired killing with drug combinations compared with the effect of a single antibiotic was suggested in 4 strains. The majority of strains were indifferent to the combinations. Similarly, variable results were observed when the combination of trimethoprim and cephapirin was tested against eight A. actinomycetemcomitans strains. Our data suggest that rifampin and cephapirin are the most active of the 11 antibiotics studied against A. actinomycetemcomitans. In addition, in vitro synergism between rifampin and other antibiotics or between trimethoprim and cephapirin was not consistently demonstrable.

  14. Characterization of a polysaccharide antigen from Bacteroides gingivalis.

    PubMed

    Schifferle, R E; Reddy, M S; Zambon, J J; Genco, R J; Levine, M J

    1989-11-01

    A polysaccharide Ag (PS) was isolated from the phenol-water extract of Bacteroides gingivalis strain A7A1-28 and separated from LPS by Sephacryl S-400 HR chromatography. The PS was composed of glucose, glucosamine, galactosamine, and galactosaminuronic acid, while the LPS contained rhamnose, mannose, galactose, glucose, glucosamine, galactosamine, phosphate, and lipid, but not galactosaminuronic acid. The PS and LPS were immunochemically distinct by immunoelectrophoresis in agarose with homologous rabbit antiserum. The phenol-water extract from strain A7A1-28 was immunoreactive by immunoelectrophoresis against antisera to three additional strains of B. gingivalis, however, the PS was only reactive with homologous serum. Immunochemical characterization of decarboxylated and deacetylated PS derivatives suggest that the acetylation of the amino sugars, but not the presence of the carboxylate residue on galactosaminuronic acid contributes to major immunodeterminant expression.

  15. Membrane Association and Destabilization by Aggregatibacter actinomycetemcomitans Leukotoxin Requires Changes in Secondary Structures

    PubMed Central

    Walters, Michael J.; Brown, Angela C.; Edrington, Thomas C.; Baranwal, Somesh; Du, Yurong; Lally, Edward T.; Boesze-Battaglia, Kathleen

    2013-01-01

    SUMMARY Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis in children, and localized aggressive periodontitis. A. actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and/or destabilization. In this study, we tested this hypothesis by analyzing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies thus provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans. PMID:23678967

  16. Development of an Animal Model for Aggregatibacter Actinomycetemcomitans Biofilm-Mediated Oral Osteolytic Infection: A Preliminary Study

    PubMed Central

    Freire, Marcelo O.; Sedghizadeh, Parish P.; Schaudinn, Christoph; Gorur, Amita; Downey, Jennifer S.; Choi, Jeong-Ho; Chen, Weizhen; Kook, Joong-Ki; Chen, Casey; Goodman, Steven D.; Zadeh, Homayoun H.

    2012-01-01

    Background Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)–biofilm colonizing titanium implants. Methods Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume. Results Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100%of biofilm-inoculated implants for up to 3 weeks and 25%for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks. Conclusions These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and

  17. Inhibitory activity of Aloe vera gel on some clinically isolated cariogenic and periodontopathic bacteria.

    PubMed

    Fani, Mohammadmehdi; Kohanteb, Jamshid

    2012-03-01

    Aloe vera is a medicinal plant with anti-inflammatory, antimicrobial, antidiabetic and immune-boosting properties. In the present study we investigated the inhibitory activities of Aloe vera gel on some cariogenic (Streptococcus mutans), periodontopathic (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis) and an opportunistic periodontopathogen (Bacteroides fragilis) isolated from patients with dental caries and periodontal diseases. Twenty isolates of each of these bacteria were investigated for their sensitivity to Aloe vera gel using the disk diffusion and microdilution methods. S. mutans was the species most sensitive to Aloe vera gel with a MIC of 12.5 µg/ml, while A. actinomycetemcomitans, P. gingivalis, and B. fragilis were less sensitive, with a MIC of 25-50 µg/ml (P < 0.01). Based on our present findings it is concluded that Aloe vera gel at optimum concentration could be used as an antiseptic for prevention of dental caries and periodontal diseases.

  18. Detection of the amoeba Entamoeba gingivalis in periodontal pockets

    PubMed Central

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly – if not exclusively – belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis. PMID:24983705

  19. Detection of the amoeba Entamoeba gingivalis in periodontal pockets.

    PubMed

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly - if not exclusively - belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis.

  20. Essential Oils from Ugandan Aromatic Medicinal Plants: Chemical Composition and Growth Inhibitory Effects on Oral Pathogens.

    PubMed

    Ocheng, Francis; Bwanga, Freddie; Joloba, Moses; Softrata, Abier; Azeem, Muhammad; Pütsep, Katrin; Borg-Karlson, Anna-Karin; Obua, Celestino; Gustafsson, Anders

    2015-01-01

    The study assessed the growth inhibitory effects of essential oils extracted from ten Ugandan medicinal plants (Bidens pilosa, Helichrysum odoratissimum, Vernonia amygdalina, Hoslundia opposita, Ocimum gratissimum, Cymbopogon citratus, Cymbopogon nardus, Teclea nobilis, Zanthoxylum chalybeum, and Lantana trifolia) used traditionally in the management of oral diseases against oral pathogens. Chemical compositions of the oils were explored by GC-MS. Inhibitory effects of the oils were assessed on periodontopathic Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans and cariogenic Streptococcus mutans and Lactobacillus acidophilus using broth dilution methods at concentrations of 1%, 0.1%, and 0.01%. The most sensitive organism was A. actinomycetemcomitans. Its growth was markedly inhibited by six of the oils at all the concentrations tested. Essential oil from C. nardus exhibited the highest activity with complete growth inhibition of A. actinomycetemcomitans and P. gingivalis at all the three concentrations tested, the major constituents in the oil being mainly oxygenated sesquiterpenes. Most of the oils exhibited limited effects on L. acidophilus. We conclude that essential oils from the studied plants show marked growth inhibitory effects on periodontopathic A. actinomycetemcomitans and P. gingivalis, moderate effects on cariogenic S. mutans, and the least effect on L. acidophilus. The present study constitutes a basis for further investigations and development of certain oils into alternative antiplaque agents. PMID:26170872

  1. Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T.; Ihalin, Riikka

    2013-01-01

    Aggregatibacteractinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1

  2. Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis.

    PubMed

    Haubek, Dorte; Johansson, Anders

    2014-01-01

    For decades, Aggregatibacter actinomycetemcomitans has been associated with aggressive forms of periodontitis in adolescents. In the middle of the 1990s, a specific JP2 clone of A. actinomycetemcomitans, belonging to the cluster of serotype b strains of A. actinomycetemcomitans and having a number of other characteristics, was found to be strongly associated with aggressive forms of periodontitis, particularly in North Africa. Although several longitudinal studies still point to the bacterial species, A. actinomycetemcomitans as a risk factor of aggressive periodontitis, it is now also widely accepted that the highly leukotoxic JP2 clone of A. actinomycetemcomitans is implicated in rapidly progressing forms of aggressive periodontitis. The JP2 clone strains are highly prevalent in human populations living in Northern and Western parts of Africa. These strains are also prevalent in geographically widespread populations that have originated from the Northwest Africa. Only sporadic signs of a dissemination of the JP2 clone strains to non-African populations have been found despite Africans living geographically widespread for hundreds of years. It remains an unanswered question if a particular host tropism exists as a possible explanation for the frequent colonization of the Northwest African population with the JP2 clone. Two exotoxins of A. actinomycetemcomitans are known, leukotoxin (LtxA) and cytolethal distending toxin (Cdt). LtxA is able to kill human immune cells, and Cdt can block cell cycle progression in eukaryotic cells and thus induce cell cycle arrest. Whereas the leukotoxin production is enhanced in JP2 clone strains thus increasing the virulence potential of A. actinomycetemcomitans, it has not been possible so far to demonstrate such a role for Cdt. Lines of evidence have led to the understanding of the highly leukotoxic JP2 clone of A. actinomycetemcomitans as an aetiological factor of aggressive periodontitis. Patients, who are colonized with the JP2

  3. Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis

    PubMed Central

    Haubek, Dorte; Johansson, Anders

    2014-01-01

    For decades, Aggregatibacter actinomycetemcomitans has been associated with aggressive forms of periodontitis in adolescents. In the middle of the 1990s, a specific JP2 clone of A. actinomycetemcomitans, belonging to the cluster of serotype b strains of A. actinomycetemcomitans and having a number of other characteristics, was found to be strongly associated with aggressive forms of periodontitis, particularly in North Africa. Although several longitudinal studies still point to the bacterial species, A. actinomycetemcomitans as a risk factor of aggressive periodontitis, it is now also widely accepted that the highly leukotoxic JP2 clone of A. actinomycetemcomitans is implicated in rapidly progressing forms of aggressive periodontitis. The JP2 clone strains are highly prevalent in human populations living in Northern and Western parts of Africa. These strains are also prevalent in geographically widespread populations that have originated from the Northwest Africa. Only sporadic signs of a dissemination of the JP2 clone strains to non-African populations have been found despite Africans living geographically widespread for hundreds of years. It remains an unanswered question if a particular host tropism exists as a possible explanation for the frequent colonization of the Northwest African population with the JP2 clone. Two exotoxins of A. actinomycetemcomitans are known, leukotoxin (LtxA) and cytolethal distending toxin (Cdt). LtxA is able to kill human immune cells, and Cdt can block cell cycle progression in eukaryotic cells and thus induce cell cycle arrest. Whereas the leukotoxin production is enhanced in JP2 clone strains thus increasing the virulence potential of A. actinomycetemcomitans, it has not been possible so far to demonstrate such a role for Cdt. Lines of evidence have led to the understanding of the highly leukotoxic JP2 clone of A. actinomycetemcomitans as an aetiological factor of aggressive periodontitis. Patients, who are colonized with the JP2

  4. Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-05-01

    To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria.

  5. Molecular Characterization of an Outer Membrane Protein of Actinobacillus actinomycetemcomitans Belonging to the OmpA Family

    PubMed Central

    White, Peter A.; Nair, Sean P.; Kim, Mi-Jurng; Wilson, Michael; Henderson, Brian

    1998-01-01

    The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34. PMID:9423883

  6. Serotype-dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains.

    PubMed

    Kikuchi, Haruko; Fujise, Osamu; Miura, Mayumi; Tanaka, Ayako; Hisano, Kyoko; Haraguchi, Akira; Hamachi, Takafumi; Maeda, Katsumasa

    2012-10-01

    Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two-phase separation system using the detergent Triton X-114, crude LPS extracts of the study strains were separated into detergent-phase LPS (DP-LPS) and aqueous-phase LPS (AP-LPS). Repeated treatment of the aqueous phase with the two-phase separation system produced only a slight decrease in AP-LPS, suggesting that AP-LPS was resistant to the detergent and thus distinguishable from DP-LPS. The presence of divalent cations increased the yield of AP-LPS. AP-LPS expression patterns were serotype-dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP-LPS, suggesting involvement of the LPS structure in the generation of AP-LPS. The two-phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP-LPS likely represents stabilized LPS aggregates, whereas DP-LPS might be derived from non-stabilized aggregates. Furthermore, time-dependent expression of stabilized LPS aggregates was found to be serotype-dependent in A. actinomycetemcomitans.

  7. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

    PubMed

    Velusamy, Senthil Kumar; Sampathkumar, Vandana; Godboley, Dipti; Fine, Daniel H

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  8. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments

    PubMed Central

    Godboley, Dipti; Fine, Daniel H.

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  9. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

    PubMed

    Velusamy, Senthil Kumar; Sampathkumar, Vandana; Godboley, Dipti; Fine, Daniel H

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  10. Aggregatibacter actinomycetemcomitans leukotoxin: a powerful tool with capacity to cause imbalance in the host inflammatory response.

    PubMed

    Johansson, Anders

    2011-03-01

    Aggregatibacter actinomycetemcomitans has been described as a member of the indigenous oral microbiota of humans, and is involved in the pathology of periodontitis and various non-oral infections. This bacterium selectively kills human leukocytes through expression of leukotoxin, a large pore-forming protein that belongs to the Repeat in Toxin (RTX) family. The specificity of the toxin is related to its prerequisite for a specific target cell receptor, LFA-1, which is solely expressed on leukocytes. The leukotoxin causes death of different leukocyte populations in a variety of ways. It activates a rapid release of lysosomal enzymes and MMPs from neutrophils and causes apoptosis in lymphocytes. In the monocytes/macrophages, the toxin activates caspase-1, a cysteine proteinase, which causes a proinflammatory response by the activation and secretion of IL-1β and IL-18. A specific clone (JP2) of A. actinomycetemcomitans with enhanced leukotoxin expression significantly correlates to disease onset in infected individuals. Taken together, the mechanisms by which this toxin kills leukocytes are closely related to the pathogenic mechanisms of inflammatory disorders, such as periodontitis. Therapeutic strategies targeting the cellular and molecular inflammatory host response in periodontal diseases might be a future treatment alternative.

  11. Purification and characterization of the serotype c antigen from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Zambon, J J; Slots, J; Miyasaki, K; Linzer, R; Cohen, R; Levine, M; Genco, R J

    1984-01-01

    The serotype c antigen from Actinobacillus actinomycetemcomitans was purified with fractional ethanol precipitation of cell-free culture supernatant, sequential ion-exchange chromatography, and gel filtration chromatography. The preparation obtained demonstrated a single precipitin line in immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis when rabbit antisera to serotype c whole bacterial cells were used. No immunological reaction was detected with antisera to serotype c lipopolysaccharide, indicating that lipopolysaccharide was not present in the preparation. The serotype c antigen was composed of 95% carbohydrate, 2% protein, and 3.1% phosphate. Gas chromatographic analysis of the antigen obtained from growth in either complex or chemically defined media revealed that the carbohydrate constituent was composed of 84 to 90.1% mannose, 4.8 to 16% glucose, 1.9% N-acetylglucosamine, 1.4% fucose, and 0.2% galactose. The present data suggest that A. actinomycetemcomitans serotype c antigen is predominantly a mannose-containing carbohydrate suggestive of a mannan. Images PMID:6423542

  12. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    SciTech Connect

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul

    2008-05-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.

  13. Photosensitization of Aggregatibacter actinomycetemcomitans with methylene blue: a microbiological and spectroscopic study

    NASA Astrophysics Data System (ADS)

    Yamada Júnior, Aécio M.; Prates, Renato A.; Cai, Silvana; Ribeiro, Martha S.

    2008-03-01

    The aim of this study was to determinate the efficiency of methylene blue (MB) to kill cultures of Aggregatibacter actinomycetemcomitans under red light and to investigate MB photobleaching by optical absorption spectroscopy. Bacteria were diluted in aqueous solution, putted in glass tubes and distributed in 5 groups: (L-MB-) control group; (L+MB-) laser alone by 5min; (L-MB+) MB alone through 5min; (3L+MB+) MB+laser 3min; (5L+MB+) MB+laser 5min. Laser parameters were P=30mW, λ=660nm, E=9J in 5min and E=5.4J in 3min. The samples were diluted and bacterial colonies were counted and converted into colony forming units (CFU). Absorption spectra of the MB-stained bacterial suspension and photosensitized bacterial suspension were obtained. Groups L-MB-, L+MB-, and L-MB+ did not show a decrease in CFU/mL. L+MB+ groups showed a significant decrease in CFU/mL but no statistically significant differences were observed between 3min and 5min. Spectroscopy showed that MB is photodegraded after irradiation and that dimer species are more notably consumed than monomeric species. These results suggest that MB is a suitable photosensitizer to reduce A. actinomycetemcomitans, and that 3min of irradiation are enough to produce a significant effect. Due to the spectral changes observed on MB solution after irradiation a type I mechanism may be involved.

  14. Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Shanmugam, Mayilvahanan; El Abbar, Faiha; Ramasubbu, Narayanan

    2015-01-01

    Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans. PMID:26221956

  15. Serotypes of Aggregatibacter actinomycetemcomitans in relation to periodontal status and geographic origin of individuals-a review of the literature

    PubMed Central

    da Silveira, Virginia R S.; Rego, Rodrigo O.; Nogueira, Nádia A P.

    2014-01-01

    Objectives: Several studies have focused on the relationship among serotype distribution, ethnical status and geographic populations, and periodontal conditions. Studies that have investigated the prevalence and the distribution of A. actinomycetemcomitans serotypes and the relation between the different serotypes of the bacterium and periodontal status were reviewed. Material and Methods: A systematic literature search for publications regarding the distribution of A. actinomycetemcomitans serotypes in subgingival samples of periodontitis patients and periodontally healthy subjects by employing polymerase chain reaction (PCR) was conducted. Results: From the 85 studies identified in the first analysis, only 12 met all inclusion and exclusion criteria. Clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Serotypes a, b and c were largely found, and serotype c was the most prevalent. They were isolated from various periodontal conditions, including aggressive periodontitis. Conclusions: The available literature suggests that serotypes a, b, and c are globally dominant, serotypes d and e are rare, and the prevalence of the most recently identified serotype fis still unknown. It is widely accepted that distribution patterns of A. actinomycetemcomitans vary among subjects of different ethnicity and geographic regions. The correlation of different serotypes with various periodontal conditions remains unclear. Key words:Aggregatibacter actinomycetemcomitans, serotypes, periodontal disease, prevalence. PMID:24316700

  16. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

    PubMed Central

    ALVAREZ, Carla; BENÍTEZ, Alvaro; ROJAS, Leticia; PUJOL, Myriam; CARVAJAL, Paola; DÍAZ-ZÚÑIGA, Jaime; VERNAL, Rolando

    2015-01-01

    In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased towards a

  17. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

    PubMed Central

    ALVAREZ, Carla; BENÍTEZ, Alvaro; ROJAS, Leticia; PUJOL, Myriam; CARVAJAL, Paola; DÍAZ-ZÚÑIGA, Jaime; VERNAL, Rolando

    2015-01-01

    ABSTRACT In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased

  18. Lethal photosensitization for decontamination of implant surfaces in the treatment of peri-implantitis.

    PubMed

    Dörtbudak, O; Haas, R; Bernhart, T; Mailath-Pokorny, G

    2001-04-01

    Peri-implantitis is considered to be a multifactorial process involving bacterial contamination of the implant surface. A previous study demonstrated that a combination of toluidine blue O (100 microgram/ml) and irradiation with a diode soft laser with a wavelength of 905 nm results in an elimination of Porphyromonas gingivalis (P. gingivalis), Prevotella intermedia (P. intermedia), and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) on different implant surfaces (machined, plasma-flame-sprayed, etched, hydroxyapatite-coated). The aim of this study was to examine the laser effect in vivo. In 15 patients with IMZ implants who showed clinical and radiographic signs of peri-implantitis, toluidine blue O was applied to the implant surface for 1 min and the surface was then irradiated with a diode soft laser with a wavelength of 690 nm for 60 s. Bacterial samples were taken before and after application of the dye and after lasing. The cultures were evaluated semiquantitatively for A. actinomycetemcomitans, P. gingivalis, and P. intermedia. It was found that the combined treatment reduced the bacterial counts by 2 log steps on average. The application of TBO and laser resulted in a significant reduction (P<0.0001) of the initial values in all 3 groups of bacteria. Complete elimination of bacteria was not achieved.

  19. Effects of ozone nano-bubble water on periodontopathic bacteria and oral cells - in vitro studies

    NASA Astrophysics Data System (ADS)

    Hayakumo, Sae; Arakawa, Shinichi; Takahashi, Masayoshi; Kondo, Keiko; Mano, Yoshihiro; Izumi, Yuichi

    2014-10-01

    The aims of the present study were to evaluate the bactericidal activity of a new antiseptic agent, ozone nano-bubble water (NBW3), against periodontopathogenic bacteria and to assess the cytotoxicity of NBW3 against human oral cells. The bactericidal activities of NBW3 against representative periodontopathogenic bacteria, Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) were evaluated using in vitro time-kill assays. The cytotoxicity of NBW3 was evaluated using three-dimensional human buccal and gingival tissue models. The numbers of colony forming units (CFUs)/mL of P. gingivalis and A. actinomycetemcomitans exposed to NBW3 dropped to below the lower limit of detection (<10 CFUs mL-1) after only 0.5 min of exposure. There were only minor decreases in the viability of oral tissue cells after 24 h of exposure to NBW3. These results suggest that NBW3 possesses potent bactericidal activity against representative periodontopathogenic bacteria and is not cytotoxic to cells of human oral tissues. The use of NBW3 as an adjunct to periodontal therapy would be promising.

  20. [USE OF THE REAL-TIME PCR FOR STUDY OF THE PERIODONTAL MICROBIOME IN PATIENTS WITH COMBINED PATHOLOGY OF GASTRODUODENAL ZONE AND CHRONIC PERIODONTITIS].

    PubMed

    Shibaeva, A V; Ayvazova, R A; Rebrikov, D V; Trubnikova, E V; Kudykina, Yu K; Belyakova, A V; Zaripova, R S; Shevelev, A B

    2016-01-01

    The total of 54 patients with chronic periodontitis of different severity was tested using real-time PCR (Dentoflor kit). The group included 38 patients with chronic gastritis. For the first time, a higher prevalence of Treponema denticola in periodontium of males in comparison with females was demonstrated. The patients with chronic gastritis had more human genome DNA at their periodontium than healthy individuals. Non-parametric statistical analysis demonstrated high association of periodontium colonization with. T. forsythensis and T. denticola (but not Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia) with the severity of the chronic periodontitis. PMID:27183718

  1. Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway.

    PubMed

    Oliveira, Tatiane; Figueiredo, Camila A; Brito, Carlos; Stavroullakis, Alexander; Ferreira, Ana Carolina; Nogueira-Filho, Getulio; Prakki, Anuradha

    2015-01-01

    Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE) and quercetin (Qt) on osteoclastogenesis under inflammatory conditions (LPS-induced). Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL), and treated with AcE (50-1000 µg/mL) or Qt (1.25, 2.5, or 5 µM). Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced) via attenuation of RANKL/PgLPS-induced NF-κB activation. PMID:26273314

  2. Allium cepa L. and Quercetin Inhibit RANKL/Porphyromonas gingivalis LPS-Induced Osteoclastogenesis by Downregulating NF-κB Signaling Pathway

    PubMed Central

    Oliveira, Tatiane; Figueiredo, Camila A.; Brito, Carlos; Stavroullakis, Alexander; Ferreira, Ana Carolina; Nogueira-Filho, Getulio; Prakki, Anuradha

    2015-01-01

    Objectives. We evaluated the in vitro modulatory effects of Allium cepa L. extract (AcE) and quercetin (Qt) on osteoclastogenesis under inflammatory conditions (LPS-induced). Methods. RAW 264.7 cells were differentiated with 30 ng/mL of RANKL, costimulated with PgLPS (1 µg/mL), and treated with AcE (50–1000 µg/mL) or Qt (1.25, 2.5, or 5 µM). Cell viability was determined by alamarBlue and protein assays. Nuclei morphology was analysed by DAPI staining. TRAP assays were performed as follows: p-nitrophenyl phosphate was used to determine the acid phosphatase activity of the osteoclasts and TRAP staining was used to evaluate the number and size of TRAP-positive multinucleated osteoclast cells. Von Kossa staining was used to measure osteoclast resorptive activity. Cytokine levels were measured on osteoclast precursor cell culture supernatants. Using western blot analysis, p-IκBα and IκBα degradation, inhibitor of NF-kappaB, were evaluated. Results. Both AcE and Qt did not affect cell viability and significantly reduced osteoclastogenesis compared to control. We observed lower production of IL-6 and IL-1α and an increased production of IL-3 and IL-4. AcE and Qt downregulated NF-κB pathway. Conclusion. AcE and Qt may be inhibitors of osteoclastogenesis under inflammatory conditions (LPS-induced) via attenuation of RANKL/PgLPS-induced NF-κB activation. PMID:26273314

  3. Actinobacillus actinomycetemcomitans in Brazilian insulin-dependent individuals with diabetes mellitus.

    PubMed

    Feitosa, A C; de Uzeda, M; Novaes, A B

    1992-01-01

    The prevalence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque specimens from 26 insulin-dependent diabetes mellitus patients, 11-25 years of age, was determined between January 1987 and December 1989. One hundred and thirty subgingival plaque samples were collected with sterile periodontal curettes. The specimens were weighted, diluted, inoculated on trypticase-soy-serum-bacitracin-vancomycin agar medium (TSBV) and incubated under microacrophilic conditions. Aa was isolated from 2.3% of healthy periodontal areas in these patients, while the microorganism was found in 12.5% of the sites with gingivitis and in 2.6% of the periodontal pockets examined. Although biochemical tests used for the characterization of Aa strains showed homogeneous results, different biotypes were isolated from one or more periodontal sites in the same patient.

  4. Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed Central

    Holt, S C; Tanner, A C; Socransky, S S

    1980-01-01

    Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells. Images Fig. 6 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:7439996

  5. Characterization of the lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4 and N27.

    PubMed Central

    Kiley, P; Holt, S C

    1980-01-01

    The lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans strains Y4 and N27 was isolated by the phenol-water procedure. Morphologically, the molecule consisted of ribbon and branched filaments which comprised 3% of the cellular dry weight. Chemical analysis of the isolated and purified LPSs of both strains showed them to consist of carbohydrate, lipid, 2-keto-3-deoxyoctonate, heptose, hexosamine, and phosphate. The major fatty acids of the lipid A moiety were saturated C14 and beta-OH C14 compounds. Rhamnose, fucose, galactose, glucose, heptose, glucosamine, and galactosamine comprised the monosaccharide portion of the LPS. Biological activity studies revealed both LPS molecules to be active in the Schwartzman reaction and in in vitro 45Ca bone resorption, as well as in macrophage activation and lethality and in platelet aggregation. Images Fig. 1 Fig. 2 Fig. 4 PMID:7228391

  6. First evidence of genetic intraspecific variability and occurrence of Entamoeba gingivalis in HIV(+)/AIDS.

    PubMed

    Cembranelli, Sibeli B S; Souto, Fernanda O; Ferreira-Paim, Kennio; Richinho, Túlio T; Nunes, Poliana L; Nascentes, Gabriel A N; Ferreira, Thatiana B; Correia, Dalmo; Lages-Silva, Eliane

    2013-01-01

    Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4(+) lymphocyte count (≤200 cells/mm(3) (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(-) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4(+) lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis

  7. First Evidence of Genetic Intraspecific Variability and Occurrence of Entamoeba gingivalis in HIV(+)/AIDS

    PubMed Central

    Cembranelli, Sibeli B. S.; Souto, Fernanda O.; Ferreira-Paim, Kennio; Richinho, Túlio T.; Nunes, Poliana L.; Nascentes, Gabriel A. N.; Ferreira, Thatiana B.; Correia, Dalmo; Lages-Silva, Eliane

    2013-01-01

    Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4+ lymphocyte count (≤200 cells/mm3 (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(−) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4+ lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis isolated

  8. Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

    SciTech Connect

    Schwarz, S.; Ellen, R.P.; Grove, D.A.

    1987-10-01

    There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of /sup 3/H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.

  9. Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures.

    PubMed Central

    Nishihara, T; Ueda, N; Amano, K; Ishihara, Y; Hayakawa, H; Kuroyanagi, T; Ohsaki, Y; Nagata, K; Noguchi, T

    1995-01-01

    The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A

  10. Genetic and Functional Analyses of the Actinobacillus actinomycetemcomitans AfeABCD Siderophore-Independent Iron Acquisition System

    PubMed Central

    Rhodes, Eric R.; Tomaras, Andrew P.; McGillivary, Glen; Connerly, Pamela L.; Actis, Luis A.

    2005-01-01

    The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions. PMID:15908408

  11. The pathogenic persona of community-associated oral streptococci.

    PubMed

    Whitmore, Sarah E; Lamont, Richard J

    2011-07-01

    The mitis group streptococci (MGS) are widespread in the oral cavity and are traditionally associated with oral health. However, these organisms have many attributes that contribute to the development of pathogenic oral communities. MGS adhere rapidly to saliva-coated tooth surfaces, thereby providing an attachment substratum for more overtly pathogenic organisms such as Porphyromonas gingivalis, and the two species assemble into heterotypic communities. Close physical association facilitates physiologic support, and pathogens such as Aggregatibacter actinomycetemcomitans display resource partitioning to favour carbon sources generated by streptococcal metabolism. MGS exchange information with community members through a number of interspecies signalling systems including AI-2 and contact dependent mechanisms. Signal transduction systems induced in P. gingivalis are based on protein dephosphorylation mediated by the tyrosine phosphatase Ltp1, and converge on a LuxR-family transcriptional regulator, CdhR. Phenotypic responses in P. gingivalis include regulation of hemin uptake systems and gingipain activity, processes that are intimately linked to the virulence of the organism. Furthermore, communities of S. gordonii with P. gingivalis or with A. actinomycetemcomitans are more pathogenic in animal models than the constituent species alone. We propose that MGS should be considered accessory pathogens, organisms whose pathogenic potential only becomes evident in the context of a heterotypic microbial community.

  12. Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

    PubMed

    Izawa, A; Ishihara, Y; Mizutani, H; Kobayashi, S; Goto, H; Okabe, E; Takeda, H; Ozawa, Y; Kamiya, Y; Sugita, Y; Kubo, K; Kamei, H; Kikuchi, T; Mitani, A; Hayashi, J; Nishihara, T; Maeda, H; Noguchi, T

    2014-05-01

    The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

  13. Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

    PubMed

    Izawa, A; Ishihara, Y; Mizutani, H; Kobayashi, S; Goto, H; Okabe, E; Takeda, H; Ozawa, Y; Kamiya, Y; Sugita, Y; Kubo, K; Kamei, H; Kikuchi, T; Mitani, A; Hayashi, J; Nishihara, T; Maeda, H; Noguchi, T

    2014-05-01

    The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease. PMID:24566623

  14. Bacteriocin production by Actinobacillus actinomycetemcomitans isolated from the oral cavity of humans with periodontal disease, periodontally healthy subjects and marmosets.

    PubMed

    Lúcia, Lima Francisca; Farias, Flávio F; Eustáquio, Costa José; Auxiliadora, Maria; Carvalho, R; Alviano, Celuta S; Farias, Luiz M

    2002-01-01

    The ability of Actinobacillus actinomycetemcomitans to produce bacteriocin has rarely been reported. Antagonistic substance production may confer an important ecological advantage for the producer microorganisms, especially in a competitive ecosystem such as the oral cavity. In the present study, 75 A. actinomycetemcomitans strains isolated from the oral cavity of human patients with periodontal disease, periodontally healthy subjects and marmosets, as well as two reference strains (A. actinomycetemcomitans ATCC 29523 and FDC Y4) were evaluated for auto-, iso-, and heteroantagonistic activity. Fifty-one (68.00%) strains exhibited antagonistic activity; heteroantagonism was observed more often than isoantagonism. Isolated strains antagonized 17 different species of gram-positive and gram-negative bacteria from the oral and nonoral microbiota. Sensitivity to heat and to proteolytic enzymes constituted strong evidence that the antagonistic substance has a proteic nature. Taken together, our data enabled us to confirm that the antagonistic substance detected was a bacteriocin. The wide spectrum of activity indicates the possibility that more than one antagonistic substance is produced and that these substances play an important role in the ecological balance of the oral ecosystem.

  15. 16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains.

    PubMed

    Ihalin, Riikka; Asikainen, Sirkka

    2006-06-01

    Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes. Contrary to the strains of serotypes c, d, and e, strains of serotypes a, b, and f consistently demonstrated intra-serotype migration patterns similar to each other. Since the migration patterns differed between serotype c and b strains a strain of each was used to determine their proportional representation in a strain mixture. The strains were distinguishable from each other above the 5% PCR-DGGE detection level (12.5 ng DNA/1.5 x 10(6) cells). DGGE provides a promising tool for in vitro studies on the coexistence of different genetic lineages of A. actinomycetemcomitans.

  16. Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide.

    PubMed

    Wilson, M E; Schifferle, R E

    1991-04-01

    A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism.

  17. Microbiological and serological investigations of oral lesions in Papillon-Lefèvre syndrome.

    PubMed Central

    Clerehugh, V; Drucker, D B; Seymour, G J; Bird, P S

    1996-01-01

    Microbiological and serological (enzyme linked immunosorbent assay) investigations were carried out, including karyotyping, on two Asian children with Papillon-Lefèvre syndrome. In case 1, a girl aged four years, the most prevalent putative periodontopathogens were Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia (deciduous dentition) and Bacteroides gracilis, E corrodens and F nucleatum (permanent dentition). In case 2, a boy aged nine years, they were F nucleatum, P intermedia and P loeschii and E corrodens. Serum from case 2 showed a raised specific IgG antibody response to Actinomyces actino-mycetemcomitans serotype b. Thus, a wider range of species than hitherto reported may be associated with Papillon-Lefèvre syndrome, including A actino-mycetemcomitans and F nucleatum. PMID:8675741

  18. A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160.

    PubMed

    Obradović, Davor; Gašperšič, Rok; Caserman, Simon; Leonardi, Adrijana; Jamnik, Maja; Podlesek, Zdravko; Seme, Katja; Anderluh, Gregor; Križaj, Igor; Maček, Peter; Butala, Matej

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116-188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116-188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained. PMID:27414641

  19. A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160

    PubMed Central

    Obradović, Davor; Gašperšič, Rok; Caserman, Simon; Leonardi, Adrijana; Jamnik, Maja; Podlesek, Zdravko; Seme, Katja; Anderluh, Gregor; Križaj, Igor; Maček, Peter; Butala, Matej

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116–188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116–188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained. PMID:27414641

  20. Immunodominant antigen of Actinobacillus actinomycetemcomitans Y4 in high-responder patients.

    PubMed Central

    Califano, J V; Schenkein, H A; Tew, J G

    1989-01-01

    This study was undertaken to look for characteristics of the immunodominant antigen(s) of Actinobacillus actinomycetemcomitans Y4 that might help explain the high antibody titers in periodontitis patients. Radioimmunoassays (RIA) were performed on sera from 481 patients; sera from the 32 patients with the highest anti-Y4 titers (above 128,000 RIA U/ml) were further analyzed. Y4 antigen was boiled for 45 min or treated with papain, and antibody responses were analyzed by RIA and Western blotting (immunoblotting). In addition, carbohydrate was purified from Y4 and examined by Western blotting. The results indicated that the immunodominant antigen of Y4 in high responders was stable after papain treatment or boiling for 45 min. Papain or boiling eliminated protein bands but a large diffuse band persisted on Western blots. With increasing dilutions of sera, bands on Western blots corresponding to protein antigens disappeared, while the large diffuse band resembling that of carbohydrate persisted. Partially purified Y4 carbohydrate contained the large diffuse band. Double-immunodiffusion analysis indicated that rabbit serotype b-specific antiserum and patient sera recognized the same antigen. When the carbohydrate extract was passed over a lipid A-binding column to remove lipopolysaccharide, the smear corresponding to the immunodominant antigen was still present on Western blots. The immunodominant antigen of Y4 in high-responder individuals appears to be a carbohydrate and is possibly the capsular polysaccharide. Images PMID:2496034

  1. Draft genome sequences of 26 porphyromonas strains isolated from the canine oral microbiome.

    PubMed

    Coil, David A; Alexiev, Alexandra; Wallis, Corrin; O'Flynn, Ciaran; Deusch, Oliver; Davis, Ian; Horsfall, Alexander; Kirkwood, Nicola; Jospin, Guillaume; Eisen, Jonathan A; Harris, Stephen; Darling, Aaron E

    2015-01-01

    We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease. PMID:25858832

  2. Augmentation of Actinobacillus actinomycetemcomitans Invasion of Human Oral Epithelial Cells and Up-Regulation of Interleukin-8 Production by Saliva CD14

    PubMed Central

    Takayama, Atsuko; Satoh, Aya; Ngai, Tomoko; Nishimura, Takashi; Ikawa, Keiji; Matsuyama, Takami; Shimauchi, Hidetoshi; Takada, Haruhiko; Sugawara, Shunji

    2003-01-01

    It has recently been shown that human salivary glands constitutively express CD14, an important molecule in innate immunity, and that a soluble form of CD14 is secreted in saliva. The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva, although the physiological function of saliva CD14 remained unclear. Actinobacillus actinomycetemcomitans is a periodontopathic bacterium and is able to invade oral epithelial cells. The present study showed that upon exposure to live A. actinomycetemcomitans Y4 for 2 h, human oral epithelial HSC-2 cells produced interleukin-8 (IL-8) for a further 24 h and whole saliva augmented the production induced by A. actinomycetemcomitans Y4. Parotid saliva showed a more pronounced effect on the production of IL-8 than whole saliva. Neither saliva preparation itself had IL-8-inducing activity. Parotid saliva exhibited antibacterial activity against a low concentration of A. actinomycetemcomitans Y4, but recombinant CD14 did not show the activity. The internalization of A. actinomycetemcomitans Y4 into HSC-2 cells was inhibited by cytochalasin B, indicating that the process was actin dependent, and depletion of CD14 from parotid saliva inhibited the invasion and, as a consequence, inhibited production of IL-8. Furthermore, human recombinant CD14 augmented invasion and IL-8 production. These results suggest that saliva CD14 promoted the invasion of oral epithelial cells by A. actinomycetemcomitans and consequently augmented the production of IL-8, playing an important role in innate immunity in the oral cavity. PMID:14500479

  3. Aspartame as a source of essential phenylalanine for the growth of oral anaerobes.

    PubMed

    Wyss, C

    1993-04-15

    Phenylalanine and aspartic acid requirements were determined for 13 species of oral bacteria using the chemically defined medium OMIZ-W1. None of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Eikenella corrodens, Selenomonas sputigena, Treponema pectinovorum, T. socranskii, or Wolinella recta required either of these amino acid constituents of aspartame (L-aspartyl-L-phenylalanine methylester). Phenylalanine was essential for the growth of Capnocytophaga gingivalis, Eubacterium timidum, Fusobacterium nucleatum, Porphyromonas gingivalis, T. denticola, and T. vincentii, while aspartic acid was not required. With the exception of E. timidum, all phenylalanine-dependent strains could grow when the free amino acid was replaced by aspartame at concentrations at least 10-fold lower than those used for aspartame as an artificial sweetener.

  4. Physicochemical and structural investigation of the surfaces of some anaerobic subgingival bacteria.

    PubMed Central

    Cowan, M M; van der Mei, H C; Rouxhet, P G; Busscher, H J

    1992-01-01

    The surfaces of nine clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Peptostreptococcus micros and that of laboratory strain P. gingivalis W83 were studied by using contact angle measurements, X-ray photoelectron spectroscopy, infrared spectroscopy, microelectrophoresis of whole cells, and transmission electron microscopy of whole and sectioned cells. P. intermedia strains were hydrophilic, as judged from their small water contact angles, and had highly negative zeta potentials, consistent with the presence of a prominent ruthenium red (RR)-staining layer and fibrillar appendages which are probably partly carbohydrate. The two clinical isolates of P. gingivalis were also hydrophilic and highly negatively charged despite the presence of prominent fibrils, which usually yield less negative zeta potentials. This finding suggests that the RR-staining layer dominates the suspension characteristics of P. gingivalis and P. intermedia strains. P. gingivalis W83 had no demonstrable fibrils and a morphologically distinct RR-staining layer, and it was more hydrophobic than the two clinical isolates of P. gingivalis. P. micros isolates were hydrophobic and much less negatively charged than the other species. The A. actinomycetemcomitans strains displayed long, prominent fibrils and a very thin RR-staining layer, which resulted in high hydrophobicity but distinctly different zeta potentials for the two. Physicochemical data on microbial cell surfaces usually have clear and predictable relationships with each other. For the strains in this study that did not follow these relationships, their aberrant behavior could be explained as due to a masking effect caused by specific surface architecture. We conclude that this combined analysis provides a detailed image of subgingival bacterial surface architecture. Images PMID:1599251

  5. Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

    PubMed Central

    Jiang, Y; Russell, T R; Graves, D T; Cheng, H; Nong, S H; Levitz, S M

    1996-01-01

    Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment. PMID:8890191

  6. Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.

    PubMed

    Ragunath, C; DiFranco, K; Shanmugam, M; Gopal, P; Vyas, V; Fine, D H; Cugini, C; Ramasubbu, N

    2016-08-01

    Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-β-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae β-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms. PMID:26280561

  7. Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human β2 integrins and erythrocytes.

    PubMed

    Reinholdt, Jesper; Poulsen, Knud; Brinkmann, Christel R; Hoffmann, Søren V; Stapulionis, Romualdas; Enghild, Jan J; Jensen, Uffe B; Boesen, Thomas; Vorup-Jensen, Thomas

    2013-02-01

    Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)β(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β(2) integrin. Cells expressing α(M)β(2) (CD11b/CD18) or α(X)β(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)β(2). Erythrocytes, which do not express β(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)β(2) and α(X)β(2) as novel receptors for LtxA.

  8. Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT

    PubMed Central

    Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Bueno-Silva, Bruno; DiRienzo, Joseph M.; Mayer, Marcia P. A.

    2016-01-01

    The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP+ cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP+ cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP+ cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis. PMID:27064424

  9. Photocatalytical Antibacterial Activity of Mixed-Phase TiO2 Nanocomposite Thin Films against Aggregatibacter actinomycetemcomitans.

    PubMed

    Yeniyol, Sinem; Mutlu, Ilven; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Karabuda, Zihni Cüneyt; Basegmez, Cansu; Ricci, John Lawrence

    2015-01-01

    Mixed-phase TiO2 nanocomposite thin films consisting of anatase and rutile prepared on commercially pure Ti sheets via the electrochemical anodization and annealing treatments were investigated in terms of their photocatalytic activity for antibacterial use around dental implants. The resulting films were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). The topology was assessed by White Light Optical Profiling (WLOP) in the Vertical Scanning Interferometer (VSI) mode. Representative height descriptive parameters of roughness R a and R z were calculated. The photocatalytic activity of the resulting TiO2 films was evaluated by the photodegradation of Rhodamine B (RhB) dye solution. The antibacterial ability of the photocatalyst was examined by Aggregatibacter actinomycetemcomitans suspensions in a colony-forming assay. XRD showed that anatase/rutile mixed-phase TiO2 thin films were predominantly in anatase and rutile that were 54.6 wt% and 41.9 wt%, respectively. Craters (2-5 µm) and protruding hills (10-50 µm) on Ti substrates were produced after electrochemical anodization with higher R a and R z surface roughness values. Anatase/rutile mixed-phase TiO2 thin films showed 26% photocatalytic decolorization toward RhB dye solution. The number of colonizing bacteria on anatase/rutile mixed-phase TiO2 thin films was decreased significantly in vitro. The photocatalyst was effective against A. actinomycetemcomitans colonization. PMID:26576430

  10. Photocatalytical Antibacterial Activity of Mixed-Phase TiO2 Nanocomposite Thin Films against Aggregatibacter actinomycetemcomitans

    PubMed Central

    Yeniyol, Sinem; Mutlu, Ilven; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Karabuda, Zihni Cüneyt; Basegmez, Cansu; Ricci, John Lawrence

    2015-01-01

    Mixed-phase TiO2 nanocomposite thin films consisting of anatase and rutile prepared on commercially pure Ti sheets via the electrochemical anodization and annealing treatments were investigated in terms of their photocatalytic activity for antibacterial use around dental implants. The resulting films were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). The topology was assessed by White Light Optical Profiling (WLOP) in the Vertical Scanning Interferometer (VSI) mode. Representative height descriptive parameters of roughness Ra and Rz were calculated. The photocatalytic activity of the resulting TiO2 films was evaluated by the photodegradation of Rhodamine B (RhB) dye solution. The antibacterial ability of the photocatalyst was examined by  Aggregatibacter actinomycetemcomitans suspensions in a colony-forming assay. XRD showed that anatase/rutile mixed-phase TiO2 thin films were predominantly in anatase and rutile that were 54.6 wt% and 41.9 wt%, respectively. Craters (2–5 µm) and protruding hills (10–50 µm) on Ti substrates were produced after electrochemical anodization with higher Ra and Rz surface roughness values. Anatase/rutile mixed-phase TiO2 thin films showed 26% photocatalytic decolorization toward RhB dye solution. The number of colonizing bacteria on anatase/rutile mixed-phase TiO2 thin films was decreased significantly in vitro. The photocatalyst was effective against A. actinomycetemcomitans colonization. PMID:26576430

  11. First report of fatal systemic Halicephalobus gingivalis infection in two Lipizzaner horses from Romania: clinical, pathological, and molecular characterization.

    PubMed

    Taulescu, Marian A; Ionicã, Angela M; Diugan, Eva; Pavaloiu, Alexandra; Cora, Roxana; Amorim, Irina; Catoi, Cornel; Roccabianca, Paola

    2016-03-01

    Halicephalobus gingivalis (H. gingivalis) causes a rare and fatal infection in horses and humans. Despite the zoonotic potential and severity of the disease, the epidemiology and pathogenesis of halicephalobiasis are still poorly understood. Several European cases of equine halicephalobiasis have been documented; however, in South-Eastern European countries, including Romania, equine neurohelminthiasis caused by H. gingivalis has not been previously described. Two Lipizzaner horses with a clinical history of progressive neurological signs were referred to the Pathology Department of the Cluj-Napoca (Romania) for necropsy. Both horses died with severe neurological signs. Gross examination and cytological, histological, and molecular analyses were performed. The stallions came from two different breeding farms. No history of traveling outside Romania was recorded. At necropsy, granulomatous and necrotizing lesions were observed in the kidneys, lymph nodes, brain, retroperitoneal adipose tissue, and lungs, indicating a systemic infection. Parasitological and histopathological analyses evidenced larval and adult forms of rhabditiform nematodes consistent with Halicephalobus species. Parasites were observed in both lymph and blood vessels of different organs and were also identified in urine samples. A subunit of the large-subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified from lesions in both horses.To the authors' knowledge, this is the first report of equine systemic H. gingivalis infection in Romania and in South-Eastern Europe. Our findings provide new insights into the geographic distribution of specific genetic lineages of H. gingivalis, while also raising public health awareness, as the parasite is zoonotic.

  12. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  13. Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis.

    PubMed

    Senhorinho, Gerusa N A; Nakano, Viviane; Liu, Chengxu; Song, Yuli; Finegold, Sydney M; Avila-Campos, Mario J

    2012-08-01

    The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis.

  14. Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism.

    PubMed

    Weigel, W A; Demuth, D R; Torres-Escobar, A; Juárez-Rodríguez, M D

    2015-10-01

    Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment.

  15. Al(III), Pd(II), and Zn(II) phthalocyanines for inactivation of dental pathogen Aggregatibacter actinomycetemcomitans as planktonic and biofilm-cultures

    NASA Astrophysics Data System (ADS)

    Kussovski, V.; Mantareva, V.; Angelov, I.; Avramov, L.; Popova, E.; Dimitrov, S.

    2012-06-01

    The Gram-negative, oral bacterium Aggregatibacter actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. The new periodontal disease treatments are emergence in order to prevent infection progression. Antimicrobial photodynamic therapy (a-PDT) can be a useful tool for this purpose. It involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent in the presence of oxygen for eradication of target cells, and appears effective in photoinactivation of microorganisms. The phthalocyanine metal complexes of Pd(II)- (PdPcC) and Al(III)- (AlPc1) were evaluated as photodynamic sensitizers towards a dental pathogen A. actinomycetemcomitans in comparison to the known methylpyridyloxy-substituted Zn(II) phthalocyanine (ZnPcMe). The planktonic and biofilm-cultivated species of A. actinomycetemcomitans were treated. The photophysical results showed intensive and far-red absorbance with high tendency of aggregation for Pd(II)-phthalocyanine. The dark toxicities of both photosensitizers were negligible at concentrations used (< 0.5 log decrease of viable cells). The photodynamic response for planktonic cultured bacteria was full photoinactivation after a-PDT with ZnPcMe. In case of the newly studied complexes, the effect was lower for PdPcC (4 log) as well as for AlPc1 (1.5-2 log). As it is known the bacterial biofilms were more resistant to a-PDT, which was confirmed for A. actinomycetemcomitans biofilms with 3 log reductions of viable cells after treatment with ZnPcMe and approximately 1 log reduction of biofilms after PdPcC and AlPc1. The initial results suggest that a-PDT can be useful for effective inactivation of dental pathogen A. actinomycetemcomitans.

  16. Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism

    PubMed Central

    Weigel, WA; Demuth, DR; Torres-Escobar, A; Juárez-Rodríguez, MD

    2015-01-01

    Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment. PMID:25923132

  17. Inactivation of Aggregatibacter actinomycetemcomitans by two different modalities of photodynamic therapy using Toluidine blue O or Radachlorin as photosensitizers: an in vitro study.

    PubMed

    Moslemi, Neda; Soleiman-Zadeh Azar, Pardis; Bahador, Abbas; Rouzmeh, Nina; Chiniforush, Nasim; Paknejad, Mojgan; Fekrazad, Reza

    2015-01-01

    Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is one of the periodontopathogens strongly associated with aggressive periodontitis. The aim of this investigation was to compare the effect of laser and light-emitting diode on the photodynamic inactivation of A. actinomycetemcomitans. Eighty-four samples of bacterial suspensions (200 μL) were prepared and divided in seven groups: control group (no treatment), laser group (indium-gallium-aluminum-phosphate laser with wavelength of 662 ± 0.1 nm, energy density of 6 j/cm(2), and irradiation time of 34 s), light-emitting diode (LED) group (wavelength 625-635 nm, energy density 6 j/cm(2), time of irradiation 30 s), Toluidine blue O (TBO) group (0.1 mg/mL), Radachlorin group (0.1 %), Radachlorin + laser group (after pre-irradiation time of 10 min, laser was irradiated), and TBO + LED group (after preirradiation time of 10 min, LED was irradiated). Then, 100 μL of each sample was cultured in brain heart infusion (BHI) plates and incubated for 48-72 h in microaerophilic atmosphere for colony counting. Application of Radachlorin + laser resulted in a significant decrease in the concentration of A. actinomycetemcomitans (P values <0.05). Photodynamic therapy with laser + Radachlorin was more effective than that of LED + TBO in suppression of this microorganism (P value <0.05). Within the limits of this study, it can be concluded that photodynamic inactivation using laser and Radachlorin was more effective than that of LED and TBO in eradication of A. actinomycetemcomitans. PMID:24981641

  18. Site-specific subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients.

    PubMed

    Paolantonio, M; Festa, F; di Placido, G; D'Attilio, M; Catamo, G; Piccolomini, R

    1999-04-01

    A high prevalence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque in patients for orthodontia already has been observed. The present study had the following aims: 1) to ascertain a direct relationship between the orthodontic appliance placement and the subgingival colonization by Aa, and 2) to determine whether the Aa growth specifically occurred on teeth with braces attached or whether the presence of orthodontic appliances could also cause the isolation of Aa in teeth free from therapeutic appliances. Twenty-four young systemically and periodontally healthy subjects with malaligned and crowded teeth in the anterior sextants of both dental arches participated in this study. After 1 session of ultrasonic scaling with oral hygiene instructions during the first experimental session, the mesiobuccal sites of the first molars and the distobuccal sites of the lateral incisors in both dental arches in each participant were subjected to clinical and microbiologic examination for the recovery of Aa. Clinical examination consisted of recording the presence of plaque and the examination of gingival bleeding on probing and probing depth. Microbiologic sampling was obtained with the insertion of 3 sterile paper points at the deepest part of each gingival sulcus. Altogether, 192 periodontal sites were examined. After the examinations, the patients received fixed orthodontic appliances in only 1 dental arch (test sites) and the other one was left free from appliances (control sites). Clinical examination and microbiologic sampling were repeated in the same experimental test and control sites after 4, 8, and 12 weeks. At the 12-week session, the orthodontic appliance was removed from the test arch, and, 4 weeks later, a further clinical and microbiologic examination was performed. The results showed that, during the period with orthodontic appliances, the presence of plaque scores and the gingival bleeding on probing scores were increased significantly and that

  19. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.

    PubMed Central

    Carlsson, J; Herrmann, B F; Höfling, J F; Sundqvist, G K

    1984-01-01

    Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin. Images PMID:6198282

  20. ygiW and qseBC are co-expressed in Aggregatibacter actinomycetemcomitans and regulate biofilm growth.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-06-01

    The quorum-sensing Escherichia coli regulators B and C (QseBC) two-component system were previously shown to regulate biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans and to be essential for virulence. In this study, we use RT-PCR to show that an open reading frame, ygiW, residing upstream of qseBC and encoding a hypothetical protein is co-expressed with qseBC. In addition, using a series of lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA Ends (RACE), the promoter that drives expression of the ygiW-qseBC operon and the transcriptional start site was mapped to the 372 bp intergenic region upstream from ygiW. No internal promoters drive qseBC expression independently from ygiW. However, qseBC expression is attenuated by approximately ninefold by a putative attenuator stem-loop (ΔG = -77.0 KJ/mol) that resides in the 137 bp intergenic region between ygiW and qseB. The QseB response regulator activates expression of the ygiW-qseBC operon and transcription from the ygiW promoter is drastically reduced in ΔqseB and ΔqseBC mutants of A. actinomycetemcomitans. In addition, transcriptional activity of the ygiW promoter is significantly reduced in a mutant expressing an in-frame deletion of qseC that lacks the sensor domain of QseC, suggesting that a periplasmic signal is required for QseB activation. Finally, a non-polar in-frame deletion in ygiW had little effect on biofilm depth but caused a significant increase in surface coverage relative to wild-type. Complementation of the mutant with a plasmid-borne copy of ygiW reduced surface coverage back to wild-type levels. Interestingly, deletion of the sensor domain of QseC or of the entire qseC open reading frame resulted in significant reductions in biofilm depth, biomass and surface coverage, indicating that the sensor domain is essential for optimal biofilm formation by A. actinomycetemcomitans. Thus, although ygiW and qseBC are co-expressed, they regulate biofilm

  1. Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

    PubMed

    Im, Jintaek; Baik, Jung Eun; Kim, Kyoung Whun; Kang, Seok-Seong; Jeon, Jun Ho; Park, Ok-Jin; Kim, Hyun Young; Kum, Kee-Yeon; Yun, Cheol-Heui; Han, Seung Hyun

    2015-08-01

    Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

  2. Aggregatibacter actinomycetemcomitans leukotoxin (LtxA; Leukothera) induces cofilin dephosphorylation and actin depolymerization during killing of malignant monocytes.

    PubMed

    Kaur, Manpreet; Kachlany, Scott C

    2014-11-01

    Leukotoxin (LtxA; Leukothera), a protein toxin secreted by the oral bacterium Aggregatibacter actinomycetemcomitans, specifically kills white blood cells (WBCs). LtxA binds to the receptor known as lymphocyte function associated antigen-1 (LFA-1), a β2 integrin expressed only on the surface of WBCs. LtxA is being studied as a virulence factor that helps A. actinomycetemcomitans evade host defences and as a potential therapeutic agent for the treatment of WBC diseases. LtxA-mediated cell death in monocytes involves both caspases and lysosomes; however, the signalling proteins that regulate and mediate cell death remain largely unknown. We used a 2D-gel proteomics approach to analyse the global protein expression changes that occur in response to LtxA. This approach identified the protein cofilin, which underwent dephosphorylation upon LtxA treatment. Cofilin is a ubiquitous actin-binding protein known to regulate actin dynamics and is regulated by LIM kinase (LIMK)-mediated phosphorylation. LtxA-mediated cofilin dephosphorylation was dependent on LFA-1 and cofilin dephosphorylation did not occur when LFA-1 bound to its natural ligand, ICAM-1. Treatment of cells with an inhibitor of LIMK (LIMKi) also led to cofilin dephosphorylation and enhanced killing by LtxA. This enhanced sensitivity to LtxA coincided with an increase in lysosomal disruption, and an increase in LFA-1 surface expression and clustering. Both LIMKi and LtxA treatment also induced actin depolymerization, which could play a role in trafficking and surface distribution of LFA-1. We propose a model in which LtxA-mediated cofilin dephosphorylation leads to actin depolymerization, LFA-1 overexpression/clustering, and enhanced lysosomal-mediated cell death.

  3. A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis

    PubMed Central

    Savage, Justin R.; Pulsipher, Abigail; Rao, Narayanam V.; Kennedy, Thomas P.; Prestwich, Glenn D.; Ryan, Maria E.; Lee, Won Yong

    2016-01-01

    Background Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. Methods We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. Results (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1–10 ng/mL vs. > 100 μg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. Conclusions We report that GM-0111 inhibits multiple molecular events involved in

  4. Association between periodontal disease and plasma levels of cholesterol and triglycerides

    PubMed Central

    Lafaurie, Gloria Inés; Millán, Lina Viviana; Ardila, Carlos Martin; Duque, Andrés; Novoa, Camilo; López, Diego; Contreras, Adolfo

    2013-01-01

    Objective: untreated periodontal disease seems to cause low grade systemic inflammation and blood lipid alteration leading to increased cardiovascular disease risk. To start testing this hypothesis in colombian patients, a multicentre study was conducted including the three main state capitals: bogota, medellin and cali. Methods: in this study 192 (28.4%) advanced and 256 (37.8%) moderate periodontitis patients were investigated for socio-demographic variables, city of precedence, periodontal parameters, smoking, red complex periodontopathic bacteria, serum antibodies against porphyromonas gingivalis and aggregatibacter actinomycetemcomitans and blood lipids including total cholesterol, hdl, ldl and triglycerides (tg). Those parameters were compared to 229 (33.8%) controls having periodontal health or gingivitis. Results: advanced periodontitis had worst periodontal indexes, than moderate periodontitis and controls. Interestingly, higher hdl and tg levels were present in periodontitis. Bmi <30 and smoking were associated with increased hdl, hdl-35, ldl and tg, while glycemia >100 mg/dl associated with hdl, hdl-35 and tg. Tannerella forsythia showed a significant association with hdl-35 in bivariate analysis and serum igg1 against p. Gingivalis associated with hdl-35 and serum igg1 against t. Forsythia associated with tg and serum igg2 against a. Actinomycetemcomitans correlated with levels of hdl y hdl-35. In logistic regression the periodontitis patients from cali presented reduced hdl levels as compared to bogota and medellin patients. Presence of igg1 antibodies against p. Gingivalis and a. Actinomycetemcomitans correlated with reduced hdl levels. Conclusion: this study confirmed that untreated periodontitis generates alteration in serum lipid levels and systemic bacterial exposure against important periodontopathic bacteria could be the biological link. PMID:24892452

  5. Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

    SciTech Connect

    Layman, D.L.; Diedrich, D.L.

    1987-06-01

    Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by /sup 3/H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in /sup 3/H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin.

  6. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

    PubMed Central

    Rashidi Maybodi, Fahimeh; Haerian Ardakani, Ahmad; Fattahi Bafghi, Ali; Haerian Ardakani, Alireza; Zafarbakhsh, Akram

    2016-01-01

    Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis. PMID:27602391

  7. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

    PubMed Central

    Rashidi Maybodi, Fahimeh; Haerian Ardakani, Ahmad; Fattahi Bafghi, Ali; Haerian Ardakani, Alireza; Zafarbakhsh, Akram

    2016-01-01

    Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis.

  8. Effectiveness of ozone against periodontal pathogenic microorganisms.

    PubMed

    Huth, Karin C; Quirling, Martina; Lenzke, Stefanie; Paschos, Ekaterini; Kamereck, Klaus; Brand, Korbinian; Hickel, Reinhard; Ilie, Nicoleta

    2011-06-01

    Ozone has been proposed as an adjunct antiseptic in periodontitis therapy. The aim of this study was to investigate the antimicrobial effectiveness of gaseous/aqueous ozone, in comparison with that of the established antiseptic chlorhexidine digluconate (CHX), against periodontal microorganisms. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Parvimonas micra in planktonic or biofilm cultures were exposed, for 1 min, to gaseous ozone, aqueous ozone, CHX, or phosphate-buffered saline (control). None of the agents was able to substantially reduce the A. actinomycetemcomitans count in biofilm cultures. In contrast, P. gingivalis, T. forsythia, and P. micra could be eliminated by 2% CHX or by ozone gas at 53 gm(-3) . Significantly greater antimicrobial effects were observed against planktonic cultures than against biofilm-associated bacteria. The rate of killing was influenced by the species of bacteria, and by the type and concentration of agent. There were no significant differences in the effectiveness of aqueous ozone (20 μg ml(-1) ) or gaseous ozone (≥ 4 gm(-3) ) compared with 2% CHX but they were more effective than 0.2% CHX. Therefore, high-concentrated gaseous and aqueous ozone merit further investigation as antiseptics in periodontitis therapy. A safe system for applying gaseous ozone into the periodontal pocket that avoids inhalation still needs to be developed.

  9. Determination of periodontopathogens in patients with Cri du chat syndrome

    PubMed Central

    Ballesta-Mudarra, Sofía; Torres-Lagares, Daniel; Rodríguez-Caballero, Ángela; Yáñez-Vico, Rosa M.; Solano-Reina, Enrique; Perea-Pérez, Evelio

    2013-01-01

    Objectives: Cri du chat syndrome is a genetic alteration associated with some oral pathologies. However, it has not been described previously any clinical relationship between the periodontal disease and the syndrome. The purpose of this comparative study was to compare periodontopathogenic flora in a group with Cri du chat syndrome and another without the síndrome, to assess a potential microbiological predisposition to suffer a periodontitis. Study Design: The study compared nineteen subjects with Cri du chat Syndrome with a control group of nineteen patients without it. All patients were clinically evaluated by periodontal probing, valuing the pocket depth, the clinical attachmente level and bleeding on probing. There were no significant differences between both groups. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola were detected by multiplex-PCR using 16S rDNA (microIDENT). Results: When A. actinomycetemcomitans, P. gingivalis, P. intermedia and T. denticola were compared, no statistically significant differences were found between the two groups (p>0.05). The value of T. forsythia was significantly higher for Cri du chat syndrome (31.6%) than for the control group (5.3%). The odds ratio for T. forsythia was 8.3. Conclusions: In the present study T. forsythia is associated with Cri du chat syndrome subjects and not with healthy subjects. Key words:Cri du Chat syndrome, periodontal health, microbiology, special care dentistry. PMID:24121919

  10. The Comparative Evaluation of the Antimicrobial Effect of Propolis with Chlorhexidine against Oral Pathogens: An In Vitro Study

    PubMed Central

    Akca, Gülçin; Topçu, Fulya Toksoy; Macit, Enis; Pikdöken, Levent; Özgen, I. Şerif

    2016-01-01

    This study aimed to compare the antimicrobial effectiveness of ethanolic extract of propolis (EEP) to chlorhexidine gluconate (CHX) on planktonic Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus salivarius subsp. salivarius, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Staphylococcus aureus, Enterococcus faecalis, Actinomyces israelii, Candida albicans, and their single-species biofilms by agar dilution and broth microdilution test methods. Both agents inhibited the growth of all planktonic species. On the other hand, CHX exhibited lower minimum bactericidal concentrations than EEP against biofilms of A. actinomycetemcomitans, S. aureus, and E. faecalis whereas EEP yielded a better result against Lactobacilli and P. intermedia. The bactericidal and fungicidal concentrations of both agents were found to be equal against biofilms of Streptecocci, P. gingivalis, A. israelii, and C. albicans. The results of this study revealed that propolis was more effective in inhibiting Gram-positive bacteria than the Gram-negative bacteria in their planktonic state and it was suggested that EEP could be as effective as CHX on oral microorganisms in their biofilm state. PMID:26949701

  11. Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

    PubMed Central

    Yamaguchi, N; Yamashita, Y; Ikeda, D; Koga, T

    1996-01-01

    Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes. PMID:8698480

  12. Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans.

    PubMed

    Helgeland, K; Nordby, O

    1993-05-01

    A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired. PMID:8496779

  13. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    PubMed Central

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution. Images PMID:2895100

  14. Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

    PubMed Central

    Abrahamson, M; Wikström, M; Potempa, J; Renvert, S; Hall, A

    1997-01-01

    AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in

  15. Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50.

    PubMed Central

    McDermid, A S; McKee, A S; Marsh, P D

    1988-01-01

    Since the pH of the gingival crevice increases from below neutrality in health to above pH 8 in disease, we decided to investigate the effect of environmental pH on the growth and enzyme activity of Bacteroides gingivalis W50. Cells were grown in a chemostat under hemin-excess conditions over a range of pH values; stable growth was observed only between pH 6.7 and 8.3, with the maximum yields obtained between pH 7.0 and 8.0. The enzyme profile of cells varied markedly with pH. Enzymes with a specificity for gingival connective tissue (collagenase, hyaluronidase) were produced optimally at or below neutral pH, whereas trypsinlike activity increased with the growth pH and was maximal at pH 8.0. Chymotrypsinlike activity was generally low, although its activity was highest at the extremes of growth pH, i.e., at pH 6.7 and 8.3. Inhibitor studies provided evidence that the breakdown of collagen involved the concerted action of both a collagenase and the trypsinlike enzyme. The ratio of trypsin to collagenolytic activity rose from 1:1 during growth at neutral pH and below to almost 7:1 during growth at pH 8.3. Thus B. gingivalis appears to be uniquely adapted as a periodontopathic organism in that under environmental conditions likely to prevail during the initial stages of pocket development it produces maximally those enzymes with a tissue-damaging potential. Then, as the pH of the pocket rises during the host inflammatory response, the activity of the trypsinlike enzyme increases markedly, which may enable cells to inactivate key components of the host defenses such as immunoglobulins and complement. PMID:3281900

  16. Antimicrobial Activity of Protamine against Oral Microorganisms.

    PubMed

    Kim, Yeon-Hee; Kim, Sang Moo; Lee, Si Young

    2015-01-01

    Protamine is an arginine-rich polycationic protein extracted from sperm cells of vertebrates including fishes such as salmon. The purpose of this study was to investigate the suppressive effects of protamine on the growth of oral pathogens for possible usage in dental materials. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the microdilution method. Twelve strains of oral viridans streptococci, Actinomyces naeslundii, Actinomyces odontolyticus, Enterococcus faecalis, Lactobacillus acidophilus, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Candida albicans were suppressed by protamine. MIC and MBC values were between 0.009 ~ 20 mg/mL and 0.019 ~ 80 mg/mL, respectively. The bactericidal activities of protamine against susceptible bacterial species were dependent on the concentration of protamine and incubation time. Based on the results of this study, protamine would be a useful compound for the development of antimicrobial agents against oral pathogens in dental materials.

  17. Antibacterial Efficacy of Exogenous Nitric Oxide on Periodontal Pathogens

    PubMed Central

    Backlund, C.J.; Sergesketter, A.R.; Offenbacher, S.; Schoenfisch, M.H.

    2014-01-01

    Current treatments for periodontitis (e.g., scaling/root planing and chlorhexidine) have limited efficacy since they fail to suppress microbial biofilms satisfactorily over time, and the use of adjunctive antimicrobials can promote the emergence of antibiotic-resistant organisms. Herein, we report the novel application of nitric oxide (NO)-releasing scaffolds (i.e., dendrimers and silica particles) as anti-periodontopathogenic agents. The effectiveness of macromolecular NO release was demonstrated by a 3-log reduction in periodontopathogenic Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis viability. In contrast, Streptococcus mutans and Streptococcus sanguinis, caries-associated organisms, were substantially less sensitive to NO treatment. Both dendrimer- and silica-based NO release exhibited substantially less toxicity to human gingival fibroblasts at concentrations necessary to eradicate periodontopathogens than did clinical concentrations of chlorhexidine. These results suggest the potential utility of macromolecular NO-release scaffolds as a novel platform for the development of periodontal disease therapeutics. PMID:25139363

  18. Evaluation of antimicrobial action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans a major periodontal pathogen using polymerase chain reaction

    PubMed Central

    Kush, Anil; Thakur, Rachna; Patil, Sandya Devi S.; Paul, Santhosh T.; Kakanur, Madhu

    2015-01-01

    Background: In the present scenario, we are made available with chemomechanical caries removal system containing a natural proteolytic enzyme for the ease in the excavation of infected dentine. The additive action for these agents is providing antimicrobial and anti-inflammatory properties. Aim: This study was undertaken for assessing the action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans. Materials and Methods: The samples were collected for cultivation of the periodontal pathogen from the clinical periodontal pockets using sterile paper points. The samples cultured under suitable conditions were analyzed with quantitative polymerase chain reaction targeting 16s r-DNA. The samples were divided into three groups namely, Group A: Control, Group B: With Papacarie Duo, Group C: With Carie Care. The pathogen inoculums plugs were inserted in the petri dishes containing chemically defined medium and the experimental gels at different concentrations and were incubated under optimal conditions. The inhibition of growth of the pathogen was studied visually. Results: There was visual inhibition of growth for Group B and C and also exhibited a dose-dependent effect also. Conclusion: Based on the results of the present study, Carie Care™ gel demonstrated better antimicrobial action against A. actinomycetemcomitans which is a major periodontal disease causing pathogen. PMID:26681861

  19. Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization - mass spectrometry

    NASA Astrophysics Data System (ADS)

    Bregy, Lukas; Müggler, Annick R.; Martinez-Lozano Sinues, Pablo; García-Gómez, Diego; Suter, Yannick; Belibasakis, Georgios N.; Kohler, Malcolm; Schmidlin, Patrick R.; Zenobi, Renato

    2015-10-01

    The detection of bacterial-specific volatile metabolites may be a valuable tool to predict infection. Here we applied a real-time mass spectrometric technique to investigate differences in volatile metabolic profiles of oral bacteria that cause periodontitis. We coupled a secondary electrospray ionization (SESI) source to a commercial high-resolution mass spectrometer to interrogate the headspace from bacterial cultures and human saliva. We identified 120 potential markers characteristic for periodontal pathogens Aggregatibacter actinomycetemcomitans (n = 13), Porphyromonas gingivalis (n = 70), Tanerella forsythia (n = 30) and Treponema denticola (n = 7) in in vitro cultures. In a second proof-of-principle phase, we found 18 (P. gingivalis, T. forsythia and T. denticola) of the 120 in vitro compounds in the saliva from a periodontitis patient with confirmed infection with P. gingivalis, T. forsythia and T. denticola with enhanced ion intensity compared to two healthy controls. In conclusion, this method has the ability to identify individual metabolites of microbial pathogens in a complex medium such as saliva.

  20. Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization - mass spectrometry.

    PubMed

    Bregy, Lukas; Müggler, Annick R; Martinez-Lozano Sinues, Pablo; García-Gómez, Diego; Suter, Yannick; Belibasakis, Georgios N; Kohler, Malcolm; Schmidlin, Patrick R; Zenobi, Renato

    2015-01-01

    The detection of bacterial-specific volatile metabolites may be a valuable tool to predict infection. Here we applied a real-time mass spectrometric technique to investigate differences in volatile metabolic profiles of oral bacteria that cause periodontitis. We coupled a secondary electrospray ionization (SESI) source to a commercial high-resolution mass spectrometer to interrogate the headspace from bacterial cultures and human saliva. We identified 120 potential markers characteristic for periodontal pathogens Aggregatibacter actinomycetemcomitans (n = 13), Porphyromonas gingivalis (n = 70), Tanerella forsythia (n = 30) and Treponema denticola (n = 7) in in vitro cultures. In a second proof-of-principle phase, we found 18 (P. gingivalis, T. forsythia and T. denticola) of the 120 in vitro compounds in the saliva from a periodontitis patient with confirmed infection with P. gingivalis, T. forsythia and T. denticola with enhanced ion intensity compared to two healthy controls. In conclusion, this method has the ability to identify individual metabolites of microbial pathogens in a complex medium such as saliva.

  1. Antimicrobial efficacy of Tulsi leaf (Ocimum sanctum) extract on periodontal pathogens: An in vitro study

    PubMed Central

    Mallikarjun, Sajjanshetty; Rao, Ashwini; Rajesh, Gururaghavendran; Shenoy, Ramya; Pai, Mithun

    2016-01-01

    Background: Periodontitis is an infection of the periodontal complex with severe forms of disease associated with specific bacteria colonizing the subgingival area. Widespread use of drugs has resulted in the emergence of side effects, uncommon infections, and resistance. Plant medicine like Tulsi has been used in many clinical conditions, and it appears to be a suitable alternative to manage conditions affecting the oral cavity. Hence, the objective was to assess the in vitro antimicrobial activity of Tulsi leaves extract (Ocimum sanctum) on periodontal pathogens with doxycycline as standard, as doxycycline has been used as an adjunct to nonsurgical therapy in periodontitis patients. Materials and Methods: Ethanolic extract of Tulsi was prepared by cold extraction method. Extract was diluted with an inert solvent, dimethyl formamide, to obtain five different concentrations (0.5%, 1%, 2%, 5%, and 10%). Doxycycline was used as a positive control and dimethyl formamide, as a negative control. The extract and controls were subjected to the microbiological investigation against Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Porphyromonas gingivalis. Agar well diffusion method was employed to determine the concentration at which Tulsi gave an inhibition zone, similar to doxycycline. Data were analyzed using one-way analysis of variance and Tukey post-hoc test was used for inter- and intra-group comparisons. Results: At 5% and 10% concentrations, Tulsi extracts demonstrated antimicrobial activity against A. actinomycetemcomitans, similar to doxycycline with similar inhibition zones (P > 0.05). P. gingivalis and P. intermedia, however, exhibited resistance to Tulsi extract that showed significantly smaller inhibition zones (P < 0.05). Conclusions: Tulsi demonstrated effective antimicrobial property against A. actinomycetemcomitans, suggesting its possible use as an effective and affordable “adjunct” along with the standard care in the management of

  2. Colonization and Persistence of Labeled and “Foreign” Strains of Aggregatibacter actinomycetemcomitans Inoculated into the Mouths of Rhesus Monkeys

    PubMed Central

    Fine, Daniel H.; Karched, Maribasappa; Furgang, David; Sampathkumar, Vandana; Velusamy, Senthil; Godboley, Dipti

    2015-01-01

    Aggregatibacter actinomycetemcomitans (Aa) is a pathobiont and part of a consortium of bacteria that can lead to periodontitis in humans. Our aim was to develop a model for oral inoculation of labeled Aa into a suitable host in order to study Aa traits and ecological factors that either enhance or repress its persistence. Primate species were screened for Aa to select a host for colonization studies. Macaca mulatta (Rhesus/Rh) was selected. Rh Aa strains were isolated, subjected to sequencing and functional analysis for comparison to human strains. “Best” methods for microbial decontamination prior to inoculation were assessed. Three groups were studied; Group 1 (N=5) was inoculated with Aa Spectinomycin resistant (SpecR) Rh strain 4.35, Group 2 (N=5) inoculated with Aa SpecR human strain IDH 781, and Group 3 (N=5) the un-inoculated control. Repeated feeding with pancakes spiked with SpecRAa followed high dose oral inoculation. Cheek, tongue, and plaque samples collected at baseline 1, 2, 3, and 4 weeks after inoculation were plated on agar; 1) selective for Aa, 2) enriched for total counts, and 3) containing 50 µg/ml of Spec. Aa was identified by colonial morphology and DNA analysis. Rh and human Aa had > 93–98 % genome identity. Rh Aa attached to tissues better than IDH 781 in vitro (p < 0.05). SpecR IDH 781 was not recovered from any tissue at any time; whereas, RhSpecR 4.35 was detected in plaque, but never tongue or cheek, in all monkeys at all times (> 1 × 105 colonies/ml; p < 0.001). In conclusion, the primate model provides a useful platform for studying integration of Aa strains into a reduced but established oral habitat. Primate derived SpecRAa was consistently detected in plaque at all collection periods; however, human derived Aa was never detected. The model demonstrated both microbial as well as tissue specificity. PMID:26213715

  3. Phototoxic effect of blue light on the planktonic and biofilm state of anaerobic periodontal pathogens

    PubMed Central

    Song, Hyun-Hwa; Lee, Jae-Kwan; Um, Heung-Sik; Lee, Si-Young; Lee, Min-Ku

    2013-01-01

    Purpose The purpose of this study was to compare the phototoxic effects of blue light exposure on periodontal pathogens in both planktonic and biofilm cultures. Methods Strains of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis, in planktonic or biofilm states, were exposed to visible light at wavelengths of 400.520 nm. A quartz-tungsten-halogen lamp at a power density of 500 mW/cm2 was used for the light source. Each sample was exposed to 15, 30, 60, 90, or 120 seconds of each bacterial strain in the planktonic or biofilm state. Confocal scanning laser microscopy (CSLM) was used to observe the distribution of live/dead bacterial cells in biofilms. After light exposure, the bacterial killing rates were calculated from colony forming unit (CFU) counts. Results CLSM images that were obtained from biofilms showed a mixture of dead and live bacterial cells extending to a depth of 30-45 µm. Obvious differences in the live-to-dead bacterial cell ratio were found in P. gingivalis biofilm according to light exposure time. In the planktonic state, almost all bacteria were killed with 60 seconds of light exposure to F. nucleatum (99.1%) and with 15 seconds to P. gingivalis (100%). In the biofilm state, however, only the CFU of P. gingivalis demonstrated a decreasing tendency with increasing light exposure time, and there was a lower efficacy of phototoxicity to P. gingivalis as biofilm than in the planktonic state. Conclusions Blue light exposure using a dental halogen curing unit is effective in reducing periodontal pathogens in the planktonic state. It is recommended that an adjunctive exogenous photosensitizer be used and that pathogens be exposed to visible light for clinical antimicrobial periodontal therapy. PMID:23678390

  4. Nucleotide sequence of the SrRNA gene of Entamoeba gingivalis: applications for construction of a species-specific DNA probe and phylogenetic analysis.

    PubMed

    Yamamoto, A; Kikuta, N; Hashimoto, T; Oyaizu, H; Goto, N

    1995-01-01

    The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozons or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.

  5. Isolation and some properties of exohemagglutinin from the culture medium of Bacteroides gingivalis 381.

    PubMed Central

    Inoshita, E; Amano, A; Hanioka, T; Tamagawa, H; Shizukuishi, S; Tsunemitsu, A

    1986-01-01

    Exohemagglutinin was found in the culture medium of Bacteroides gingivalis 381. Exohemagglutinin was purified 3,150-fold from culture fluid by ultracentrifugation followed by gel filtration on Sepharose CL-4B and by affinity chromatography on arginine-agarose. Examination of the final preparation of exohemagglutinin by biochemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the isolated exohemagglutinin contained three major proteins but not a detectable lipopolysaccharide. Hemagglutination inhibition experiments showed that the activity of exohemagglutinin was inhibited by L-arginine and the arginine-containing peptides, although the activity was unaffected by the sugars tested. Some protein and glycoproteins that were examined also exhibited the inhibitory activity. When the bovine submaxillary mucin was chemically modified by beta-elimination and bovine serum albumin was modified by guanidination, the inhibitory effects on hemagglutination were significantly enhanced. These results suggest that the hemagglutination of the isolated exohemagglutinin may be involved in arginine residues as components of ligand-binding sites on erythrocytes. Images PMID:3699890

  6. Lipopolysaccharide of Aggregatibacter actinomycetemcomitans induces the expression of chemokines MCP-1, MIP-1α, and IP-10 via similar but distinct signaling pathways in murine macrophages.

    PubMed

    Park, Ok-Jin; Cho, Min-Kyung; Yun, Cheol-Heui; Han, Seung Hyun

    2015-09-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium frequently isolated from lesions of patients with localized aggressive periodontitis. Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS from A. actinomycetemcomitans (AaLPS) and investigated its ability to induce the expression of chemokines, which play an important role in recruitment of leukocytes to the infection site. AaLPS induced the expression of chemokines, MCP-1, MIP-1α, and IP-10 in murine macrophages, leading to the infiltration of peripheral blood mononuclear cells in a transwell system. Although TLR4 was essential for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α expressions were MyD88-dependent, but IP-10 expression was MyD88-independent, as determined using macrophages from mice deficient in TLR4 or MyD88. Furthermore, the activation of ERK and JNK were necessary for the expression of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were required for IP-10 expression. In addition, IFN-β/STAT1 signaling was exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α expression. AaLPS also activated the transcription factors, NF-κB, AP-1, NF-IL6, and ISRE, all of which are involved in chemokine gene expression. These results suggest that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and IP-10 through TLR4 in murine macrophages. Further, the induction of MCP-1 and MIP-1α requires MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 MAP kinase, and IFN-β/STAT1.

  7. Correlation between Either Cupriavidus or Porphyromonas and Primary Pulmonary Tuberculosis Found by Analysing the Microbiota in Patients’ Bronchoalveolar Lavage Fluid

    PubMed Central

    Zhou, Yuhua; Lin, Feishen; Cui, Zelin; Zhang, Xiangrong; Hu, Chunmei; Shen, Tian; Chen, Chunyan; Zhang, Xia; Guo, Xiaokui

    2015-01-01

    Pulmonary tuberculosis (TB) has gained attention in recent decades because of its rising incidence trend; simultaneously, increasing numbers of studies have identified the relationship between microbiota and chronic infectious diseases. In our work, we enrolled 32 patients with primary TB characterised by unilateral TB lesion formation diagnosed by chest radiographic exam. Bronchoalveolar lavage fluid was taken from both lungs. Twenty-four healthy people were chosen as controls. Pyrosequencing was performed on the V3 hypervariable region of 16S rDNA in all bacterial samples and used as a culture-independent method to describe the phylogenetic composition of the microbiota. Through pyrosequencing, 271,764 amplicons were detected in samples and analysed using tools in the Ribosomal Database Project (RDP) and bioinformatics. These analyses revealed significant differences in the microbiota in the lower respiratory tract (LRT) of TB patients compared with healthy controls; in contrast, the microbiota of intra/extra-TB lesions were similar. These results showed that the dominant bacterial genus in the LRT of TB patients was Cupriavidus and not Streptococcus, which resulted in a significant change in the microbiota in TB patients. The abundance of Mycobacteria and Porphyromonas significantly increased inside TB lesions when compared with non-lesion-containing contralateral lungs. From these data, it can be concluded that Cupriavidus plays an important role in TB’s secondary infection and that in addition to Mycobacteria, Porphyromonas may also be a co-factor in lesion formation. The mechanisms underlying this connection warrant further research. PMID:26000957

  8. Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

    PubMed Central

    Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

    1991-01-01

    The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

  9. Antibacterial activity of moxifloxacin on bacteria associated with periodontitis within a biofilm.

    PubMed

    Tsaousoglou, Phoebus; Nietzsche, Sandor; Cachovan, Georg; Sculean, Anton; Eick, Sigrun

    2014-02-01

    The activity of moxifloxacin was compared with ofloxacin and doxycycline against bacteria associated with periodontitis within a biofilm (single strain and mixed population) in vitro. MICs and minimal bactericidal concentrations (MBCs) of moxifloxacin, ofloxacin and doxycyline were determined against single strains and mixed populations in a planktonic state. Single-species biofilms of two Porphyromonas gingivalis and two Aggregatibacter actinomycetemcomitans strains and a multispecies biofilm consisting of 12 species were formed for 3 days. The minimal biofilm eradication concentrations (MBECs) were determined after exposing the biofilms to the antibacterials (0.002-512 µg ml(-1)) for 18 h, addition of nutrient broth for 3 days and subsequent subcultivation. Photographs were taken using confocal laser-scanning microscopy and scanning electron microscopy. The MICs and MBCs did not differ between ofloxacin and moxifloxacin against A. actinomycetemcomitans, whilst moxifloxacin was more active than the other tested antibacterials against anaerobes and the mixed population. The single-species biofilms were eradicated by moderate concentrations of the antibacterials, and the lowest MBECs were always found for moxifloxacin (2-8 µg ml(-1)). MBECs against the multispecies biofilms were 128, >512 and >512 µg ml(-1) for moxifloxacin, ofloxacin and doxycycline, respectively. In summary, moxifloxacin in a topical formulation may have potential as an adjunct to mechanical removal of the biofilms.

  10. Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

    PubMed

    Fyrestam, Jonas; Bjurshammar, Nadja; Paulsson, Elin; Johannsen, Annsofi; Östman, Conny

    2015-09-01

    Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans. PMID:26168965

  11. Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

    PubMed

    Fyrestam, Jonas; Bjurshammar, Nadja; Paulsson, Elin; Johannsen, Annsofi; Östman, Conny

    2015-09-01

    Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylpo