Science.gov

Sample records for porphyromonas gingivalis haemoglobin

  1. Neutralization of toxic haem by Porphyromonas gingivalis haemoglobin receptor.

    PubMed

    Nhien, Nguyen Thanh Thuy; Huy, Nguyen Tien; Naito, Mariko; Oida, Tatsuo; Uyen, Dinh Thanh; Huang, Mingguo; Kikuchi, Mihoko; Harada, Shigeharu; Nakayama, Koji; Hirayama, Kenji; Kamei, Kaeko

    2010-03-01

    Free haem is known to be toxic to organs, tissues and cells. It enhances permeability by binding to a cell membrane, which leads to cell death, and damages lipids, proteins and DNA through the generation of reactive oxygen species. Lysine- and arginine-specific gingipains (Kgp and RgpA/B) are major proteinases that play an important role in the pathogenicity of a black-pigmented periodontopathogen named Porphyromonas gingivalis. One of the adhesin domains of gingipain, HbR could bind haem as an iron nutrient source for P. gingivalis. Using erythrocyte and its membrane as a model, results from the present study demonstrate that recombinant HbR expressed in Escherichia coli could inhibit haem-induced haemolysis, probably through removing haem from the haem-membrane complex and lowering free haem toxicity by mediating dimerization of haem molecules. The ability to protect a cell membrane from haem toxicity is a new function for HbR. PMID:19861401

  2. Genetic manipulation of Porphyromonas gingivalis.

    PubMed

    Bélanger, Myriam; Rodrigues, Paulo; Progulske-Fox, Ann

    2007-06-01

    Porphyromonas gingivalis, an oral anaerobic bacterium, is an important etiological agent of periodontal disease and may contribute to cardiovascular disease, preterm birth, and diabetes as well. Therefore, genetic studies are of crucial importance in investigating molecular mechanisms of P. gingivalis virulence. Although molecular genetic tools have been available for many bacterial species for some time, genetic manipulations of Porphyromonas species were not developed until more recently and remain limited. In this unit, current molecular genetic approaches for mutant construction in P. gingivalis using the suicide vector pPR-UF1 and the transposon Tn4351 are described, as are protocols for performing electroporation and conjugation. Furthermore, a technique to restore the wild-type phenotype of the mutant by complementation using vector pT-COW is provided. Finally, a description of a noninvasive reporter system allowing the study of gene expression and regulation in P. gingivalis completes this unit. PMID:18770611

  3. Gingipain aminopeptidase activities in Porphyromonas gingivalis

    PubMed Central

    Veillard, Florian; Potempa, Barbara; Poreba, Marcin; Drag, Marcin; Potempa, Jan

    2014-01-01

    Bestatin, a specific inhibitor of metalloaminopeptidases, inhibits the growth of Porphyromonas gingivalis. To identify its target enzyme, a library of fluorescent substrates was used but no metalloaminopeptidase activity was found. All aminopeptidase activity of P. gingivalis was bestatin-insensitive and directed exclusively toward N-terminal arginine and lysine substrates. Class-specific inhibitors and gingipain-null mutants showed that gingipains were the only enzymes responsible for this activity. The kinetic constants obtained for Rgps were comparable to those of human aminopeptidases but Kgp aminopeptidase activity was weaker. This finding reveals a new role for gingipains as aminopeptidases in degradation of proteins and peptides P. gingivalis. PMID:23667904

  4. Functional Advantages of Porphyromonas gingivalis Vesicles

    PubMed Central

    Ho, Meng-Hsuan; Chen, Chin-Ho; Goodwin, J. Shawn; Wang, Bing-Yan; Xie, Hua

    2015-01-01

    Porphyromonas gingivalis is a keystone pathogen of periodontitis. Outer membrane vesicles (OMVs) have been considered as both offense and defense components of this bacterium. Previous studies indicated that like their originating cells, P. gingivalis vesicles, are able to invade oral epithelial cells and gingival fibroblasts, in order to promote aggregation of some specific oral bacteria and to induce host immune responses. In the present study, we investigated the invasive efficiency of P. gingivalis OMVs and compared results with that of the originating cells. Results revealed that 70–90% of human primary oral epithelial cells, gingival fibroblasts, and human umbilical vein endothelial cells carried vesicles from P. gingivalis 33277 after being exposed to the vesicles for 1 h, while 20–50% of the host cells had internalized P. gingivalis cells. We also detected vesicle-associated DNA and RNA and a vesicle-mediated horizontal gene transfer in P. gingivalis strains, which represents a novel mechanism for gene transfer between P. gingivalis strains. Moreover, purified vesicles of P. gingivalis appear to have a negative impact on biofilm formation and the maintenance of Streptococcus gordonii. Our results suggest that vesicles are likely the best offence weapon of P. gingivalis for bacterial survival in the oral cavity and for induction of periodontitis. PMID:25897780

  5. 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

  6. 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

  7. 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

  8. Invasion of Porphyromonas gingivalis strains into vascular cells and tissue

    PubMed Central

    Olsen, Ingar; Progulske-Fox, Ann

    2015-01-01

    Porphyromonas gingivalis is considered a major pathogen in adult periodontitis and is also associated with multiple systemic diseases, for example, cardiovascular diseases. One of its most important virulence factors is invasion of host cells. The invasion process includes attachment, entry/internalization, trafficking, persistence, and exit. The present review discusses these processes related to P. gingivalis in cardiovascular cells and tissue. Although most P. gingivalis strains invade, the invasion capacity of strains and the mechanisms of invasion including intracellular trafficking among them differ. This is consistent with the fact that there are significant differences in the pathogenicity of P. gingivalis strains. P. gingivalis invasion mechanisms are also dependent on types of host cells. Although much is known about the invasion process of P. gingivalis, we still have little knowledge of its exit mechanisms. Nevertheless, it is intriguing that P. gingivalis can remain viable in human cardiovascular cells and atherosclerotic plaque and later exit and re-enter previously uninfected host cells. PMID:26329158

  9. 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

  10. Porphyromonas gingivalis-host interactions in a Drosophila melanogaster model.

    PubMed

    Igboin, Christina O; Tordoff, Kevin P; Moeschberger, Melvin L; Griffen, Ann L; Leys, Eugene J

    2011-01-01

    Porphyromonas gingivalis is a Gram-negative obligate anaerobe that has been implicated in the etiology of adult periodontitis. We recently introduced a Drosophila melanogaster killing model for examination of P. gingivalis-host interactions. In the current study, the Drosophila killing model was used to characterize the host response to P. gingivalis infection by identifying host components that play a role during infection. Drosophila immune response gene mutants were screened for altered susceptibility to killing by P. gingivalis. The Imd signaling pathway was shown to be important for the survival of Drosophila infected by nonencapsulated P. gingivalis strains but was dispensable for the survival of Drosophila infected by encapsulated P. gingivalis strains. The P. gingivalis capsule was shown to mediate resistance to killing by Drosophila antimicrobial peptides (Imd pathway-regulated cecropinA and drosocin) and human beta-defensin 3. Drosophila thiol-ester protein II (Tep II) and Tep IV and the tumor necrosis factor (TNF) homolog Eiger were also involved in the immune response against P. gingivalis infection, while the scavenger receptors Eater and Croquemort played no roles in the response to P. gingivalis infection. This study demonstrates that the Drosophila killing model is a useful high-throughput model for characterizing the host response to P. gingivalis infection and uncovering novel interactions between the bacterium and the host. PMID:21041486

  11. Functional properties of nonhuman primate antibody to Porphyromonas gingivalis.

    PubMed Central

    Anderson, D M; Ebersole, J L; Novak, M J

    1995-01-01

    The nonhuman primate (NHP) serves as a useful model for examining the host-parasite interactions in Porphyromonas gingivalis-associated periodontal disease. This study determined the influence of NHP sera on (i) the direct killing of P. gingivalis, (ii) P. gingivalis-induced superoxide anion (O2-) release from human polymorphonuclear leukocytes (PMNs), and (iii) the ability of PMNs to bind and phagocytize P. gingivalis. Three types of NHP sera were utilized: (i) normal or baseline sera; (ii) sera obtained after ligature-induced periodontitis; and (iii) sera obtained following active immunization with formalinized P. gingivalis. All assays were performed with or without the addition of human complement. Significantly more (P < 0.01) direct killing of P. gingivalis occurred with immunized sera and complement than with any of the other treatments. The sera from ligature-induced periodontitis NHPs had significantly less (P < 0.03) killing capacity than the baseline sera, which contained natural antibody produced to P. gingivalis colonization. Sera from immunized NHPs were used to opsonize P. gingivalis and caused significantly greater (P < 0.01) levels of O2- release from PMNs. Finally, the sera from immunized NHPs significantly enhanced (P < 0.009) the uptake of P. gingivalis by PMNs, although binding of the bacteria to PMNs was similar among all three serum types. Active immunization of NHPs with P. gingivalis elicited a functional antibody that enhanced direct killing, positively influenced the activation of PMNs, and enhanced the ability of PMNs to phagocytize P. gingivalis. Moreover, antibody produced as a sequela of progressing periodontitis appeared to lack these functions. A wide variability in functional capacity of the sera from individual NHPs, which may contribute to an individual's susceptibility to P. gingivalis-induced disease, was noted. This variability suggested that results from functional tests of serum antibody may aid in predicting host

  12. Porphyromonas gingivalis Fim-A genotype distribution among Colombians

    PubMed Central

    Jaramillo, Adriana; Parra, Beatriz; Botero, Javier Enrique; Contreras, Adolfo

    2015-01-01

    Introduction: Porphyromonas gingivalis is associated with periodontitis and exhibit a wide array of virulence factors, including fimbriae which is encoded by the FimA gene representing six known genotypes. Objetive: To identify FimA genotypes of P. gingivalis in subjects from Cali-Colombia, including the co-infection with Aggregatibacter actinomycetemcomitans, Treponema denticola, and Tannerella forsythia. Methods: Subgingival samples were collected from 151 people exhibiting diverse periodontal condition. The occurrence of P. gingivalis, FimA genotypes and other bacteria was determined by PCR. Results: P. gingivalis was positive in 85 patients. Genotype FimA II was more prevalent without reach significant differences among study groups (54.3%), FimA IV was also prevalent in gingivitis (13.0%). A high correlation (p= 0.000) was found among P. gingivalis, T. denticola, and T. forsythia co-infection. The FimA II genotype correlated with concomitant detection of T. denticola and T. forsythia. Conclusions: Porphyromonas gingivalis was high even in the healthy group at the study population. A trend toward a greater frequency of FimA II genotype in patients with moderate and severe periodontitis was determined. The FimA II genotype was also associated with increased pocket depth, greater loss of attachment level, and patients co-infected with T. denticola and T. forsythia. PMID:26600627

  13. 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

  14. Novel antimicrobial peptide specifically active against Porphyromonas gingivalis.

    PubMed

    Suwandecha, T; Srichana, T; Balekar, N; Nakpheng, T; Pangsomboon, K

    2015-09-01

    Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles, lipopolysaccharides, hemolysins and proteinases. Antimicrobial peptides (AMPs) including bacteriocins have been found to inhibit the growth of P. gingivalis; however, these peptides are relatively large molecules. Hence, it is difficult to synthesize them by a scale-up production. Therefore, this study aimed to synthesize a shorter AMP that was still active against P. gingivalis. A peptide that contained three cationic amino acids (Arg, His and Lys), two anionic amino acids (Glu and Asp), hydrophobic amino acids residues (Leu, Ile, Val, Ala and Pro) and hydrophilic residues (Ser and Gly) was obtained and named Pep-7. Its bioactivity and stability were tested after various treatments. The mechanism of action of Pep-7 and its toxicity to human red blood cells were investigated. The Pep-7 inhibited two pathogenic P. gingivalis ATCC 33277 and P. gingivalis ATCC 53978 (wp50) strains at a minimum bactericidal concentration (MBC) of 1.7 µM, but was ineffective against other oral microorganisms (P. intermedia, Tannerella forsythensis, Streptococcus salivarius and Streptococcus sanguinis). From transmission electron microscopy studies, Pep-7 caused pore formation at the poles of the cytoplasmic membranes of P. gingivalis. A concentration of Pep-7 at four times that of its MBC induced some hemolysis but only at 0.3%. The Pep-7 was heat stable under pressure (autoclave at 110 and 121 °C) and possessed activity over a pH range of 6.8-8.5. It was not toxic to periodontal cells over a range of 70.8-4.4 μM and did not induce toxic pro-inflammatory cytokines. The Pep-7 showed selective activity against Porphyromonas sp. by altering the permeability barriers of P. gingivalis. The Pep-7 was not mutagenic in vitro. This work highlighted the potential for the use of this synthetic Pep-7 against P. gingivalis. PMID:26041027

  15. Phylogeny of Porphyromonas gingivalis by Ribosomal Intergenic Spacer Region Analysis

    PubMed Central

    Rumpf, Robert W.; Griffen, Ann L.; Leys, Eugene J.

    2000-01-01

    Periodontitis has been associated with the presence of Porphyromonas gingivalis, and previous studies have shown phenotypic differences in the pathogenicities of strains of P. gingivalis. An accurate and comprehensive phylogeny of strains of P. gingivalis would be useful in determining if there is an evolutionary basis to pathogenicity in this species. Previous phylogenies of P. gingivalis strains based on random amplified polymorphic DNA (RAPD) analysis and multilocus enzyme electrophoresis (MLEE) show little agreement. While the 16S ribosomal gene is the standard for phylogenetic reconstruction among bacterial species, it is insufficiently variable for this purpose. In the present study, the phylogeny of P. gingivalis was constructed on the basis of the sequence of the most variable region of the ribosomal operon, the intergenic spacer region (ISR). Heteroduplex analysis of the ISR has been used to study the variability of P. gingivalis strains in periodontitis. In the present study, typing by heteroduplex analysis was compared to ISR sequence-based phylogeny and close agreement was observed. The two strains of P. gingivalis whose heteroduplex types are strongly associated with periodontitis were found to be closely related and were well separated from strains whose heteroduplex types are less strongly associated with disease, suggesting a relationship between pathogenicity and phylogeny. PMID:10790104

  16. Suppression of T-Cell Chemokines by Porphyromonas gingivalis

    PubMed Central

    Jauregui, Catherine E.; Wang, Qian; Wright, Christopher J.; Takeuchi, Hiroki; Uriarte, Silvia M.

    2013-01-01

    Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with immune dysbiosis. In this study, we found that P. gingivalis did not induce the expression of the T-cell chemokine IP-10 (CXCL10) from neutrophils, peripheral blood mononuclear cells (PBMCs), or gingival epithelial cells. Furthermore, P. gingivalis suppressed gamma interferon (IFN-γ)-stimulated release of IP-10, ITAC (CXCL11), and Mig (CXCL9) from epithelial cells and inhibited IP-10 secretion in a mixed infection with the otherwise stimulatory Fusobacterium nucleatum. Inhibition of chemokine expression occurred at the level of gene transcription and was associated with downregulation of interferon regulatory factor 1 (IRF-1) and decreased levels of Stat1. Ectopic expression of IRF-1 in epithelial cells relieved P. gingivalis-induced inhibition of IP-10 release. Direct contact between P. gingivalis and epithelial cells was not required for IP-10 inhibition. These results highlight the immune-disruptive potential of P. gingivalis. Suppression of IP-10 and other Th1-biasing chemokines by P. gingivalis may perturb the balance of protective and destructive immunity in the periodontal tissues and facilitate the pathogenicity of oral microbial communities. PMID:23589576

  17. An unusual presentation of subdural empyema caused by Porphyromonas gingivalis

    PubMed Central

    Rasheed, Ahmed; Khawchareonporn, Thana; Muengtaweepongsa, Sombat; Suebnukarn, Siriwan

    2013-01-01

    Subdural empyema is an uncommon clinical entity. The first case of Porphyromonas gingivalis subdural empyema is reported. We report a case of 34-year-old male who presented with subdural empyema and sinusitis. Through the utilization of polymerase chain reaction (PCR) tests on subdural pus, we were able to confirm the diagnosis and institute appropriate treatment. Early surgical intervention and intravenous antibiotics meant that the patient recovered fully. Infections caused by P. gingivalis should be considered in differential diagnoses of central nervous system (CNS) abscesses or subdural empyema especially in patients with precedent periodontal diseases and sinusitis. PMID:24339621

  18. Local Chemokine Paralysis, a Novel Pathogenic Mechanism for Porphyromonas gingivalis

    PubMed Central

    Darveau, Richard P.; Belton, Carol M.; Reife, Robert A.; Lamont, Richard J.

    1998-01-01

    Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated with Porphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by both P. gingivalis and commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast, P. gingivalis was found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion by P. gingivalis. Furthermore, a noninvasive mutant of P. gingivalis was unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites of P. gingivalis colonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue. PMID:9529095

  19. Invasion of Aortic and Heart Endothelial Cells by Porphyromonas gingivalis

    PubMed Central

    Deshpande, Rajashri G.; Khan, Mahfuz B.; Attardo Genco, Caroline

    1998-01-01

    Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen Porphyromonas gingivalis, to readily enter the bloodstream. However, the ability of P. gingivalis to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that P. gingivalis can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy. P. gingivalis A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0.3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by P. gingivalis A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by P. gingivalis A7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, and P. gingivalis proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for P. gingivalis invasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by P. gingivalis. P. gingivalis 381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding fimA mutant DPG3, which

  20. Prevalence of Porphyromonas gingivalis Four rag Locus Genotypes in Patients of Orthodontic Gingivitis and Periodontitis

    PubMed Central

    Liu, Yi; Zhang, Yujie; Wang, Lili; Guo, Yang; Xiao, Shuiqing

    2013-01-01

    Porphyromonas gingivalis is considered as a major etiological agent in periodontal diseases and implied to result in gingival inflammation under orthodontic appliance. rag locus is a pathogenicity island found in Porphyromonas gingivalis. Four rag locus variants are different in pathogenicity of Porphyromonas gingivalis. Moreover, there are different racial and geographic differences in distribution of rag locus genotypes. In this study, we assessed the prevalence of Porphyromonas gingivalis and rag locus genotypes in 102 gingival crevicular fluid samples from 57 cases of gingivitis patients with orthodontic appliances, 25 cases of periodontitis patients and 20 cases of periodontally healthy people through a 16S rRNA-based PCR and a multiplex PCR. The correlations between Porphyromona.gingivalis/rag locus and clinical indices were analyzed. The prevalence of Porphyromonas gingivalis and rag locus genes in periodontitis group was the highest among three groups and higher in orthodontic gingivitis than healthy people (p<0.01). An obviously positive correlation was observed between the prevalence of Porphyromonas gingivalis/rag locus and gingival index. rag-3 and rag-4 were the predominant genotypes in the patients of orthodontic gingivitis and mild-to-moderate periodontitis in Shandong. Porphyromonas.gingivalis carrying rag-1 has the strong virulence and could be associated with severe periodontitis. PMID:23593379

  1. Prevalence of Porphyromonas gingivalis four rag locus genotypes in patients of orthodontic gingivitis and periodontitis.

    PubMed

    Liu, Yi; Zhang, Yujie; Wang, Lili; Guo, Yang; Xiao, Shuiqing

    2013-01-01

    Porphyromonas gingivalis is considered as a major etiological agent in periodontal diseases and implied to result in gingival inflammation under orthodontic appliance. rag locus is a pathogenicity island found in Porphyromonas gingivalis. Four rag locus variants are different in pathogenicity of Porphyromonas gingivalis. Moreover, there are different racial and geographic differences in distribution of rag locus genotypes. In this study, we assessed the prevalence of Porphyromonas gingivalis and rag locus genotypes in 102 gingival crevicular fluid samples from 57 cases of gingivitis patients with orthodontic appliances, 25 cases of periodontitis patients and 20 cases of periodontally healthy people through a 16S rRNA-based PCR and a multiplex PCR. The correlations between Porphyromona.gingivalis/rag locus and clinical indices were analyzed. The prevalence of Porphyromonas gingivalis and rag locus genes in periodontitis group was the highest among three groups and higher in orthodontic gingivitis than healthy people (p<0.01). An obviously positive correlation was observed between the prevalence of Porphyromonas gingivalis/rag locus and gingival index. rag-3 and rag-4 were the predominant genotypes in the patients of orthodontic gingivitis and mild-to-moderate periodontitis in Shandong. Porphyromonas.gingivalis carrying rag-1 has the strong virulence and could be associated with severe periodontitis. PMID:23593379

  2. 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

  3. 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

  4. Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells.

    PubMed

    Sakanaka, Akito; Takeuchi, Hiroki; Kuboniwa, Masae; Amano, Atsuo

    2016-05-01

    Porphyromonas gingivalis is deeply involved in the pathogenesis of marginal periodontitis, and recent findings have consolidated its role as an important and unique pathogen. This bacterium has a unique dual lifestyle in periodontal sites including subgingival dental plaque (biofilm) and gingival cells, as it has been clearly shown that P. gingivalis is able to exert virulence using completely different tactics in each environment. Inter-bacterial cross-feeding enhances the virulence of periodontal microflora, and such metabolic and adhesive interplay creates a supportive environment for P. gingivalis and other species. Human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm. P. gingivalis can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. Subsequently, from its intracellular position, the pathogen exploits cellular recycling pathways to exit invaded cells, by which it is able to control its population in infected tissues, allowing for persistent infection in gingival tissues. Here, we outline the dual lifestyle of P. gingivalis in subgingival areas and its effects on the pathogenesis of periodontitis. PMID:26456558

  5. Porphyromonas gingivalis invades human pocket epithelium in vitro.

    PubMed

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

    1994-01-01

    The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis. PMID:8113953

  6. Mast Cells Contribute to Porphyromonas gingivalis-induced Bone Loss.

    PubMed

    Malcolm, J; Millington, O; Millhouse, E; Campbell, L; Adrados Planell, A; Butcher, J P; Lawrence, C; Ross, K; Ramage, G; McInnes, I B; Culshaw, S

    2016-06-01

    Periodontitis is a chronic inflammatory and bone-destructive disease. Development of periodontitis is associated with dysbiosis of the microbial community, which may be caused by periodontal bacteria, such as Porphyromonas gingivalis Mast cells are sentinels at mucosal surfaces and are a potent source of inflammatory mediators, including tumor necrosis factors (TNF), although their role in the pathogenesis of periodontitis remains to be elucidated. This study sought to determine the contribution of mast cells to local bone destruction following oral infection with P. gingivalis Mast cell-deficient mice (Kit(W-sh/W-sh)) were protected from P. gingivalis-induced alveolar bone loss, with a reduction in anti-P. gingivalis serum antibody titers compared with wild-type infected controls. Furthermore, mast cell-deficient mice had reduced expression of Tnf, Il6, and Il1b mRNA in gingival tissues compared with wild-type mice. Mast cell-engrafted Kit(W-sh/W-sh) mice infected with P. gingivalis demonstrated alveolar bone loss and serum anti-P. gingivalis antibody titers equivalent to wild-type infected mice. The expression of Tnf mRNA in gingival tissues of Kit(W-sh/W-sh) mice was elevated following the engraftment of mast cells, indicating that mast cells contributed to the Tnf transcript in gingival tissues. In vitro, mast cells degranulated and released significant TNF in response to oral bacteria, and neutralizing TNF in vivo abrogated alveolar bone loss following P. gingivalis infection. These data indicate that mast cells and TNF contribute to the immunopathogenesis of periodontitis and may offer therapeutic targets. PMID:26933137

  7. 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

  8. Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Sharma, Ashu; Genco, Robert J.

    2002-01-01

    The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial

  9. LuxS signaling in Porphyromonas gingivalis-host interactions.

    PubMed

    Scheres, Nina; Lamont, Richard J; Crielaard, Wim; Krom, Bastiaan P

    2015-10-01

    Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1β, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482. PMID:25434960

  10. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis.

    PubMed

    Lamont, R J; Jenkinson, H F

    1998-12-01

    Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a

  11. Life Below the Gum Line: Pathogenic Mechanisms of Porphyromonas gingivalis

    PubMed Central

    Lamont, Richard J.; Jenkinson, Howard F.

    1998-01-01

    Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a

  12. Interactions of Porphyromonas gingivalis with oxyhaemoglobin and deoxyhaemoglobin.

    PubMed Central

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

    2002-01-01

    When grown on blood-containing solid media, the anaerobic periodontal pathogen Porphyromonas gingivalis produces a haem pigment, the major component of which is the mu-oxo bishaem of iron protoporphyrin IX [Smalley, Silver, Marsh and Birss (1998) Biochem. J. 331, 681-685]. In this study, mu-oxo bishaem generation by P. gingivalis from oxy- and deoxyhaemoglobin was examined. Bacterial cells were shown to convert oxyhaemoglobin into methaemoglobin, which was degraded progressively, generating a mixture of both monomeric and mu-oxo dimeric iron protoporphyrin IX. The rate of methaemoglobin formation was accelerated in the presence of bacterial cells, but was inhibited by N-ethylmaleimide and tosyl-lysylchloromethylketone. Interaction of cells with deoxyhaemoglobin resulted in formation of an iron(III) haem species (Soret gamma(max), 393 nm), identified as pure mu-oxo bishaem. PMID:11829761

  13. LPS from Porphyromonas gingivalis Sensitizes Capsaicin-Sensitive Nociceptors

    PubMed Central

    Ferraz, Caio Cezar Randi; Diógenes, Aníbal; Henry, Michael A.; Hargreaves, Kenneth M.

    2010-01-01

    Although odontogenic infections are often accompanied by pain, little is known about the potential mechanisms mediating this effect. In this study, we tested the hypothesis that trigeminal nociceptive neurons are directly sensitized by lipopolysaccharide (LPS) isolated from an endodontic pathogen, Porphyromonas gingivalis (P. gingivalis). In vitro studies conducted with cultures of rat trigeminal neurons demonstrated that pretreatment with LPS produced a significant increase in the capsaicin-evoked release of calcitonin gene-related peptide (CGRP) when compared to vehicle pretreatment, thus showing sensitization of the capsaicin receptor, TRPV1, by LPS. Furthermore, confocal microscopic examination of human tooth pulp samples showed the colocalization of the LPS receptor (toll-like receptor 4; TLR4) with CGRP containing nerve fibers. Collectively, these results suggest the direct sensitization of nociceptors by LPS at concentrations found in infected canal systems as one mechanism responsible for the pain associated with bacterial infections. PMID:21146075

  14. Interactions of Porphyromonas gingivalis with oxyhaemoglobin and deoxyhaemoglobin.

    PubMed

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

    2002-02-15

    When grown on blood-containing solid media, the anaerobic periodontal pathogen Porphyromonas gingivalis produces a haem pigment, the major component of which is the mu-oxo bishaem of iron protoporphyrin IX [Smalley, Silver, Marsh and Birss (1998) Biochem. J. 331, 681-685]. In this study, mu-oxo bishaem generation by P. gingivalis from oxy- and deoxyhaemoglobin was examined. Bacterial cells were shown to convert oxyhaemoglobin into methaemoglobin, which was degraded progressively, generating a mixture of both monomeric and mu-oxo dimeric iron protoporphyrin IX. The rate of methaemoglobin formation was accelerated in the presence of bacterial cells, but was inhibited by N-ethylmaleimide and tosyl-lysylchloromethylketone. Interaction of cells with deoxyhaemoglobin resulted in formation of an iron(III) haem species (Soret gamma(max), 393 nm), identified as pure mu-oxo bishaem. PMID:11829761

  15. Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

    PubMed

    Pezzotti, Giuseppe; Bock, Ryan M; McEntire, Bryan J; Jones, Erin; Boffelli, Marco; Zhu, Wenliang; Baggio, Greta; Boschetto, Francesco; Puppulin, Leonardo; Adachi, Tetsuya; Yamamoto, Toshiro; Kanamura, Narisato; Marunaka, Yoshinori; Bal, B Sonny

    2016-03-29

    Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment. PMID:26948186

  16. Chemical structure and immunobiological activity of Porphyromonas gingivalis lipid A.

    PubMed

    Ogawa, Tomohiko; Asai, Yasuyuki; Makimura, Yutaka; Tamai, Riyoko

    2007-01-01

    In 1933, Boivin et al. extracted an endotoxin from Salmonella typhimurium for the first time, after which a variety of chemical and biological studies on endotoxins have been performed. In 1952, the structural and functional properties of endotoxic lipopolysaccharide (LPS), extracted by a hot phenol and water method devised by Westphal et al., were reported, which led to a number of studies of Gram-negative bacteria in regards to the host defense mechanism. Since 1960, the unique chemical structure and biological activity of Bacteroides species LPS have received a great deal of attention, and there is a long history of such studies. In addition, among oral bacterial strains that have received attention as causative periodontopathic bacteria, many have been classified as Bacteroides species. In particular, a number of researchers have investigated whether LPS of Porphyromonas gingivalis (formerly Bacteroides gingivalis), a black-pigmented oral anaerobic rod, is a virulent factor of the bacterium. The active center of the LPS of these Bacteroides species, the lipid A molecule, is known to be an active participant in endotoxic activation, though its other biological activities are weak, due to its unique chemical structure and action as an antagonist of LPS. On the other hand, many reports have noted that the LPS of those species activate cells in C3H/HeJ mice, which generally do not respond to LPS. We were the first to reveal the chemical structure of P. gingivalis lipid A and, together with other researchers, reported that P. gingivalis LPS and its lipid A have activities toward C3H/HeJ mice. Since that time, because of the popularity of Toll-like receptor (TLR) studies, a great deal of evidence has been reported indicating that P. gingivalis LPS and its lipid A are ligands that act on TLR2. In order to solve such problems as heterogeneity and contamination of the biologically active components of P. gingivalis lipid A, we produced a chemical synthesis counterpart

  17. Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis.

    PubMed

    Arimoto, Takafumi; Ansai, Toshihiro; Yu, Weixian; Turner, Anthony J; Takehara, Tadamichi

    2002-01-22

    We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability. PMID:11886747

  18. Bacterial Adhesion of Porphyromonas Gingivalis on Provisional Fixed Prosthetic Materials

    PubMed Central

    Zortuk, Mustafa; Kesim, Servet; Kaya, Esma; Özbilge, Hatice; Kiliç, Kerem; Çölgeçen, Özlem

    2010-01-01

    Background: When provisional restorations are worn for long term period, the adhesion of bacteria becomes a primary factor in the development of periodontal diseases. The aims of this study were to evaluate the surface roughness and bacterial adhesion of four different provisional fixed prosthodon-tic materials. Methods: Ten cylindrical specimens were prepared from bis-acrylic composites (PreVISION CB and Protemp 3 Garant), a light-polymerized composite (Revotek LC), and a polymethyl methacrylate-based (Dentalon) provisional fixed prosthodontic materials. Surface roughness was assessed by profilometry. The bacterial adhesion test was applied using Porphyromonas gingivalis (P. gingivalis) and spectro-fluorometric method. Statistical analysis was performed using ANOVA and Dunnett t-tests. Results: All tested materials were significantly rougher than glass (P < 0.05). Revotek LC had the greatest fluorescence intensity, PreVISION and Protemp 3 Garant had moderate values and all of them had significantly more bacterial adhesion compared to glass (P < 0.05). Dentalon had the lowest fluorescence intensity among the provisional fixed prosthodontic materials. Conclusion: The quantity of bacterial adhesion and surface roughness differed among the assessed provisional fixed prosthodontic materials. The light-polymerized provisional material Revotek LC had rougher surface and more bacterial adhesion compared with the others. PMID:21448445

  19. Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis.

    PubMed Central

    Amano, A; Sojar, H T; Lee, J Y; Sharma, A; Levine, M J; Genco, R J

    1994-01-01

    Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces. Images PMID:8039907

  20. Genomic Loci of the Porphyromonas gingivalis Insertion Element IS1126

    PubMed Central

    Dong, Hong; Chen, Tsute; Dewhirst, Floyd E.; Fleischmann, Robert D.; Fraser, Claire M.; Duncan, Margaret J.

    1999-01-01

    The Porphyromonas gingivalis genome contains multiple copies of insertion element IS1126. When chromosomal DNA digests of different strains were probed with IS1126, between 25 and 35 hybridizing fragments per genome were detected, depending on the strain. Unrelated strains had very different restriction fragment length polymorphism (RFLP) patterns. When different laboratory copies of a specific strain were examined, the IS1126 RFLP patterns were very similar but small differences were observed, indicating that element-associated changes had occurred during laboratory passage. Within the next year, genome sequencing, assembly, and annotation for P. gingivalis W83 will be completed. Because repetitive elements complicate the assembly of randomly sequenced DNA fragments, we isolated and sequenced the flanking regions of IS1126 copies in strain W83. We also isolated and sequenced the flanking regions of IS1126 copies in strain ATCC 33277 in order to compare insertion sites in phylogenetically divergent strains. We identified 37 new sequences flanking IS1126 from strain ATCC 33277 and 30 from strain W83. The insertion element was found between genes except where it transposed into another insertion element. Examination of identifiable flanking genes or open reading frames indicated that the insertion sites were different in the two strains, except that both strains possess an insertion adjacent to the Lys-gingipain gene (J. P. Lewis and F. L. Macrina, Infect. Immun. 66:3035–3042, 1998). Most of the genes or sequences flanking IS1126 in ATCC 33277 were present in W83 but were contiguous and not insertion element associated. Thus, where genes were identified in both strains, their order was maintained, indicating that the two genomes are organized similarly, but the loci of IS1126 are different. In both strains, insertion element-associated duplicated target sites were lost from several copies of IS1126, providing evidence of homologous recombination between elements

  1. 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

  2. Porphyromonas gulae Has Virulence and Immunological Characteristics Similar to Those of the Human Periodontal Pathogen Porphyromonas gingivalis.

    PubMed

    Lenzo, Jason C; O'Brien-Simpson, Neil M; Orth, Rebecca K; Mitchell, Helen L; Dashper, Stuart G; Reynolds, Eric C

    2016-09-01

    Periodontitis is a significant problem in companion animals, and yet little is known about the disease-associated microbiota. A major virulence factor for the human periodontal pathogen Porphyromonas gingivalis is the lysyl- and arginyl-specific proteolytic activity of the gingipains. We screened several Porphyromonas species isolated from companion animals-P. asaccharolytica, P. circumdentaria, P. endodontalis, P. levii, P. gulae, P. macacae, P. catoniae, and P. salivosa-for Lys- and Arg-specific proteolytic activity and compared the epithelial and macrophage responses and induction of alveolar bone resorption of the protease active species to that of Porphyromonas gingivalis Only P. gulae exhibited Lys-and Arg-specific proteolytic activity. The genes encoding the gingipains (RgpA/B and Kgp) were identified in the P. gulae strain ATCC 51700 and all publicly available 12 draft genomes of P. gulae strains. P. gulae ATCC 51700 induced levels of alveolar bone resorption in an animal model of periodontitis similar to those in P. gingivalis W50 and exhibited a higher capacity for autoaggregation and binding to oral epithelial cells with induction of apoptosis. Macrophages (RAW 264.7) were found to phagocytose P. gulae ATCC 51700 and the fimbriated P. gingivalis ATCC 33277 at similar levels. In response to P. gulae ATCC 51700, macrophages secreted higher levels of cytokines than those induced by P. gingivalis ATCC 33277 but lower than those induced by P. gingivalis W50, except for the interleukin-6 response. Our results indicate that P. gulae exhibits virulence characteristics similar to those of the human periodontal pathogen P. gingivalis and therefore may play a key role in the development of periodontitis in companion animals. PMID:27354442

  3. Infection with Porphyromonas gingivalis Exacerbates Endothelial Injury in Obese Mice

    PubMed Central

    Inubushi, Toshihiro; Kitagawa, Masae; Furusho, Hisako; Ando, Toshinori; Ayuningtyas, Nurina Febriyanti; Nagasaki, Atsuhiro; Ishihara, Kazuyuki; Tahara, Hidetoshi; Kozai, Katsuyuki; Takata, Takashi

    2014-01-01

    Background A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. Methods In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. Results Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. Conclusions Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury. PMID:25334003

  4. Functional differences of Porphyromonas gingivalis Fimbriae in determining periodontal disease pathogenesis: a literature review

    PubMed Central

    Contreras, Adolfo

    2013-01-01

    Porphyromonas gingivalis is implicated in chronic and aggressive periodontitis. This bacterium has numerous virulence factors and one is the Fimbriae, which is quite important for bacterial colonization. Fimbriae are appendices that anchor to the bacterial wall and are comprised of the protein FimBriline encoded by the FimA gene. Thus far, six genotypes have been identified, FimA I to V and Ib. Genotypes II and IV are associated with periodontal disease, while genotype I is related to gingival health. Genotype identification of P. gingivalis FimA in periodontitis would be important to confirm the pathogenic genotypes and to establish risk at population level. This review is about the P. gingivalis FimA genotype prevalence worldwide. A systematic search using Pubmed, Hinary, and Science Direct within the following descriptors: Porphyromonas gingivalis, bacterial adhesion, periodontitis, Fimbriae, FimA, genotipification was performed to April 2011. PMID:24892323

  5. The Cytochrome bd Oxidase of Porphyromonas gingivalis Contributes to Oxidative Stress Resistance and Dioxygen Tolerance

    PubMed Central

    Leclerc, Julia; Rosenfeld, Eric; Trainini, Mathieu; Martin, Bénédicte; Meuric, Vincent; Bonnaure-Mallet, Martine; Baysse, Christine

    2015-01-01

    Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans. The disease is associated with the formation of a mixed oral biofilm which is exposed to oxygen and environmental stress, such as oxidative stress. To investigate possible roles for cytochrome bd oxidase in the growth and persistence of this anaerobic bacterium inside the oral biofilm, mutant strains deficient in cytochrome bd oxidase activity were characterized. This study demonstrated that the cytochrome bd oxidase of Porphyromonas gingivalis, encoded by cydAB, was able to catalyse O2 consumption and was involved in peroxide and superoxide resistance, and dioxygen tolerance. PMID:26629705

  6. Oral Immunization with Recombinant Streptococcus gordonii Expressing Porphyromonas gingivalis FimA Domains

    PubMed Central

    Sharma, Ashu; Honma, Kiyonobu; Evans, Richard T.; Hruby, Dennis E.; Genco, Robert J.

    2001-01-01

    Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis. PMID:11292708

  7. 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. PMID:27007570

  8. 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

  9. Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

    PubMed

    Gorman, Michael A; Seers, Christine A; Michell, Belinda J; Feil, Susanne C; Huq, N Laila; Cross, Keith J; Reynolds, Eric C; Parker, Michael W

    2015-01-01

    The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis. PMID:25327141

  10. Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health

    PubMed Central

    Gorman, Michael A; Seers, Christine A; Michell, Belinda J; Feil, Susanne C; Huq, N Laila; Cross, Keith J; Reynolds, Eric C; Parker, Michael W

    2015-01-01

    The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis. PMID:25327141

  11. Sensitive detection of Porphyromonas gingivalis based on magnetic capture and upconversion fluorescent identification with multifunctional nanospheres.

    PubMed

    Qin, Wei; Zheng, Bin; Yuan, Yuan; Li, Meng; Bai, Yang; Chang, Jin; Wang, Hanjie; Wang, Yonglan

    2016-08-01

    A specific and sensitive detection system was designed to detect Porphyromonas gingivalis, a major periodontal pathogen, in mixed bacterial fluids. This new detection system was based on the use of fluorescent and magnetic encoding nanospheres that were conjugated with monoclonal antibodies specific to P. gingivalis, thus enabling rapid detection of the target bacterium. This strategy simplifies the detection process and improves the sensitivity compared with conventional methods, with a detection limit of approximately 10 colony-forming units (CFU) ml(-1) . This new method shows strong anti-interference ability and excellent selectivity and specificity to detect P. gingivalis in mixed solutions. PMID:27334431

  12. Periodontitis and Porphyromonas gingivalis in Preclinical Stage of Arthritis Patients

    PubMed Central

    Hashimoto, Motomu; Yamazaki, Toru; Hamaguchi, Masahide; Morimoto, Takeshi; Yamori, Masashi; Asai, Keita; Isobe, Yu; Furu, Moritoshi; Ito, Hiromu; Fujii, Takao; Terao, Chikashi; Mori, Masato; Matsuo, Takashi; Yoshitomi, Hiroyuki; Yamamoto, Keiichi; Yamamoto, Wataru; Bessho, Kazuhisa; Mimori, Tsuneyo

    2015-01-01

    Purpose To determine whether the presence of periodontitis (PD) and Porphyromonas gingivalis (Pg) in the subgingival biofilm associates with the development of rheumatoid arthritis (RA) in treatment naïve preclinical stage of arthritis patients. Methods We conducted a prospective cohort study of 72 consecutive patients with arthralgia who had never been treated with any anti-rheumatic drugs or glucocorticoids. Periodontal status at baseline was assessed by dentists. PD was defined stringently by the maximal probing depth≧4 mm, or by the classification by the 5th European Workshop in Periodontology (EWP) in 2005 using attachment loss. Up to eight plaque samples were obtained from each patient and the presence of Pg was determined by Taqman PCR. The patients were followed up for 2 years and introduction rate of methotrexate (MTX) treatment on the diagnosis of RA was compared in patients with or without PD or Pg. Results Patients with PD (probing depth≧4mm) had higher arthritis activity (p = 0.02) and higher risk for future introduction of MTX treatment on the diagnosis of RA during the follow up than patients without PD (Hazard ratio 2.68, p = 0.03). Arthritis activity and risk for MTX introduction increased with the severity of PD assessed by EWP, although not statistically significant. On the other hand, presence of Pg was not associated with arthritis activity (p = 0.72) or the risk for MTX introduction (p = 0.45). Conclusion In treatment naïve arthralgia patients, PD, but not the presence of Pg, associates with arthritis activity and future requirement of MTX treatment on the diagnosis of RA. PMID:25849461

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

    PubMed

    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

  14. 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

  15. 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

  16. Activation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated atherosclerosis.

    PubMed

    Yamaguchi, Yohei; Kurita-Ochiai, Tomoko; Kobayashi, Ryoki; Suzuki, Toshihiko; Ando, Tomohiro

    2015-06-01

    Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in hyperlipidemic animals. Atherosclerosis is a disease characterized by inflammation of the arterial wall. Recent studies have suggested that the NLRP3 inflammasome plays an important role in the development of vascular inflammation and atherosclerosis. Herein, we investigated a possible association between the inflammasome in atherosclerosis and periodontal disease induced by P. gingivalis infection using apolipoprotein E-deficient, spontaneously hyperlipidemic (Apoe(shl)) mice. Oral infection with wild-type (WT) P. gingivalis significantly increased the area of aortic sinus covered with atherosclerotic plaque and alveolar bone loss, compared with KDP136 (gingipain-null mutant) or KDP150 (FimA-deficient mutant) challenge. WT challenge also increased IL-1β, IL-18 and TNF-α production in peritoneal macrophages, and gingival or aortic gene expression of Nod-like receptor family, pyrin domain containing 3 (NLRP3), pro-IL-1β, pro-IL-18 and pro-caspase-1. Porphyromonas gingivalis genomic DNA was detected more in the aorta, gingival tissue, liver and spleen of WT-challenged mice than those in KDP136- or KDP150-challenged mice. We conclude that WT P. gingivalis activates innate immune cells through the NLRP3 inflammasome compared with KDP136 or KDP150. The NLRP3 inflammasome may play a critical role in periodontal disease and atherosclerosis induced by P. gingivalis challenge through sustained inflammation. PMID:25663345

  17. Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells.

    PubMed

    Sztukowska, Maryta N; Ojo, Akintunde; Ahmed, Saira; Carenbauer, Anne L; Wang, Qian; Shumway, Brain; Jenkinson, Howard F; Wang, Huizhi; Darling, Douglas S; Lamont, Richard J

    2016-06-01

    The oral anaerobe Porphyromonas gingivalis is associated with the development of cancers including oral squamous cell carcinoma (OSCC). Here, we show that infection of gingival epithelial cells with P. gingivalis induces expression and nuclear localization of the ZEB1 transcription factor, which controls epithelial-mesenchymal transition. P. gingivalis also caused an increase in ZEB1 expression as a dual species community with Fusobacterium nucleatum or Streptococcus gordonii. Increased ZEB1 expression was associated with elevated ZEB1 promoter activity and did not require suppression of the miR-200 family of microRNAs. P. gingivalis strains lacking the FimA fimbrial protein were attenuated in their ability to induce ZEB1 expression. ZEB1 levels correlated with an increase in expression of mesenchymal markers, including vimentin and MMP-9, and with enhanced migration of epithelial cells into matrigel. Knockdown of ZEB1 with siRNA prevented the P. gingivalis-induced increase in mesenchymal markers and epithelial cell migration. Oral infection of mice by P. gingivalis increased ZEB1 levels in gingival tissues, and intracellular P. gingivalis were detected by antibody staining in biopsy samples from OSCC. These findings indicate that FimA-driven ZEB1 expression could provide a mechanistic basis for a P. gingivalis contribution to OSCC. PMID:26639759

  18. 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

  19. Transposition of the Endogenous Insertion Sequence Element IS1126 Modulates Gingipain Expression in Porphyromonas gingivalis

    PubMed Central

    Simpson, Waltena; Wang, Chin-Yen; Mikolajczyk-Pawlinska, Jowita; Potempa, Jan; Travis, James; Bond, Vincent C.; Genco, Caroline Attardo

    1999-01-01

    We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459–2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determined that IS1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (kgp) and the arginine-specific proteinase, gingipain R2 (rgpB). Sequence analysis of P. gingivalis MSM-3 revealed that Tn4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS1126. Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS1126 compared to the parental strain A7436. Examination of the first additional IS1126 element, IS11261, indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS1126 element, IS11262, was found to have inserted upstream of a newly identified gene, hmuR, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function

  20. Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae.

    PubMed

    Maeda, Kazuhiko; Nagata, Hideki; Nonaka, Aya; Kataoka, Kosuke; Tanaka, Muneo; Shizukuishi, Satoshi

    2004-11-01

    Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization. PMID:15488735

  1. 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

  2. A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

    PubMed Central

    Njoroge, T; Genco, R J; Sojar, H T; Hamada, N; Genco, C A

    1997-01-01

    Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells. PMID:9125593

  3. Secreted gingipains from Porphyromonas gingivalis colonies exert potent immunomodulatory effects on human gingival fibroblasts.

    PubMed

    Bengtsson, Torbjörn; Khalaf, Atika; Khalaf, Hazem

    2015-09-01

    Periodontal pathogens, including Porphyromonas gingivalis, can form biofilms in dental pockets and cause inflammation, which is one of the underlying mechanisms involved in the development of periodontal disease, ultimately leading to tooth loss. Although P. gingivalis is protected in the biofilm, it can still cause damage and modulate inflammatory responses from the host, through secretion of microvesicles containing proteinases. The aim of this study was to evaluate the role of cysteine proteinases in P. gingivalis colony growth and development, and subsequent immunomodulatory effects on human gingival fibroblast. By comparing the wild type W50 with its gingipain deficient strains we show that cysteine proteinases are required by P. gingivalis to form morphologically normal colonies. The lysine-specific proteinase (Kgp), but not arginine-specific proteinases (Rgps), was associated with immunomodulation. P. gingivalis with Kgp affected the viability of gingival fibroblasts and modulated host inflammatory responses, including induction of TGF-β1 and suppression of CXCL8 and IL-6 accumulation. These results suggest that secreted products from P. gingivalis, including proteinases, are able to cause damage and significantly modulate the levels of inflammatory mediators, independent of a physical host-bacterial interaction. This study provides new insight of the pathogenesis of P. gingivalis and suggests gingipains as targets for diagnosis and treatment of periodontitis. PMID:26302843

  4. Potent In Vitro and In Vivo Activity of Plantibody Specific for Porphyromonas gingivalis FimA.

    PubMed

    Choi, Young-Suk; Moon, Ji-Hoi; Kim, Tae-Geum; Lee, Jin-Yong

    2016-04-01

    Fimbrial protein fimbrillin (FimA), a major structural subunit of Porphyromonas gingivalis, has been suggested as a vaccine candidate to control P. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA from P. gingivalis 2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) of P. gingivalis in vitro and in vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD = 0.11 nM). Binding of P. gingivalis (10(8) cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 μg/ml plantibody. Anti-FimA plantibody (10 μg/ml) reduced invasion of periodontal ligament cells by P. gingivalis (multiplicity of infection, 100) by 68.3%. Intracellular killing of P. gingivalis opsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverable P. gingivalis cells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. These in vitro and in vivo effects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies with P. gingivalis strains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to control P. gingivalis-induced periodontal disease. PMID:26865596

  5. Resistance of a Tn4351-generated polysaccharide mutant of Porphyromonas gingivalis to polymorphonuclear leukocyte killing.

    PubMed

    Genco, C A; Schifferle, R E; Njoroge, T; Forng, R Y; Cutler, C W

    1995-02-01

    In this study, we describe the development of an efficient transpositional mutagenesis system for Porphyromonas gingivalis using the Bacteroides fragilis transposon Tn4351. Using this system, we have isolated and characterized a Tn4351-generated mutant of P. gingivalis A7436, designated MSM-1, which exhibits enhanced resistance to polymorphonuclear leukocyte (PMN) phagocytosis and killing. P. gingivalis MSM-1 was initially selected based on its colony morphology; MSM-1 appeared as a mucoid, beige-pigmented colony. Analysis of P. gingivalis MSM-1 by electron microscopy and staining with ruthenium red revealed the presence of a thick ruthenium red-staining layer that was twice the thickness of this layer observed in the parent strain. P. gingivalis MSM-1 was found to be more hydrophilic than strain A7436 by hydrocarbon partitioning. Analysis of phenol-water extracts prepared from P. gingivalis A7436 and MSM-1 by Western (immunoblot) analysis and immunodiffusion with hyperimmune sera raised against A7436 and MSM-1 revealed the loss of a high-molecular-weight anionic polysaccharide component in extracts prepared from MSM-1. P. gingivalis MSM-1 was also found to be more resistant to PMN phagocytosis and intracellular killing than the parent strain, as assessed in a fluorochrome phagocytosis microassay. These differences were statistically significant (P < 0.05) when comparing PMN phagocytosis in nonimmune serum and intracellular killing in nonimmune and immune sera. P. gingivalis MSM-1 was also more resistant to killing by crude granule extracts from PMNs than was P. gingivalis A7436. These results indicate that the increased evasion of PMN phagocytosis and killing exhibited by P. gingivalis MSM-1 may result from alterations in polysaccharide-containing antigens. PMID:7822002

  6. Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis

    PubMed Central

    Sheets, Shaun M.; Robles-Price, Antonette G.; McKenzie, Rachelle M. E.; Casiano, Carlos A.; Fletcher, Hansel M.

    2012-01-01

    Porphyromonas gingivalis, a major periodontal pathogen, must acquire nutrients from host derived substrates, overcome oxidative stress and subvert the immune system. These activities can be coordinated via the gingipains which represent the most significant virulence factor produced by this organism. In the context of our contribution to this field, we will review the current understanding of gingipain biogenesis, glycosylation, and regulation, as well as discuss their role in oxidative stress resistance and apoptosis. We can postulate a model, in which gingipains may be part of the mechanism for P. gingivalis virulence. PMID:18508429

  7. Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice

    PubMed Central

    Ao, Min; Miyauchi, Mutsumi; Furusho, Hisako; Inubushi, Toshihiro; Kitagawa, Masae; Nagasaki, Atsuhiro; Sakamoto, Shinichi; Kozai, Katsuyuki; Takata, Takashi

    2015-01-01

    Background Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW. Methods To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy. Results Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further

  8. Porphyromonas gingivalis invades oral epithelial cells in vitro.

    PubMed

    Sandros, J; Papapanou, P; Dahlén, G

    1993-05-01

    The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells. PMID:8388449

  9. 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

  10. Role of the Amino-Terminal Region of Porphyromonas gingivalis Fimbriae in Adherence to Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Han, Yiping; Hamada, Nobushiro; Sharma, Ashu; Genco, Robert J.

    1999-01-01

    Porphyromonas gingivalis fimbriae elicit many responses in eukaryotic cells, including mitogenicity, cytokine production, epithelial cell invasion, and cellular immune response. Specific domains of the major fimbrial protein (FimA) have been shown to be important in triggering some of these functions. The goal of the present study was to identify the domain(s) of P. gingivalis FimA responsible for specific interaction with human mucosal epithelial cells. Fimbriated P. gingivalis strains have been shown to bind to buccal epithelial cells, whereas nonfimbriated strains bind at low levels or not at all. This and other studies provide evidence that FimA mediates the adherence of P. gingivalis to oral epithelial cells. To determine the specific region(s) of P. gingivalis FimA involved in epithelial cell binding, specific antipeptide antibodies were used to inhibit the binding of iodinated purified fimbriae as well as the binding of P. gingivalis cells to epithelial cells. Antibodies directed against peptides 49 to 68 (VVMANTAGAMELVGKTLAEVK) and 69 to 90 (ALTTELTAENQEAAGLIMTAEP) were found to highly inhibit both the binding of fimbriae and the binding of P. gingivalis cells to epithelial cells. The antibody against FimA peptides 69 to 90 also reacted with P. gingivalis fimbriae in immunogold labeling and immunoblot analysis, thereby indicating that this peptide domain is exposed on the surface of fimbriae. Our results suggest that the amino-terminal domain corresponding to amino acid residues 49 to 90 of the fimbrillin protein is a major epithelial cell binding domain of P. gingivalis fimbriae. PMID:10531284

  11. 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

    2016-01-01

    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. PMID:27056232

  12. 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

  13. 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. PMID:25341202

  14. Deep Sequencing of Porphyromonas gingivalis and Comparative Transcriptome Analysis of a LuxS Mutant

    PubMed Central

    Hirano, Takanori; Beck, David A. C.; Demuth, Donald R.; Hackett, Murray; Lamont, Richard J.

    2012-01-01

    Porphyromonas gingivalis is a major etiological agent in chronic and aggressive forms of periodontal disease. The organism is an asaccharolytic anaerobe and is a constituent of mixed species biofilms in a variety of microenvironments in the oral cavity. P. gingivalis expresses a range of virulence factors over which it exerts tight control. High-throughput sequencing technologies provide the opportunity to relate functional genomics to basic biology. In this study we report qualitative and quantitative RNA-Seq analysis of the transcriptome of P. gingivalis. We have also applied RNA-Seq to the transcriptome of a ΔluxS mutant of P. gingivalis deficient in AI-2-mediated bacterial communication. The transcriptome analysis confirmed the expression of all predicted ORFs for strain ATCC 33277, including 854 hypothetical proteins, and allowed the identification of hitherto unknown transcriptional units. Twelve non-coding RNAs were identified, including 11 small RNAs and one cobalamin riboswitch. Fifty-seven genes were differentially regulated in the LuxS mutant. Addition of exogenous synthetic 4,5-dihydroxy-2,3-pentanedione (DPD, AI-2 precursor) to the ΔluxS mutant culture complemented expression of a subset of genes, indicating that LuxS is involved in both AI-2 signaling and non-signaling dependent systems in P. gingivalis. This work provides an important dataset for future study of P. gingivalis pathophysiology and further defines the LuxS regulon in this oral pathogen. PMID:22919670

  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. 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

  17. Stimulation of proteinase and amidase activities in Porphyromonas (Bacteroides) gingivalis by amino acids and dipeptides.

    PubMed Central

    Chen, Z X; Potempa, J; Polanowski, A; Renvert, S; Wikström, M; Travis, J

    1991-01-01

    Proteolytic enzymes from the organism Porphyromonas gingivalis are believed to be involved in the development of periodontitis. Studies on both crude extracts and purified trypsinlike enzymes from this organism indicate that substantial stimulation of both amidase and proteinase activities can be obtained during incubation with glycine-containing compounds. We postulate that P. gingivalis may have developed this unusual property to take advantage of the glycine-rich environment which occurs during the periodontitis-associated degradation of gingival collagen. The finding of such a stimulation in crevicular fluids from discrete periodontal sites has been correlated with the presence of P. gingivalis and could be utilized for the early detection of infection by this organism during the onset of periodontitis. PMID:1855999

  18. 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

  19. 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. PMID:24864137

  20. Antibody responses to Porphyromonas gingivalis (P. gingivalis) in subjects with rheumatoid arthritis and periodontitis

    PubMed Central

    Mikuls, Ted R.; Payne, Jeffrey B.; Reinhardt, Richard A.; Thiele, Geoffrey M.; Maziarz, Eileen; Cannella, Amy C.; Holers, V. Michael; Kuhn, Kristine A.; O'Dell, James R.

    2009-01-01

    Summary Antibody titers to P. gingivalis are increased in patients with rheumatoid arthritis and are associated with disease-specific autoimmunity. Background Periodontitis (PD) has been implicated as a risk factor for rheumatoid arthritis (RA). We sought to characterize antibody titers to P. gingivalis (a pathogen in PD) in subjects with RA, PD, and in healthy controls and to examine their relationship with disease autoantibodies. Methods P. gingivalis antibody was measured in subjects with RA (n = 78), PD (n = 39), and in controls (n = 40). Group frequencies of bacterial titer elevations were compared using the Chi-square test and antibody titers were compared using non-parametric tests. Correlations of P. gingivalis titer with C-reactive protein (CRP), antibody to cyclic citrullinated peptide (anti-CCP), and rheumatoid factor (RF) were examined in those with RA while CRP and autoantibody concentrations were compared based on seropositivity to P. gingivalis. Results Antibody titers to P. gingivalis were highest in PD, lowest in controls, and intermediate in RA (p = 0.0003). Elevations in P. gingivalis (titer ≥ 800) were more common in RA and PD (67% and 77%, respectively) than in controls (40%) (p = 0.002). In RA, there were significant correlations with P. gingivalis titer with CRP, anti-CCP-IgM, and -IgG-2. CRP (p = 0.006), anti-CCP-IgM (p = 0.01) and -IgG2 (p = 0.04) concentrations were higher in RA cases with P. gingivalis titers ≥ 800 compared to cases with titers < 800. Conclusion Antibodies to P. gingivalis are more common in RA subjects than controls, although lower than that in PD. Associations of P. gingivalis titers with RA-related autoantibody and CRP concentrations suggests that infection with this organism plays a role in disease risk and progression in RA. PMID:18848647

  1. Mice Lacking Inducible Nitric Oxide Synthase Demonstrate Impaired Killing of Porphyromonas gingivalis

    PubMed Central

    Gyurko, Robert; Boustany, Gabriel; Huang, Paul L.; Kantarci, Alpdogan; Van Dyke, Thomas E.; Genco, Caroline A.; Gibson III, Frank C.

    2003-01-01

    Porphyromonas gingivalis is a primary etiological agent of generalized severe periodontitis, and emerging data suggest the importance of reactive oxygen and nitrogen species in periodontal tissue damage, as well as in microbial killing. Since nitric oxide (NO) released from inducible NO synthase (iNOS) has been shown to possess immunomodulatory, cytotoxic, and antibacterial effects in experimental models, we challenged iNOS-deficient (iNOS−/−) mice with P. gingivalis by using a subcutaneous chamber model to study the specific contribution of NO to host defense during P. gingivalis infection. iNOS−/− mice inoculated with P. gingivalis developed skin lesions and chamber rejection with higher frequency and to a greater degree than similarly challenged C57BL/6 wild-type (WT) mice. Chamber fluid from iNOS−/− mice possessed significantly more P. gingivalis than that of WT mice. The immunoglobulin G responses to P. gingivalis in serum was similar in WT and iNOS−/− mice, and the inductions of tumor necrosis factor alpha, interleukin-1β and interleukin-6, and prostaglandin E2 were comparable between the two mouse strains. Although no differences in total leukocyte counts in chamber fluids were observed between iNOS−/− and WT mice, the percentage of dead polymorphonuclear leukocytes (PMNs) was significantly greater in iNOS−/− mouse chamber fluids than that of WT samples. Interestingly, casein-elicited PMNs from iNOS−/− mice released more superoxide than did WT PMNs when stimulated with P. gingivalis. These results indicate that modulation of superoxide levels is a mechanism by which NO influences PMN function and that NO is an important element of the host defense against P. gingivalis. PMID:12933833

  2. Porphyromonas gingivalis increases the invasiveness of oral cancer cells by upregulating IL-8 and MMPs.

    PubMed

    Ha, Na Hee; Park, Dae Gun; Woo, Bok Hee; Kim, Da Jeong; Choi, Jeom Il; Park, Bong Soo; Kim, Yong Deok; Lee, Ji Hye; Park, Hae Ryoun

    2016-10-01

    Recent studies indicate that chronic inflammation promotes the aggressiveness of cancers. However, the direct molecular mechanisms underlying a functional link between chronic periodontitis, the most common form of oral inflammatory diseases, and the malignancy of oral cancer remain unknown. To elucidate the role of chronic periodontitis in progression of oral cancer, we examined the effect of Porphyromonas gingivalis (P. gingivalis), a major pathogen that causes chronic periodontitis, on the invasiveness of oral squamous cell carcinoma (OSCC) cells, including SCC-25, OSC-20 and SAS cells. Exposures to P. gingivalis promoted the invasive ability of OSC-20 and SAS cells via the upregulation of matrix metalloproteinases (MMPs), specifically MMP-1 and MMP-2. However, P. gingivalis-infected SCC-25 cells did not exhibit changes in their invasive properties or the low expression levels of MMPs. In an effort to delineate the molecular players that control the invasiveness, we first assessed the level of interleukin-8 (IL-8), a well-known inflammatory cytokine, in P. gingivalis-infected OSCC cells. IL-8 secretion was substantially increased in the OSC-20 and SAS cells, but not in the SCC-25 cells, following P. gingivalis infection. When IL-8 was directly applied to SCC-25 cells, their invasive ability and MMP level were significantly increased. Furthermore, the downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells attenuated their invasive potentials and MMP levels. Taken together, our findings strongly suggest that P. gingivalis infection plays an important role in the promotion of the invasive potential of OSCC cells via the upregulation of IL-8 and MMPs. PMID:27468958

  3. Binding and accumulation of hemin in Porphyromonas gingivalis are induced by hemin.

    PubMed Central

    Genco, C A; Odusanya, B M; Brown, G

    1994-01-01

    Although hemin is an essential nutrient for the black-pigmented oral bacterium Porphyromonas gingivalis, the mechanisms involved in hemin binding and uptake are poorly defined. In this study, we have examined the binding of hemin and Congo red (CR) to P. gingivalis whole cells and have defined the conditions for maximal binding. Additionally, the accumulation of hemin by P. gingivalis under growing conditions has been characterized. P. gingivalis A7436 was grown under hemin- or iron-deplete conditions (basal medium [BM] or Schaedler broth with dipyridyl [SBD]) or under hemin- or iron-replete conditions (BM with hemin [BMH] or Schaedler broth [SB]), and hemin and CR binding were assessed spectrophotometrically. Binding of hemin by P. gingivalis whole cells was rapid and was observed in samples obtained from cells grown under hemin- and iron-replete and hemin-deplete conditions but was not observed in cells grown under iron limitation. We also found that P. gingivalis whole cells bound more hemin when grown in BMH or SB than cells grown in BM or SBD. Binding of CR by P. gingivalis A7436 was also enhanced when cells were grown in the presence of hemin or when cells were incubated with hemin prior to CR binding. Hemin binding and accumulation were also assessed using [14C]hemin and [59Fe]hemin under growing conditions. Both [14C]hemin and [59Fe]hemin were accumulated by P. gingivalis, indicating that iron and the porphyrin ring were taken into the cell. Binding and accumulation of hemin under growing conditions were also induced by growth of P. gingivalis in hemin-replete media. Hemin accumulation was inhibited by the addition of KCN to P. gingivalis cultures, indicating that active transport was required for hemin uptake. [14C]hemin binding and accumulation were also inhibited by the addition of either cold hemin or protoporphyrin IX. Taken together, these results indicate that P. gingivalis transports the entire hemin moiety into the cell and that the binding and

  4. Polymersome-mediated intracellular delivery of antibiotics to treat Porphyromonas gingivalis-infected oral epithelial cells.

    PubMed

    Wayakanon, Kornchanok; Thornhill, Martin H; Douglas, C W Ian; Lewis, Andrew L; Warren, Nicholas J; Pinnock, Abigail; Armes, Steven P; Battaglia, Giuseppe; Murdoch, Craig

    2013-11-01

    The gram-negative anaerobe Porphyromonas gingivalis colonizes the gingival crevice and is etiologically associated with periodontal disease that can lead to alveolar bone damage and resorption, promoting tooth loss. Although susceptible to antibiotics, P. gingivalis can evade antibiotic killing by residing within gingival keratinocytes. This provides a reservoir of organisms that may recolonize the gingival crevice once antibiotic therapy is complete. Polymersomes are nanosized amphiphilic block copolymer vesicles that can encapsulate drugs. Cells internalize polymersomes by endocytosis into early endosomes, where they are disassembled by the low pH, causing intracellular release of their drug load. In this study, polymersomes were used as vehicles to deliver antibiotics in an attempt to kill intracellular P. gingivalis within monolayers of keratinocytes and organotypic oral mucosal models. Polymersome-encapsulated metronidazole or doxycycline, free metronidazole, or doxycycline, or polymersomes alone as controls, were used, and the number of surviving intracellular P. gingivalis was quantified after host cell lysis. Polymersome-encapsulated metronidazole or doxycycline significantly (P<0.05) reduced the number of intracellular P. gingivalis in both monolayer and organotypic cultures compared to free antibiotic or polymersome alone controls. Polymersomes are effective delivery vehicles for antibiotics that do not normally gain entry to host cells. This approach could be used to treat recurrent periodontitis or other diseases caused by intracellular-dwelling organisms. PMID:23921377

  5. Identification of a Diguanylate Cyclase and Its Role in Porphyromonas gingivalis Virulence

    PubMed Central

    Chaudhuri, Swarnava; Pratap, Siddharth; Paromov, Victor; Li, Zhijun; Mantri, Chinmay K.

    2014-01-01

    Porphyromonas gingivalis is a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3′-5′) cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated in P. gingivalis, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in the pgn_1932 gene had a significant effect on the intracellular c-di-GMP level in P. gingivalis. Genetic analysis showed that expression of the fimA and rgpA genes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in the pgn_1932 mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in the pgn_1932 gene also had a significant impact on the adhesive and invasive capabilities of P. gingivalis, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion by P. gingivalis by controlling the expression and biosynthesis of FimA. PMID:24733094

  6. Evidence of Recombination in Porphyromonas gingivalis and Random Distribution of Putative Virulence Markers

    PubMed Central

    Frandsen, Ellen V. G.; Poulsen, Knud; Curtis, Michael A.; Kilian, Mogens

    2001-01-01

    The association of Porphyromonas gingivalis to periodontal disease is not clearly understood. Similar proportions of P. gingivalis may be cultivated from both inactive and actively degrading periodontal pockets. Differences in virulence among strains of P. gingivalis exist, but the molecular reason for this remains unknown. We examined the population structure of P. gingivalis to obtain a framework in which to study pathogenicity in relation to evolution. Phylogenetic trees derived from the sequencing of fragments of four housekeeping genes, ahp, thy, rmlB, and infB, in 57 strains were completely different with no correlation between clustering of strains in the four dendrograms. Combining the various alleles of the four gene fragments sequenced resulted in 41 different sequence types. The index of association, IA, based on a single representative of each sequence type was 0.143 ± 0.202, indicating a population at linkage equilibrium. Inclusion of all isolates for the calculation of IA resulted in a value of 0.206 ± 0.171. This suggests an epidemic population structure supported by the finding of genetically identical strains in different parts of the world. We observed a random distribution of two virulence-associated mobile genetic elements, the ragB locus and the insertion sequence IS1598, among 132 strains tested. In conclusion, P. gingivalis has a nonclonal population structure characterized by frequent recombination. Our study suggests that particular genotypes, possibly with increased pathogenic potential, may spread successfully in the human population. PMID:11401989

  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. PMID:25102110

  8. Porphyromonas gingivalis and Treponema denticola Mixed Microbial Infection in a Rat Model of Periodontal Disease

    PubMed Central

    Verma, Raj K.; Rajapakse, Sunethra; Meka, Archana; Hamrick, Clayton; Pola, Sheela; Bhattacharyya, Indraneel; Nair, Madhu; Wallet, Shannon M.; Aukhil, Ikramuddin; Kesavalu, Lakshmyya

    2010-01-01

    Porphyromonas gingivalis and Treponema denticola are periodontal pathogens that express virulence factors associated with the pathogenesis of periodontitis. In this paper we tested the hypothesis that P. gingivalis and T. denticola are synergistic in terms of virulence; using a model of mixed microbial infection in rats. Groups of rats were orally infected with either P. gingivalis or T. denticola or mixed microbial infections for 7 and 12 weeks. P. gingivalis genomic DNA was detected more frequently by PCR than T. denticola. Both bacteria induced significantly high IgG, IgG2b, IgG1, IgG2a antibody levels indicating a stimulation of Th1 and Th2 immune response. Radiographic and morphometric measurements demonstrated that rats infected with the mixed infection exhibited significantly more alveolar bone loss than shaminfected control rats. Histology revealed apical migration of junctional epithelium, rete ridge elongation, and crestal alveolar bone resorption; resembling periodontal disease lesion. These results showed that P. gingivalis and T. denticola exhibit no synergistic virulence in a rat model of periodontal disease. PMID:20592756

  9. The anti-bacterial activity of titanium-copper sintered alloy against Porphyromonas gingivalis in vitro.

    PubMed

    Bai, Bing; Zhang, Erlin; Liu, Junchao; Zhu, Jingtao

    2016-01-01

    This study investigates the anti-bacterial property of Ti-Cu sintered alloys against Porphyromonas gingivalis. The anti-anaerobic property of Ti-Cu sintered alloys against P. gingivalis was investigated by antibacterial activity test, DNA measurement, DAPI staining and morphology observation. The antibacterial rates of the Ti-5Cu against P. gingivalis after 18 and 24 h incubation were 36.04 and 54.39%, and those of Ti-10Cu were 68.69 and 75.39%, which were lower than their anti-aerobic abilities. The concentration of P. gingivalis DNA gradually decreased with the increasing Cu content, which was nearly 50% after 24 h incubation on Ti-10Cu. SEM results showed that the shape of P. gingivalis changed and the bacteria broke apart with the addition of Cu and the extension of the culture time. Ti-Cu sintered alloys could not only kill anaerobic bacteria but also reduce the activity of the survived bacteria. The anti-anaerobic mechanism was thought to be in associated with the Cu ion released from Ti-Cu alloy. PMID:27477233

  10. Identification of Porphyromonas gingivalis Strains by Heteroduplex Analysis and Detection of Multiple Strains

    PubMed Central

    Leys, Eugene J.; Smith, James H.; Lyons, Sharon R.; Griffen, Ann L.

    1999-01-01

    Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of Porphyromonas gingivalis. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of P. gingivalis for identification. We identified 22 distinct but closely related heteroduplex types of P. gingivalis in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which P. gingivalis was detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains of P. gingivalis in large numbers of samples. PMID:10565905

  11. Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway.

    PubMed

    Takeuchi, Hiroki; Furuta, Nobumichi; Morisaki, Ichijiro; Amano, Atsuo

    2011-05-01

    Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well-known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11- and RalA-positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell-to-cell spreading. PMID:21155963

  12. Characterization of Porphyromonas gingivalis Insertion Sequence-Like Element ISPg5

    PubMed Central

    Califano, Joseph V.; Kitten, Todd; Lewis, Janina P.; Macrina, Francis L.; Fleischmann, Robert D.; Fraser, Claire M.; Duncan, Margaret J.; Dewhirst, Floyd E.

    2000-01-01

    Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445–455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting. PMID:10948151

  13. 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

  14. 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

  15. Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis.

    PubMed

    Dou, Y; Aruni, W; Muthiah, A; Roy, F; Wang, C; Fletcher, H M

    2016-06-01

    PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis. PMID:26216199

  16. 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

  17. Sialidase and Sialoglycoproteases Can Modulate Virulence in Porphyromonas gingivalis ▿ †

    PubMed Central

    Aruni, Wilson; Vanterpool, Elaine; Osbourne, Devon; Roy, Francis; Muthiah, Arun; Dou, Yuetan; Fletcher, Hansel M.

    2011-01-01

    The Porphyromonas gingivalis recombinant VimA can interact with the gingipains and several other proteins, including a sialidase. Sialylation can be involved in protein maturation; however, its role in virulence regulation in P. gingivalis is unknown. The three sialidase-related proteins in P. gingivalis showed the characteristic sialidase Asp signature motif (SXDXGXTW) and other unique domains. To evaluate the roles of the associated genes, randomly chosen P. gingivalis isogenic mutants created by allelic exchange and designated FLL401 (PG0778::ermF), FLL402 (PG1724::ermF), and FLL403 (PG0352::ermF-ermAM) were characterized. Similar to the wild-type strain, FLL402 and FLL403 displayed a black-pigmented phenotype in contrast to FLL401, which was not black pigmented. Sialidase activity in P. gingivalis FLL401 was reduced by approximately 70% in comparison to those in FLL402 and FLL403, which were reduced by approximately 42% and 5%, respectively. Although there were no changes in the expression of the gingipain genes, their activities were reduced by 60 to 90% in all the isogenic mutants compared to that for the wild type. Immunoreactive bands representing the catalytic domains for RgpA, RgpB, and Kgp were present in FLL402 and FLL403 but were missing in FLL401. While adhesion was decreased, the capacity for invasion of epithelial cells by the isogenic mutants was increased by 11 to 16% over that of the wild-type strain. Isogenic mutants defective in PG0778 and PG0352 were more sensitive to hydrogen peroxide than the wild type. Taken together, these results suggest that the P. gingivalis sialidase activity may be involved in regulating gingipain activity and other virulence factors and may be important in the pathogenesis of this organism. PMID:21502589

  18. Cleavage of Human Transferrin by Porphyromonas gingivalis Gingipains Promotes Growth and Formation of Hydroxyl Radicals

    PubMed Central

    Goulet, Véronique; Britigan, Bradley; Nakayama, Koji; Grenier, Daniel

    2004-01-01

    Porphyromonas gingivalis, a gram-negative anaerobic bacterium associated with active lesions of chronic periodontitis, produces several proteinases which are presumably involved in host colonization, perturbation of the immune system, and tissue destruction. The aims of this study were to investigate the degradation of human transferrin by gingipain cysteine proteinases of P. gingivalis and to demonstrate the production of toxic hydroxyl radicals (HO·) catalyzed by the iron-containing transferrin fragments generated or by release of iron itself. Analysis by polyacrylamide gel electrophoresis and Western immunoblotting showed that preparations of Arg- and Lys-gingipains of P. gingivalis cleave transferrin (iron-free and iron-saturated forms) into fragments of various sizes. Interestingly, gingival crevicular fluid samples from diseased periodontal sites but not samples from healthy periodontal sites contained fragments of transferrin. By using 55Fe-transferrin, it was found that degradation by P. gingivalis gingipains resulted in the production of free iron, as well as iron bound to lower-molecular-mass fragments. Subsequent to the degradation of transferrin, bacterial cells assimilated intracellularly the radiolabeled iron. Growth of P. gingivalis ATCC 33277, but not growth of an Arg-gingipain- and Lys-gingipain-deficient mutant, was possible in a chemically defined medium containing 30% iron-saturated transferrin as the only source of iron and peptides, suggesting that gingipains play a critical role in the acquisition of essential growth nutrients. Finally, the transferrin degradation products generated by Arg-gingipains A and B were capable of catalyzing the formation of HO·, as determined by a hypoxanthine/xanthine oxidase system and spin trapping-electron paramagnetic resonance spectrometry. Our study indicates that P. gingivalis gingipains degrade human transferrin, providing sources of iron and peptides. The iron-containing transferrin fragments or the

  19. Synthesis and assembly of Porphyromonas gingivalis fimbrial protein in potato tissues.

    PubMed

    Shin, Eun-Ah; Park, Yong Keun; Lee, Kang Oh; Langridge, William H R; Lee, Jin-Yong

    2009-10-01

    Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266-337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266-337) protein. Further, a protein band of ~160 kDa was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against periodontal disease caused by P. gingivalis. PMID:19507071

  20. Sequencing of the Ribosomal Intergenic Spacer Region for Strain Identification of Porphyromonas gingivalis

    PubMed Central

    Rumpf, Robert W.; Griffen, Ann L.; Wen, Bo-Gui; Leys, Eugene J.

    1999-01-01

    The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of Porphyromonas gingivalis were amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a P. gingivalis ISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison. PMID:10405432

  1. 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

  2. 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

  3. 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. PMID:26358096

  4. Micromolar sodium fluoride mediates anti-osteoclastogenesis in Porphyromonas gingivalis-induced alveolar bone loss.

    PubMed

    Bhawal, Ujjal K; Lee, Hye-Jin; Arikawa, Kazumune; Shimosaka, Michiharu; Suzuki, Masatoshi; Toyama, Toshizo; Sato, Takenori; Kawamata, Ryota; Taguchi, Chieko; Hamada, Nobushiro; Nasu, Ikuo; Arakawa, Hirohisa; Shibutani, Koh

    2015-12-01

    Osteoclasts are bone-specific multinucleated cells generated by the differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. Periodontitis is an inflammatory disease characterized by extensive bone resorption. In this study, we investigated the effects of sodium fluoride (NaF) on osteoclastogenesis induced by Porphyromonas gingivalis, an important colonizer of the oral cavity that has been implicated in periodontitis. NaF strongly inhibited the P. gingivalis-induced alveolar bone loss. That effect was accompanied by decreased levels of cathepsin K, interleukin (IL)-1β, matrix metalloproteinase 9 (MMP9), and tartrate-resistant acid phosphatase, which were up-regulated during P. gingivalis-induced osteoclastogenesis. Consistent with the in vivo anti-osteoclastogenic effect, NaF inhibited osteoclast formation caused by the differentiation factor RANKL (receptor activator of nuclear factor κB ligand) and macrophage colony-stimulating factor (M-CSF). The RANKL-stimulated induction of the transcription factor nuclear factor of activated T cells (NFAT) c1 was also abrogated by NaF. Taken together, our data demonstrate that NaF inhibits RANKL-induced osteoclastogenesis by reducing the induction of NFATc1, ultimately leading to the suppressed expression of cathepsin K and MMP9. The in vivo effect of NaF on the inhibition of P. gingivalis-induced osteoclastogenesis strengthens the potential usefulness of NaF for treating periodontal diseases. PMID:26674426

  5. 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

  6. A Resistance-Nodulation-Cell Division Family Xenobiotic Efflux Pump in an Obligate Anaerobe, Porphyromonas gingivalis

    PubMed Central

    Ikeda, Takeshi; Yoshimura, Fuminobu

    2002-01-01

    Porphyromonas gingivalis, a gram-negative obligate anaerobe, contains two homologs of an Escherichia coli resistance-nodulation-cell division-type multidrug exporter gene, acrB, in putative operons, together with homologs of membrane fusion protein gene acrA and outer membrane channel gene tolC. MIC determination and accumulation assays with mutants with disruptions of one or more genes showed that one cluster, named xepCAB, pumped out multiple agents including rifampin, puromycin, and ethidium bromide. PMID:12234854

  7. 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.

  8. Amino acid-linked porphyrin-nitroimidazole antibiotics targeting Porphyromonas gingivalis.

    PubMed

    Dingsdag, Simon A; Yap, Benjamin C-M; Hunter, Neil; Crossley, Maxwell J

    2015-01-01

    The periodontal pathogen Porphyromonas gingivalis requires porphyrin supplementation for growth. Previously, in order to inhibit P. gingivalis growth, we synthesised very effective 'Trojan horse' ester and amide-linked deuterporphyrin-nitroimidazole (DPIX-Nim) adducts that exploited this requirement to transport metronidazole-derived antibiotics with excellent antimicrobial selectivity and recognition by the HA2 porphyrin binding site. Herein, in the context of developing topical agents to target P. gingivalis, l-amino acids are incorporated into adducts as linkers to improve uptake. Ten 13- and 17-propionic amide regioisomers of l-amino acid-linked deuterporphyrin-nitroimidazole adducts were synthesised using a peptide coupling approach. DPIX-Lys regioisomers without attached nitroimidazole were also synthesised as comparison compounds. All the porphyrin adducts bound (Kd50 7 to 20 nM) to a recombinant HA2 receptor with similar binding affinity to haem, except the lysine-proline linked DPIX-Lys(Boc)Pro-Nim adducts (Kd50 300 nM) and the DPIX-Lys(Nim)-Nim adducts (Kd50 200 nM), both of which have large appended groups. DPIX-Lys(Boc)-Nim, DPIX-Lys(OH)-Nim, and DPIX-Pro-Nim adducts were shown to be very effective against P. gingivalis. DPIX-Lys(Boc)Pro-Nim adducts and DPIX-Lys(Nim)-Nim adducts showed weak activity. Importantly, DPIX-Lys(Boc)-Nim adducts were selective for P. gingivalis and, unlike metronidazole, did not kill a range of other anaerobic bacteria isolated from the human gastrointestinal tract. PMID:25337819

  9. Attenuation of the Virulence of Porphyromonas gingivalis by Using a Specific Synthetic Kgp Protease Inhibitor

    PubMed Central

    Curtis, M. A.; Aduse Opoku, J.; Rangarajan, M.; Gallagher, A.; Sterne, J. A. C.; Reid, C. R.; Evans, H. E. A.; Samuelsson, B.

    2002-01-01

    The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar Ki was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens. PMID:12438376

  10. Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

    PubMed Central

    Kinder, S A; Holt, S C

    1993-01-01

    Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22. Images PMID:8380804

  11. 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

  12. 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

  13. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene.

    PubMed Central

    Fletcher, H M; Schenkein, H A; Morgan, R M; Bailey, K A; Berry, C R; Macrina, F L

    1995-01-01

    In a previous study we cloned and determined the nucleotide sequence of the prtH gene from Porphyromonas gingivalis W83. This gene specifies a 97-kDa protease which is normally found in the membrane vesicles produced by P. gingivalis and which cleaves the C3 complement protein under defined conditions. We developed a novel ermF-ermAM antibiotic resistance gene cassette, which was used with the cloned prtH gene to prepare an insertionally inactivated allele of this gene. This genetic construct was introduced by electroporation into P. gingivalis W83 in order to create a protease-deficient mutant by recombinational allelic exchange. The mutant strain, designated V2296, was compared with the parent strain W83 for proteolytic activity and virulence. Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83. Casein substrate zymography revealed that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant. In in vivo experiments using a mouse model, V2296 was dramatically reduced in virulence compared with the wild-type W83 strain. A molecular survey of several clinical isolates of P. gingivalis using the prtH gene as a probe suggested that prtH gene sequences were conserved and that they may have been present in multiple copies. Two of 10 isolates did not hybridize with the prtH gene probe. These strains, like the V2296 mutant, also displayed decreased virulence in the mouse model. Taken together, these results suggest an important role for P. gingivalis proteases in soft tissue infections and specifically indicate that the prtH gene product is a virulence factor. PMID:7890419

  14. A putative TetR regulator is involved in nitric oxide stress resistance in Porphyromonas gingivalis.

    PubMed

    Boutrin, M-C; Yu, Y; Wang, C; Aruni, W; Dou, Y; Shi, L; Fletcher, H M

    2016-08-01

    To survive in the periodontal pocket, Porphyromonas gingivalis, the main causative agent of periodontal disease, must overcome oxidative and nitric oxide (NO) stress. Previously, we reported that, in the presence of NO comparable to stress conditions, the transcriptome of P. gingivalis was differentially expressed, and genes belonging to the PG1178-81 cluster were significantly upregulated. To further evaluate their role(s) in NO stress resistance, these genes were inactivated by allelic exchange mutagenesis. Isogenic mutants P. gingivalis FLL460 (ΔPG1181::ermF) and FLL461 (ΔPG1178-81::ermF) were black-pigmented, with gingipain and hemolytic activities comparable to that of the wild-type strain. Whereas the recovery of these isogenic mutants from NO stress was comparable to the wild-type, there was increased sensitivity to hydrogen peroxide-induced stress. RNA-Seq analysis under conditions of NO stress showed that approximately 5 and 8% of the genome was modulated in P. gingivalis FLL460 and FLL461, respectively. The PG1178-81 gene cluster was shown to be part of the same transcriptional unit and is inducible in response to NO stress. In the presence of NO, PG1181, a putative transcriptional regulator, was shown to bind to its own promoter region and that of several other NO responsive genes including PG0214 an extracytoplasmic function σ factor, PG0893 and PG1236. Taken together, the data suggest that PG1181 is a NO responsive transcriptional regulator that may play an important role in the NO stress resistance regulatory network in P. gingivalis. PMID:26332057

  15. Xylitol Inhibits Inflammatory Cytokine Expression Induced by Lipopolysaccharide from Porphyromonas gingivalis

    PubMed Central

    Han, Su-Ji; Jeong, So-Yeon; Nam, Yun-Ju; Yang, Kyu-Ho; Lim, Hoi-Soon; Chung, Jin

    2005-01-01

    Porphyromonas gingivalis is one of the suspected periodontopathic bacteria. The lipopolysaccharide (LPS) of P. gingivalis is a key factor in the development of periodontitis. Inflammatory cytokines play important roles in the gingival tissue destruction that is a characteristic of periodontitis. Macrophages are prominent at chronic inflammatory sites and are considered to contribute to the pathogenesis of periodontitis. Xylitol stands out and is widely believed to possess anticaries properties. However, to date, little is known about the effect of xylitol on periodontitis. The aim of the present study was to determine tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression when RAW 264.7 cells were stimulated with P. gingivalis LPS (hereafter, LPS refers to P. gingivalis LPS unless stated otherwise) and the effect of xylitol on the LPS-induced TNF-α and IL-1β expression. The kinetics of TNF-α and IL-1β levels in culture supernatant after LPS treatment showed peak values at 1 h (TNF-α) and 2 to 4 h (IL-1β), respectively. NF-κB, a transcription factor, was also activated by LPS treatment. These cytokine expressions and NF-κB activation were suppressed by pretreatment with pyrrolidine dithiocarbamate (an inhibitor of NF-κB). Pretreatment with xylitol inhibited LPS-induced TNF-α and IL-1β gene expression and protein synthesis. LPS-induced mobilization of NF-κB was also inhibited by pretreatment with xylitol in a dose-dependent manner. Xylitol also showed inhibitory effect on the growth of P. gingivalis. Taken together, these findings suggest that xylitol may have good clinical effect not only for caries but also for periodontitis by its inhibitory effect on the LPS-induced inflammatory cytokine expression. PMID:16275942

  16. 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

  17. Porphyromonas gingivalis galE is involved in lipopolysaccharide O-antigen synthesis and biofilm formation.

    PubMed

    Nakao, Ryoma; Senpuku, Hidenobu; Watanabe, Haruo

    2006-11-01

    Porphyromonas gingivalis is a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides in P. gingivalis. Gene knockout P. gingivalis mutants were constructed by insertion of an ermF-ermAM cassette; among these mutants, the galE mutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, the galE mutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of the galE mutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by the galE mutant had an intensity 4.5-fold greater than those of the wild type. Further, the galE mutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of the galE mutation led to a partial recovery of the parental phenotypes. We concluded that the galE gene plays a pivotal role in the modification of LPS O antigen and biofilm formation in P. gingivalis and considered that our findings of a relationship between the function of the P. gingivalis galE gene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease. PMID:16954395

  18. 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

  19. Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis

    PubMed Central

    2013-01-01

    Introduction Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model. Methods DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression. Results Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1β, IL-6, and IL-22 in the CFA

  20. The Unique hmuY Gene Sequence as a Specific Marker of Porphyromonas gingivalis

    PubMed Central

    Mackiewicz, Paweł; Radwan-Oczko, Małgorzata; Kantorowicz, Małgorzata; Chomyszyn-Gajewska, Maria; Frąszczak, Magdalena; Bielecki, Marcin; Olczak, Mariusz; Olczak, Teresa

    2013-01-01

    Porphyromonas gingivalis, a major etiological agent of chronic periodontitis, acquires heme from host hemoproteins using the HmuY hemophore. The aim of this study was to develop a specific P. gingivalis marker based on a hmuY gene sequence. Subgingival samples were collected from 66 patients with chronic periodontitis and 40 healthy subjects and the entire hmuY gene was analyzed in positive samples. Phylogenetic analyses demonstrated that both the amino acid sequence of the HmuY protein and the nucleotide sequence of the hmuY gene are unique among P. gingivalis strains/isolates and show low identity to sequences found in other species (below 50 and 56%, respectively). In agreement with these findings, a set of hmuY gene-based primers and standard/real-time PCR with SYBR Green chemistry allowed us to specifically detect P. gingivalis in patients with chronic periodontitis (77.3%) and healthy subjects (20%), the latter possessing lower number of P. gingivalis cells and total bacterial cells. Isolates from healthy subjects possess the hmuY gene-based nucleotide sequence pattern occurring in W83/W50/A7436 (n = 4), 381/ATCC 33277 (n = 3) or TDC60 (n = 1) strains, whereas those from patients typically have TDC60 (n = 21), W83/W50/A7436 (n = 17) and 381/ATCC 33277 (n = 13) strains. We observed a significant correlation between periodontal index of risk of infectiousness (PIRI) and the presence/absence of P. gingivalis (regardless of the hmuY gene-based sequence pattern of the isolate identified [r = 0.43; P = 0.0002] and considering particular isolate pattern [r = 0.38; P = 0.0012]). In conclusion, we demonstrated that the hmuY gene sequence or its fragments may be used as one of the molecular markers of P. gingivalis. PMID:23844074

  1. Structural characterization of peptide-mediated inhibition of Porphyromonas gingivalis biofilm formation.

    PubMed

    Daep, Carlo Amorin; James, Deanna M; Lamont, Richard J; Demuth, Donald R

    2006-10-01

    Porphyromonas gingivalis is a periodontal pathogen whose primary niche is the anaerobic environment of subgingival dental plaque, but initial colonization of the oral cavity is likely to occur on supragingival surfaces that already support robust biofilm communities. Our studies have shown that P. gingivalis adheres to Streptococcus gordonii through interaction of the minor fimbrial antigen Mfa1 with a specific region of the streptococcal SspB polypeptide (residues 1167 to 1193) designated BAR. We show that a synthetic peptide comprising the BAR sequence potently inhibits P. gingivalis adherence to S. gordonii (50% inhibitory concentration = 1.3 microM) and prevents the development of P. gingivalis biofilms. However, a retroinverso peptide that possessed the same side chain topology as that of BAR was inactive, suggesting that interactions of Mfa1 with the peptide backbone of BAR are important for binding. A conformationally constrained analog of BAR inhibited P. gingivalis adherence and biofilm formation but at a lower specific activity than that of BAR. Therefore, to further define the structural features of the Mfa1-BAR interaction, we functionally screened combinatorial libraries of BAR in which active site residues (Asn1182, Thr1184, and Val1185) were replaced with each of the 19 common amino acids. Peptides containing positively charged amino acids at position 1182 or hydrophobic residues at position 1185 bound P. gingivalis more efficiently than did control peptides containing Asn and Val at these positions, suggesting that electrostatic and hydrophobic interactions may contribute to Mfa1-SspB binding. In contrast, replacement of Pro or Gly at these positions was detrimental to adherence, suggesting that perturbation of the BAR secondary structure influences activity. The net effect of substitutions for Thr1184 was less pronounced either positively or negatively than that at the other sites. These results define physicochemical characteristics of the

  2. 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

  3. Porphyromonas gingivalis peptidoglycans induce excessive activation of the innate immune system in silkworm larvae.

    PubMed

    Ishii, Kenichi; Hamamoto, Hiroshi; Imamura, Katsutoshi; Adachi, Tatsuo; Shoji, Mikio; Nakayama, Koji; Sekimizu, Kazuhisa

    2010-10-22

    Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue. PMID:20702417

  4. 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

  5. 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.

  6. VimA mediates multiple functions that control virulence in Porphyromonas gingivalis.

    PubMed

    Aruni, A W; Robles, A; Fletcher, H M

    2013-06-01

    Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is an important etiological agent of periodontal disease. Its ability to survive in the periodontal pocket and orchestrate the microbial/host activities that can lead to disease suggest that P. gingivalis possesses a complex regulatory network involving transcriptional and post-transcriptional mechanisms. The vimA (virulence modulating) gene is part of the 6.15-kb bcp-recA-vimA-vimE-vimF-aroG locus and plays a role in oxidative stress resistance. In addition to the glycosylation and anchorage of several surface proteins including the gingipains, VimA can also modulate sialylation, acetyl coenzyme A transfer, lipid A and its associated proteins and may be involved in protein sorting and transport. In this review, we examine the multifunctional role of VimA and discuss its possible involvement in a major regulatory network important for survival and virulence regulation in P. gingivalis. It is postulated that the multifunction of VimA is modulated via a post-translational mechanism involving acetylation. PMID:23279905

  7. VimA-dependent modulation of the secretome in Porphyromonas gingivalis.

    PubMed

    Osbourne, D; Aruni, A Wilson; Dou, Y; Perry, C; Boskovic, D S; Roy, F; Fletcher, H M

    2012-12-01

    The VimA protein of Porphyromonas gingivalis is a multifunctional protein involved in cell surface biogenesis. To further determine if its acetyl coenzyme A (acetyl-CoA) transfer and putative sorting functions can affect the secretome, its role in peptidoglycan biogenesis and effects on the extracellular proteins of P. gingivalis FLL92, a vimA-defective mutant, were evaluated. There were structural and compositional differences in the peptidoglycan of P. gingivalis FLL92 compared with the wild-type strain. Sixty-eight proteins were present only in the extracellular fraction of FLL92. Fifteen proteins present in the extracellular fraction of the parent strain were missing in the vimA-defective mutant. These proteins had protein sorting characteristics that included a C-terminal motif with a common consensus Gly-Gly-CTERM pattern and a polar tail consisting of aromatic amino acid residues. These observations suggest that the VimA protein is likely involved in peptidoglycan synthesis, and corroborates our previous report, which suggests a role in protein sorting. PMID:23134608

  8. Porphyromonas gingivalis Peptidoglycans Induce Excessive Activation of the Innate Immune System in Silkworm Larvae*

    PubMed Central

    Ishii, Kenichi; Hamamoto, Hiroshi; Imamura, Katsutoshi; Adachi, Tatsuo; Shoji, Mikio; Nakayama, Koji; Sekimizu, Kazuhisa

    2010-01-01

    Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue. PMID:20702417

  9. Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis.

    PubMed Central

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

    1995-01-01

    Infection with Porphyromonas gingivalis is strongly associated with adult periodontitis, and proteinases are considered to be important virulent factors of the bacterium. In order to investigate the function of proteinases in disease development we examined vesicles, a biological carrier of these enzymes, for the generation of vascular permeability enhancement (VPE) activity, believed to correlate with the exudation of gingival crevicular fluid. The vesicles generated VPE activity from human plasma in a dose-dependent manner which could be inhibited 90% by antipain, a specific inhibitor of the Arg-specific cysteine proteinases (Arg-gingipains [RGPs] from P. gingivalis. Incubation of vesicles with high-molecular-weight-kininogen (HMWK)-deficient plasma did not result in VPE activity. On this basis, RGPs associated with vesicles were assumed to be responsible for most of the VPE activity generation via plasma prekallikrein activation and subsequent bradykinin production. The secondary pathway for VPE activity production was dependent on the direct release of bradykinin from HMWK by the concerted action of RGP and a Lys-specific cysteine proteinase (Lys-gingipain [KGP]), also associated with vesicles. These results indicate that RGP and KGP are biologically important VPE factors acting either via prekallikrein activation (RGP) and/or HMWK cleavage (RGP and KGP) to release BK and, thereby, contributing to the production of gingival crevicular fluid at periodontal sites infected with P. gingivalis. PMID:7729914

  10. 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

  11. 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

  12. 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. PMID:23890177

  13. Immunohistological study of lesions induced by Porphyromonas gingivalis in a murine model.

    PubMed

    Gemmell, E; Bird, P S; Bowman, J J; Xu, L; Polak, B; Walsh, L J; Seymour, G J

    1997-10-01

    A previous study used a mouse model to demonstrate protection after challenge with Porphyromonas gingivalis ATCC 33277. In the present study, this same model was used to determine the phenotype of cells recruited into the lesions during the course of the protective immune response after immunization with this periodontal pathogen. BALB/c mice were immunized with 100 micrograms of P. gingivalis outer membrane antigens per mouse weekly for 3 weeks followed by challenge with live organisms 3 weeks after the final immunization. Hematoxylin and eosin-stained sections showed inflammatory infiltrates in all lesions from control (immunized with adjuvant only) and immunized mice. The lesions developed central necrotic cores surrounded by neutrophils, phagocytic macrophages and lymphocytes. Neutrophils were the predominant cells in the lesions 1 day after challenge with significantly more in immunized than control mice. Acid phosphatase and nonspecific esterase-positive macrophages were detected at day 4 and became the predominant cells in the healing lesions. CD4- and CD8-positive T-cells were present from day 1, and while numbers increased over time, there were no significant differences in control or immunized mice. When mice were depleted of CD4 or CD8 cells prior to immunization with P. gingivalis, fewer neutrophils were found in the lesions 1 day after challenge compared with undepleted immunized mice. Acid phosphatase and nonspecific esterase-positive macrophages were not affected by T-cell depletion. The results suggest that the P. gingivalis-induced lesion in immunized BALB/c mice is consistent with a strong innate immune response involving the recruitment of neutrophils in the first instance which may be under the control of T cells. This is followed by the infiltration of phagocytic macrophages which are involved in the healing process and do not appear to be regulated by T cells. PMID:9467382

  14. The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis

    PubMed Central

    KELLY, C G; BOOTH, V; KENDAL, H; SLANEY, J M; CURTIS, M A; LEHNER, T

    1997-01-01

    Passive immunization with the monoclonal antibody 61BG1.3 selectively prevents colonization by Porphyromonas gingivalis in humans (Booth V, Ashley FP, Lehner T. Infect Immun 1996; 64:422-7). The protective MoAb recognizes the j3 component of the RI protease of P. gingivalis which is formed by proteolytic processing of a polyprotein precursor termed PrpRl. This subunit is both a haemagglutinin and an antigen which is recognized by sera from patients with periodontitis. In this study the relationship was investigated between a colonization epitope which is recognized by the MoAb 61BG1.3, a haemagglutinating and B cell epitope which are recognized by sera from patients with periodontitis. B cell epitopes were mapped by Western blotting with a series of truncated recombinant polypeptides spanning the adhesion domain within residues 784–1130 of PrpRl and by ELISA using a panel of synthetic peptides spanning the same sequence. The epitope which is recognized by the protective MoAb was mapped within residues 907–931 of PrpRl, while serum responses of patients were directed predominantly to the adjacent carboxy-terminal sequence within residues 934–1042. The haemagglutinating epitope was mapped to residues 1073–1112. In view of our previous findings that the MoAb 61BG1.3 prevents colonization of P. gingivalis in vivo and inhibits haemagglutination, these two epitopes may be in proximity in the native protein. Active or passive immunization strategies which target the protective or haemagglutinating epitopes of the adhesion domain of PrpRl may provide a means of preventing infection with P. gingivalis. PMID:9367414

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

    PubMed

    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; Coutinho-Silva, R

    2015-09-01

    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. Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes.

    PubMed Central

    Roberts, F A; Richardson, G J; Michalek, S M

    1997-01-01

    The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte

  17. Cleavage of IgG1 in GCF is associated with presence of Porphyromonas gingivalis

    PubMed Central

    Guentsch, Arndt; Hirsch, Christiane; Pfister, Wolfgang; Vincents, Bjarne; Abrahamson, Magnus; Sroka, Aneta; Potempa, Jan; Eick, Sigrun

    2012-01-01

    Background and Objectives Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of P. gingivalis and other periodontopathogens. Material and methods GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis, and five periodontally-healthy individuals. The bacterial loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Tannerella forsythia were analysed by real-time PCR, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. Results Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in the healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. P. gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (p < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of T. forsythia and P. intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of P. gingivalis (r = 0.425, p < 0.01). The presence of Kgp (range 0.07–10.98 ng/ml) was associated with proteolytic fragments of IgG1 (p < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. Conclusion In patients with periodontitis cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by P. gingivalis. PMID:23116446

  18. 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

  19. 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

  20. 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 Central

    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

  1. Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

    SciTech Connect

    Wojtowicz, Halina; Wojaczynski, Jacek; Olczak, Mariusz; Kroliczewski, Jaroslaw; Latos-Grazynski, Lechoslaw; Olczak, Teresa

    2009-05-29

    Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and {sup 1}H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

  2. Conservation of fimbriae and the hemagglutinating adhesin HA-Ag2 among Porphyromonas gingivalis strains and other anaerobic bacteria studied by epitope mapping analysis.

    PubMed Central

    Du, L; Pellen-Mussi, P; Chandad, F; Mouton, C; Bonnaure-Mallet, M

    1997-01-01

    Monoclonal antibodies characterized as antifimbria and anti-HA-Ag2 were used in immunoblotting to examine the antigenic distribution of fimbriae and HA-Ag2 among a collection of human and animal Porphyromonas strains and human Prevotella and Bacteroides strains. The results showed that fimbrial and HA-Ag2 antigenic structures are peculiar to the species Porphyromonas gingivalis. PMID:9384294

  3. Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    PubMed Central

    Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Hörkkö, Sohvi

    2012-01-01

    Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875

  4. Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.

    PubMed

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

    2011-08-15

    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 used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward 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 given that it did not bind to the homologous gingipain HRgpA. This indicated the 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

  5. Targeted antimicrobial activity of a specific IgG-SMAP28 conjugate against Porphyromonas gingivalis in a mixed culture.

    PubMed

    Franzman, Michael R; Burnell, Kindra K; Dehkordi-Vakil, Farideh H; Guthmiller, Janet M; Dawson, Deborah V; Brogden, Kim A

    2009-01-01

    Antimicrobial peptides coupled to a ligand, receptor or antibody for a specific pathogenic bacteria could be used to develop narrow-spectrum pharmaceuticals with 'targeted' antimicrobial activity void of adverse reactions often associated with the use of broad-spectrum antibiotics. To assess the feasibility of this approach, in this study sheep myeloid antimicrobial peptide (SMAP) 28 was linked to affinity- and protein G-purified rabbit immunoglobulin G (IgG) antibodies specific to the outer surface of Porphyromonas gingivalis strain 381. The selective activity of the P. gingivalis IgG-SMAP28 conjugate was then assessed by adding it to an artificially generated microbial community containing P. gingivalis, Aggregatibacter actinomycetemcomitans and Peptostreptococcus micros. The specificity of the P. gingivalis IgG-SMAP28 conjugate in this mixed culture was concentration-dependent. The conjugate at 50 microg protein/mL lacked specificity and killed P. gingivalis, A. actinomycetemcomitans and P. micros. The conjugate at 20 microg protein/mL was more specific and killed P. gingivalis. This is an initial step to develop a selective antimicrobial agent that can eliminate a specific periodontal pathogen, such as P. gingivalis, from patients with periodontal disease without harming the normal commensal flora. PMID:18778918

  6. Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis.

    PubMed Central

    Jagels, M A; Travis, J; Potempa, J; Pike, R; Hugli, T E

    1996-01-01

    The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis. Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils. Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsullonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (Nalpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at

  7. 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

  8. 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

  9. 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

  10. Porphyromonas gingivalis infected macrophages upregulate CD36 expression via ERK/NF-κB pathway.

    PubMed

    Liang, Dong-Yu; Liu, Feng; Chen, Jian-Xia; He, Xiao-Li; Zhou, Yi-Long; Ge, Bao-Xue; Luo, Li-Jun

    2016-09-01

    CD36, a scavenger receptor, plays an important role in the progression of atherosclerosis through its interaction with oxidized low-density lipoprotein (ox-LDL). Porphyromonas gingivalis (P. gingivalis, Pg) has been shown to promote macrophage-derived foam cell formation by affecting the expression of CD36. However, the regulatory role of CD36 in macrophages infected with Pg remains largely unknown. Therefore, the aim of the present study was to explore the molecular mechanism of Pg induced CD36 expression in macrophages. Our results showed that Pg promoted ox-LDL uptake by macrophages and the formation of foam cells. Pg infection increased CD36 mRNA and protein levels in ox-LDL-untreated macrophages. Moreover, small interferon RNA (siRNA) targeting CD36 significantly reduced foam cell formation induced by Pg. Additionally, Pg stimulated nuclear translocation of p65, which directly bound to the promoters of CD36 to facilitate its transcription. Inhibition of p65, NF-κB or ERK1/2 blocked Pg-induced CD36 production; whereas, overexpression of NF-κB subunits p65 and p50 upregulated CD36. Furthermore, Ras inhibitors significantly attenuated ERK1/2 activation and CD36 expression. Taken together, the data indicated that stimulation of the ERK/NF-κB pathway by Pg led to transactivation of the CD36 promoters, thereby upregulating CD36 expression in the infected macrophages. These findings may help design new treatment strategies in atherosclerosis. PMID:27234131

  11. Modulation of inflammasome activity by Porphyromonas gingivalis in periodontitis and associated systemic diseases.

    PubMed

    Olsen, Ingar; Yilmaz, Özlem

    2016-01-01

    Inflammasomes are large multiprotein complexes localized in the cytoplasm of the cell. They are responsible for the maturation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 as well as for the activation of inflammatory cell death, the so-called pyroptosis. Inflammasomes assemble in response to cellular infection, cellular stress, or tissue damage; promote inflammatory responses and are of great importance in regulating the innate immune system in chronic inflammatory diseases such as periodontitis and several chronic systemic diseases. In addition to sensing cellular integrity, inflammasomes are involved in the homeostatic mutualism between the indigenous microbiota and the host. There are several types of inflammasomes of which NLRP3 is best characterized in microbial pathogenesis. Many opportunistic bacteria try to evade the innate immune system in order to survive in the host cells. One of these is the periodontopathogen Porphyromonas gingivalis which has been shown to have several mechanisms of modulating innate immunity by limiting the activation of the NLRP3 inflammasome. Among them, ATP-/P2X7- signaling is recently associated not only with periodontitis but also with development of several systemic diseases. The present paper reviews multiple mechanisms through which P. gingivalis can modify innate immunity by affecting inflammasome activity. PMID:26850450

  12. Acute toxicity and the effect of andrographolide on Porphyromonas gingivalis-induced hyperlipidemia in rats.

    PubMed

    Al Batran, Rami; 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 ×10(12) 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

  13. Porphyromonas gingivalis Periodontal Infection and Its Putative Links with Alzheimer's Disease

    PubMed Central

    Singhrao, Sim K.; Harding, Alice; Poole, Sophie; Kesavalu, Lakshmyya; Crean, StJohn

    2015-01-01

    Periodontal disease (PD) and Alzheimer's disease (AD) are inflammatory conditions affecting the global adult population. In the pathogenesis of PD, subgingival complex bacterial biofilm induces inflammation that leads to connective tissue degradation and alveolar bone resorption around the teeth. In health, junctional epithelium seals the gingiva to the tooth enamel, thus preventing bacteria from entering the gingivae. Chronic PD involves major pathogens (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) which have an immune armoury that can circumvent host's immune surveillance to create and maintain an inflammatory mediator rich and toxic environment to grow and survive. The neurodegenerative condition, AD, is characterised by poor memory and specific hallmark proteins; periodontal pathogens are increasingly being linked with this dementing condition. It is therefore becoming important to understand associations of periodontitis with relevance to late-onset AD. The aim of this review is to discuss the relevance of finding the keystone periodontal pathogen P. gingivalis in AD brains and its plausible contribution to the aetiological hypothesis of this dementing condition. PMID:26063967

  14. 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

  15. Modulation of inflammasome activity by Porphyromonas gingivalis in periodontitis and associated systemic diseases

    PubMed Central

    Olsen, Ingar; Yilmaz, Özlem

    2016-01-01

    Inflammasomes are large multiprotein complexes localized in the cytoplasm of the cell. They are responsible for the maturation of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 as well as for the activation of inflammatory cell death, the so-called pyroptosis. Inflammasomes assemble in response to cellular infection, cellular stress, or tissue damage; promote inflammatory responses and are of great importance in regulating the innate immune system in chronic inflammatory diseases such as periodontitis and several chronic systemic diseases. In addition to sensing cellular integrity, inflammasomes are involved in the homeostatic mutualism between the indigenous microbiota and the host. There are several types of inflammasomes of which NLRP3 is best characterized in microbial pathogenesis. Many opportunistic bacteria try to evade the innate immune system in order to survive in the host cells. One of these is the periodontopathogen Porphyromonas gingivalis which has been shown to have several mechanisms of modulating innate immunity by limiting the activation of the NLRP3 inflammasome. Among them, ATP-/P2X7- signaling is recently associated not only with periodontitis but also with development of several systemic diseases. The present paper reviews multiple mechanisms through which P. gingivalis can modify innate immunity by affecting inflammasome activity. PMID:26850450

  16. 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

  17. 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. PMID:25172523

  18. 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.

  19. 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

  20. Gingipain-dependent degradation of mTOR pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion

    PubMed Central

    Stafford, Prachi; Higham, Jon; Pinnock, Abigail; Murdoch, Craig; Douglas, C. W. Ian; Stafford, Graham P; Lambert, Daniel W

    2014-01-01

    SUMMARY Porphyromonas gingivalis and Tannerella forsythia are Gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules which may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4 hour time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggests that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection. PMID:23714361

  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. 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

  3. 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

  4. Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

    PubMed Central

    2012-01-01

    Background Periodontal disease is suggested to increase the risk of atherothrombotic disease by inducing dyslipidemia. Recently, we demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), which is known to play a critical role in the regulation of circulating low-density lipoprotein (LDL) cholesterol levels, is elevated in periodontitis patients. However, the underlying mechanisms of elevation of PCSK9 in periodontitis patients are largely unknown. Here, we explored whether Porphyromonas gingivalis, a representative periodontopathic bacterium, -induced inflammatory response regulates serum PCSK9 and cholesterol levels using animal models. Methods We infected C57BL/6 mice intraperitoneally with Porphyromonas gingivalis, a representative strain of periodontopathic bacteria, and evaluated serum PCSK9 levels and the serum lipid profile. PCSK9 and LDL receptor (LDLR) gene and protein expression, as well as liver X receptors (Lxrs), inducible degrader of the LDLR (Idol), and sterol regulatory element binding transcription factor (Srebf)2 gene expression, were examined in the liver. Results P. gingivalis infection induced a significant elevation of serum PCSK9 levels and a concomitant elevation of total and LDL cholesterol compared with sham-infected mice. The LDL cholesterol levels were significantly correlated with PCSK9 levels. Expression of the Pcsk9, Ldlr, and Srebf2 genes was upregulated in the livers of the P. gingivalis-infected mice compared with the sham-infected mice. Although Pcsk9 gene expression is known to be positively regulated by sterol regulatory element binding protein (SREBP)2 (human homologue of Srebf2), whereas Srebf2 is negatively regulated by cholesterol, the elevated expression of Srebf2 found in the infected mice is thought to be mediated by P. gingivalis infection. Conclusions P. gingivalis infection upregulates PCSK9 production via upregulation of Srebf2, independent of cholesterol levels. Further studies are required to

  5. Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

    PubMed Central

    El-Awady, Ahmed R.; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B.; Palani, Chithra D.; Arce, Roger M.; Waller, Jennifer L.; Genco, Caroline A.; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V.; Cutler, Christopher W.

    2015-01-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs. PMID:25679217

  6. Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

    PubMed Central

    Bajrami, Bekim; Clark, Robert B.; Housley, William; Yao, Xudong

    2012-01-01

    Recent reports indicate that Porphyromonas gingivalis mediates alveolar bone loss or osteoclast modulation through engagement of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopolysaccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides of P. gingivalis have been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because conflicting evidence has been reported concerning the capacity of P. gingivalis LPS or lipid A to engage TLR2 versus TLR4. In the present study, we first prepared P. gingivalis LPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphorylated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray-MS methods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived from P. gingivalis are contaminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingival plaque do not contain free lipid A constituents of P. gingivalis but contain substantial amounts of phosphorylated dihydroceramide lipids. Therefore, the free lipid A of P. gingivalis is not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A of P. gingivalis could actually be mediated by phosphorylated dihydroceramides. PMID:22144487

  7. 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

  8. 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

  9. 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

  10. Gingipains from Porphyromonas gingivalis – Complex domain structures confer diverse functions

    PubMed Central

    Li, N.

    2011-01-01

    Gingipains, a group of arginine or lysine specific cysteine proteinases (also known as RgpA, RgpB and Kgp), have been recognized as major virulence factors in Porphyromonas gingivalis. This bacterium is one of a handful of pathogens that cause chronic periodontitis. Gingipains are involved in adherence to and colonization of epithelial cells, haemagglutination and haemolysis of erythrocytes, disruption and manipulation of the inflammatory response, and the degradation of host proteins and tissues. RgpA and Kgp are multi-domain proteins composed of catalytic domains and haemagglutinin/adhesin (HA) regions. The structure of the HA regions have previously been defined by a gingipain domain structure hypothesis which is a set of putative domain boundaries derived from the sequences of fragments of these proteins extracted from the cell surface. However, multiple sequence alignments and hidden Markov models predict an alternative domain architecture for the HA regions of gingipains. In this alternate model, two or three repeats of the so-called “cleaved adhesin” domains (and one other undefined domain in some strains) are the modules which constitute the substructure of the HA regions. Recombinant forms of these putative cleaved adhesin domains are indeed stable folded protein modules and recently determined crystal structures support the hypothesis of a modular organisation of the HA region. Based on the observed K2 and K3 structures as well as multiple sequence alignments, it is proposed that all the cleaved adhesin domains in gingipains will share the same β-sandwich jelly roll fold. The new domain model of the structure for gingipains and the haemagglutinin (HagA) proteins of P. gingivalis will guide future functional studies of these virulence factors. PMID:24466435

  11. Gingipains from Porphyromonas gingivalis promote the transformation and proliferation of vascular smooth muscle cell phenotypes

    PubMed Central

    Cao, Chong; Ji, Xiaowei; Luo, Xin; Zhong, Liangjun

    2015-01-01

    The aim of the present study was to ascertain the effect of Porphyromonas gingivalis cysteine protease gingipain on the proliferation of rat aortic smooth muscle cells (RASMCs). Gingipains were isolated and purified from the supernatant of P. gingivalis W83, which was cultured under standard anaerobic conditions; primary RASMCs were also cultured. RASMCs were exposed to 200, 100, 50, 25, 12, 6, 3, 1, and 0 μg/mL activated gingipains and the proliferation was evaluated using a cell counting kit-8 (CCK-8) assay after 48 h. α-Smooth muscle actin (α-SMA) and osteopontin (OPN) expression were measured by immunohistochemical staining. In addition, RASMCs were stimulated with 5, 10, 20, and 40 μM KYT-1 (arg-gingipain inhibitor) and KYT-36 (lys-gingipain inhibitor) in combination with the gingipain extracts. Different concentrations of gingipains significantly promoted the proliferation of RASMCs, except those treated with 1 μg/mL, compared to the untreated controls. The proliferation was sustained at a concentration above 12 μg/mL. Immunohistochemical staining showed OPN expression after gingipain stimulation. The proliferative effects of gingipains on RASMCs were blocked after treatment with 10 μM KYT-1 or 10 μM KYT-36 (P < 0.0001); however, the difference between KYT-1 and KYT-36 groups was not statistically significant. These results demonstrated that gingipains can promote phenotypic transformation and proliferation of RASMCs and their effects were blocked by KYT-1 and KYT-36, which help us to ascertain whether Rgp or Kgp contributes to the development of atherosclerosis. PMID:26770435

  12. 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

  13. The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization.

    PubMed

    Lin, F-Y; Huang, C-Y; Lu, H-Y; Shih, C-M; Tsao, N-W; Shyue, S-K; Lin, C-Y; Chang, Y-J; Tsai, C-S; Lin, Y-W; Lin, S-J

    2015-06-01

    Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell-carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell-carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E-selectin expression, which is mediated by eNOS production and p38 mitogen-activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel-induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth. PMID:25220060

  14. Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K

    PubMed Central

    Ali Mohammed, Marwan Mansoor; Nerland, Audun H.; Al-Haroni, Mohammed; Bakken, Vidar

    2013-01-01

    Background Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. Methods F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. Results Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. Conclusion DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions. PMID:23372876

  15. Tissue Destruction Induced by Porphyromonas gingivalis Infection in a Mouse Chamber Model Is Associated with Host Tumor Necrosis Factor Generation

    PubMed Central

    Lin, Yuh-Yih; Huang, Jan-Hung; Lai, Yo-Yin; Huang, Han-Ching; Hu, Suh-Woan

    2005-01-01

    Intrachamber challenge with Porphyromonas gingivalis strain 381 in a mouse subcutaneous chamber model results in a local infection that progresses to exfoliation of the chambers within 15 days. This study was designed to elucidate the contribution of host reactions to tissue destruction manifested by chamber exfoliation in animals infected with P. gingivalis. Chamber fluids showed increasing levels of prostaglandin E2 with infection, and the levels of tumor necrosis factor (TNF) in chamber fluids peaked just before chamber exfoliation. Intraperitoneal injection of a TNF inhibitor, thalidomide (TH), reduced the number of exfoliated chambers, while indomethacin had no effect. Exogenous TNF in chambers without bacterial infection did not cause chamber exfoliation but induced neutrophil infiltration. In a dual-chamber model, two chambers were implanted in the same mouse. One chamber was infected with P. gingivalis, and 9 days later exogenous TNF was added to the other chamber. Altogether, 66.67% of uninfected chambers were exfoliated between day 11 and day 16, although no bacteria were recovered from uninfected chambers. TH treatment alleviated both infected and uninfected chamber exfoliation. In this study, tissue destruction caused by P. gingivalis 381 infection was due to the elevation of the TNF levels and not due to local bacterial activities. Our results further indicate that local infection by P. gingivalis 381, a nondisseminating strain, actually has systemic effects on the host pathological outcome. PMID:16299286

  16. Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY

    PubMed Central

    2010-01-01

    Background Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY. Results HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation. Conclusions Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation. PMID:20438645

  17. Chronic Oral Infection with Porphyromonas gingivalis Accelerates Atheroma Formation by Shifting the Lipid Profile

    PubMed Central

    Tabeta, Koichi; Aoki, Yukari; Miyashita, Hirotaka; Miyauchi, Sayuri; Miyazawa, Haruna; Nakajima, Takako; Yamazaki, Kazuhisa

    2011-01-01

    Background Recent studies have suggested that periodontal disease increases the risk of atherothrombotic disease. Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in the arteries. Although several studies have suggested that certain periodontopathic bacteria accelerate atherogenesis in apolipoprotein E-deficient mice, the mechanistic link between cholesterol accumulation and periodontal infection-induced inflammation is largely unknown. Methodology/Principal Findings We orally infected C57BL/6 and C57BL/6.KOR-Apoeshl (B6.Apoeshl) mice with Porphyromonas gingivalis, which is a representative periodontopathic bacterium, and evaluated atherogenesis, gene expression in the aorta and liver and systemic inflammatory and lipid profiles in the blood. Furthermore, the effect of lipopolysaccharide (LPS) from P. gingivalis on cholesterol transport and the related gene expression was examined in peritoneal macrophages. Alveolar bone resorption and elevation of systemic inflammatory responses were induced in both strains. Despite early changes in the expression of key genes involved in cholesterol turnover, such as liver X receptor and ATP-binding cassette A1, serum lipid profiles did not change with short-term infection. Long-term infection was associated with a reduction in serum high-density lipoprotein (HDL) cholesterol but not with the development of atherosclerotic lesions in wild-type mice. In B6.Apoeshl mice, long-term infection resulted in the elevation of very low-density lipoprotein (VLDL), LDL and total cholesterols in addition to the reduction of HDL cholesterol. This shift in the lipid profile was concomitant with a significant increase in atherosclerotic lesions. Stimulation with P. gingivalis LPS induced the change of cholesterol transport via targeting the expression of LDL receptor-related genes and resulted in the disturbance of regulatory mechanisms of the cholesterol level in macrophages. Conclusions

  18. Wild Bitter Melon Leaf Extract Inhibits Porphyromonas gingivalis-Induced Inflammation: Identification of Active Compounds through Bioassay-Guided Isolation.

    PubMed

    Tsai, Tzung-Hsun; Huang, Wen-Cheng; Ying, How-Ting; Kuo, Yueh-Hsiung; Shen, Chien-Chang; Lin, Yin-Ku; Tsai, Po-Jung

    2016-01-01

    Porphyromonas gingivalis has been identified as one of the major periodontal pathogens. Activity-directed fractionation and purification processes were employed to identify the anti-inflammatory active compounds using heat-killed P. gingivalis-stimulated human monocytic THP-1 cells in vitro. Five major fractions were collected from the ethanol/ethyl acetate extract of wild bitter melon (Momordica charantia Linn. var. abbreviata Ser.) leaves and evaluated for their anti-inflammatory activity against P. gingivalis. Among the test fractions, Fraction 5 effectively decreased heat-killed P. gingivalis-induced interleukin (IL)-8 and was subjected to separation and purification by using chromatographic techniques. Two cucurbitane triterpenoids were isolated from the active fraction and identified as 5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol (1) and 3β,7β,25-trihydroxycucurbita-5,23-dien-19-al (2) by comparing spectral data. Treatments of both compounds in vitro potently suppressed P. gingivalis-induced IL-8, IL-6, and IL-1β levels and the activation of mitogen-activated protein kinase (MAPK) in THP-1 cells. Both compounds effectively inhibited the mRNA levels of IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in P. gingivalis-stimulated gingival tissue of mice. These findings imply that 5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol and 3β,7β,25-trihydroxycucurbita-5,23-dien-19-al could be used for the development of novel therapeutic approaches against P. gingivalis infections. PMID:27058519

  19. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway.

    PubMed

    Okahashi, Nobuo; Inaba, Hiroaki; Nakagawa, Ichiro; Yamamura, Taihei; Kuboniwa, Masae; Nakayama, Koji; Hamada, Shigeyuki; Amano, Atsuo

    2004-03-01

    Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viable P. gingivalis on cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection with P. gingivalis induced receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-kappaB) did not prevent the RANKL expression induced by P. gingivalis. Degradation of inhibitor of NF-kappaB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced by P. gingivalis infection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection with P. gingivalis KDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest that P. gingivalis infection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production. PMID:14977979

  20. 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

  1. Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

    PubMed Central

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

    1995-01-01

    Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence of an internal transcription-translation initiation site which could permit independent expression of different regions of the polyprotein. Immunochemical analysis of P. gingivalis mid-logarithmic-phase cultures suggested that the processing of PrpRI may be closely coupled to its synthesis, with only the final stages taking place at the cell surface. Southern hybridization studies demonstrated that the prpR1 gene is widely distributed in other P. gingivalis strains and that a second homologous locus to the alpha component and at least two other homologous loci to the beta component are present on the P. gingivalis chromosome. These data indicate that the ArgI protease of P. gingivalis is a member of a family of sequence-related gene products which may share both functional and antigenic properties. PMID:7591131

  2. 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

  3. 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

  4. 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

  5. Comparative whole-genome analysis of virulent and avirulent strains of Porphyromonas gingivalis.

    PubMed

    Chen, Tsute; Hosogi, Yumiko; Nishikawa, Kiyoshi; Abbey, Kevin; Fleischmann, Robert D; Walling, Jennifer; Duncan, Margaret J

    2004-08-01

    We used Porphyromonas gingivalis gene microarrays to compare the total gene contents of the virulent strain W83 and the avirulent type strain, ATCC 33277. Signal ratios and scatter plots indicated that the chromosomes were very similar, with approximately 93% of the predicted genes in common, while at least 7% of them showed very low or no signals in ATCC 33277. Verification of the array results by PCR indicated that several of the disparate genes were either absent from or variant in ATCC 33277. Divergent features included already reported insertion sequences and ragB, as well as additional hypothetical and functionally assigned genes. Several of the latter were organized in a putative operon in W83 and encoded enzymes involved in capsular polysaccharide synthesis. Another cluster was associated with two paralogous regions of the chromosome with a low G+C content, at 41%, compared to that of the whole genome, at 48%. These regions also contained conserved and species-specific hypothetical genes, transposons, insertion sequences, and integrases and were located adjacent to tRNA genes; thus, they had several characteristics of pathogenicity islands. While this global comparative analysis showed the close relationship between W83 and ATCC 33277, the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277 is suggestive of chromosomal islands that may have been acquired by lateral gene transfer. PMID:15292149

  6. Role of Superoxide Dismutase Activity in the Physiology of Porphyromonas gingivalis

    PubMed Central

    Lynch, Michael C.; Kuramitsu, Howard K.

    1999-01-01

    Porphyromonas gingivalis is a gram-negative, obligate anaerobe strongly associated with chronic adult periodontitis. A previous study has demonstrated that this organism requires superoxide dismutase (SOD) for its modest aerotolerance. In this study, we have constructed a mutant deficient in SOD activity by insertional inactivation as well as a sod::lacZ reporter translational fusion construct to study the regulation of expression of this gene. We have confirmed that SOD is essential for tolerance to atmospheric oxygen but does not appear to be protective against hydrogen peroxide or exogenously generated reactive oxygen species. Furthermore, the sod mutant appeared to be no more sensitive to killing by neutrophils than the parental strain 381. SOD appears to be protective against oxygen-dependent DNA damage as measured by increased mutation to rifampin resistance by the sod mutant. Use of the sod::lacZ construct confirmed that SOD expression is maximal at mid-log phase and is influenced by oxygen, temperature, and pH. However, expression does not appear to be significantly affected by iron depletion, osmolarity, or nutrient depletion. The transcription start site of the sod gene was determined to be 315 bp upstream of the sod start codon and to be within an upstream open reading frame. Our studies demonstrate the essential role that SOD plays in aerotolerance of this organism as well as the selective induction of this enzyme by environmental stimuli. PMID:10377114

  7. Rapid detection of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromona gingivalis by multiplex PCR.

    PubMed

    García, L; Tercero, J C; Legido, B; Ramos, J A; Alemany, J; Sanz, M

    1998-01-01

    The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens. PMID:9524322

  8. High-density lipoprotein therapy inhibits Porphyromonas gingivalis-induced abdominal aortic aneurysm progression.

    PubMed

    Delbosc, Sandrine; Rouer, Martin; Alsac, Jean-Marc; Louedec, Liliane; Al Shoukr, Faisal; Rouzet, François; Michel, Jean-Baptiste; Meilhac, Olivier

    2016-04-01

    Clinical and experimental studies have highlighted the potential implication of periondontal bacteria contamination in the pathogenesis of abdominal aortic aneurysms (AAA). In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDLs) display multiple functions, including anti-inflammatory and lipopolysaccharide scavenging properties. Low plasma levels of HDL-cholesterol have been reported in AAA patients. We tested the effect of a HDL therapy in Sprague-Dawley rat model of AAA, obtained by intraluminal elastase infusion followed by repeated injections of Porphyromonas gingivalis (Pg). HDLs, isolated by ultracentrifugation of plasma from healthy human volunteers, were co-injected intravenously (10 mg/kg) with Pg (1.107 Colony Forming Unit) one, eight and 15 days after elastase perfusion. Rats were sacrificed one week after the last injection. Our results show that Pg injections promote the formation of a persistent neutrophil-rich thrombus associated with increased aortic diameter in this AAA model. HDLs significantly reduced the increased AAA diameter induced by Pg. Histology showed the onset of a healing process in the Pg/HDL group. HDL injections also reduced neutrophil activation in Pg-injected rats associated with decreased cytokine levels in conditioned media and plasma. Scintigraphic analysis showed an intense uptake of 99mTc-HDL by the AAA suggesting that HDLs could exert their beneficial effect by acting directly on the thrombus components. HDL supplementation may therefore constitute a new therapeutic tool for AAA treatment. PMID:26676721

  9. The Periodontal Pathogen Porphyromonas gingivalis Preferentially Interacts with Oral Epithelial Cells in S Phase of the Cell Cycle.

    PubMed

    Al-Taweel, Firas B; Douglas, C W Ian; Whawell, Simon A

    2016-07-01

    Porphyromonas gingivalis, a key periodontal pathogen, is capable of invading a variety of cells, including oral keratinocytes, by exploiting host cell receptors, including alpha-5 beta-1 (α5β1) integrin. Previous studies have shown that P. gingivalis accelerates the cell cycle and prevents apoptosis of host cells, but it is not known whether the cell cycle phases influence bacterium-cell interactions. The cell cycle distribution of oral keratinocytes was characterized by flow cytometry and BrdU (5-bromo-2-deoxyuridine) staining following synchronization of cultures by serum starvation. The effect of cell cycle phases on P. gingivalis invasion was measured by using antibiotic protection assays and flow cytometry, and these results were correlated with gene and surface expression levels of α5 integrin and urokinase plasminogen activator receptor (uPAR). There was a positive correlation (R = 0.98) between the number of cells in S phase and P. gingivalis invasion, the organism was more highly associated with cells in S phase than with cells in G2 and G1 phases, and S-phase cells contained 10 times more bacteria than did cells that were not in S phase. Our findings also show that α5 integrin, but not uPAR, was positively correlated with cells in S phase, which is consistent with previous reports indicating that P. gingivalis invasion of cells is mediated by α5 integrin. This study shows for the first time that P. gingivalis preferentially associates with and invades cells in the S phase of the cell cycle. The mechanism of targeting stable dividing cells may have implications for the treatment of periodontal diseases and may partly explain the persistence of this organism at subgingival sites. PMID:27091929

  10. Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing.

    PubMed

    Robles, A G; Reid, K; Roy, F; Fletcher, H M

    2011-06-01

    The ability for DNA mismatch repair, after oxidative stress-induced DNA damage, is critical for the persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket. Our previous report demonstrated that, in contrast to other organisms, the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by a yet-to-be described mechanism in P. gingivalis. 8-oxoG does not block DNA replication; rather, it mispairs with adenine, which can be repaired by the MutY glycosylase. To determine the function of the P. gingivalis MutY homologue in DNA repair, it was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a mutY-deficient mutant (FLL147) by allelic exchange mutagenesis. FLL147 had an increased rate of spontaneous mutation and was more sensitive to hydrogen peroxide compared with the wild-type W83 strain. DNA oligomers containing a site-specific 8-oxoG:A mispair was repaired similarly in both the P. gingivalis mutY-defective mutant and wild-type strains. The P. gingivalis mutY homologue was shown to complement the mutY mutation in Escherichia coli. In a gel mobility shift assay, the purified recombinant MutY is able to bind an oligo containing an 8-oxoG:A mispair. Taken together, MutY may play the expected role in oxidative stress resistance in P. gingivalis. However, there may exist other redundant mechanism(s) for the removal of 8-oxoG:A mismatch in this organism. PMID:21545695

  11. Expression of Porphyromonas gingivalis small RNA in response to hemin availability identified using microarray and RNA-seq analysis.

    PubMed

    Phillips, Priscilla; Progulske-Fox, Ann; Grieshaber, Scott; Grieshaber, Nicole

    2014-02-01

    There is a significant body of work suggesting that sRNA-mediated post-transcriptional regulation is a conserved mechanism among pathogenic bacteria to modulate bacterial virulence and survival. Porphyromonas gingivalis is recognized as an etiological agent of periodontitis and implicated in contributing to the development of multiple inflammatory diseases including cardiovascular disease. Using NimbleGen microarray analysis and a strand-specific method to sequence cDNA libraries of small RNA-enriched P. gingivalis transcripts using Illumina's high-throughput sequencing technology, we identified putative sRNA and generated sRNA expression profiles in response to growth phase, hemin availability after hemin starvation, or both. We identified transcripts that mapped to intergenic sequences as well as antisense transcripts that mapped to open reading frames of the annotated genome. Overall, this approach provided a comprehensive way to survey transcriptional activity to discover functionally linked RNA transcripts, responding to specific environmental cues, that merit further investigation. PMID:24245974

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

    PubMed Central

    Aoyagi, T; Yamazaki, K; Kabasawa-Katoh, Y; Nakajima, T; Yamashita, N; Yoshie, H; Hara, K

    2000-01-01

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

  13. The periodontal pathogen Porphyromonas gingivalis induces expression of transposases and cell death of Streptococcus mitis in a biofilm model.

    PubMed

    Duran-Pinedo, Ana E; Baker, Vinesha D; Frias-Lopez, Jorge

    2014-08-01

    Oral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the "keystone-pathogen" where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating that P. gingivalis induces S. mitis death and DNA fragmentation in an in vitro biofilm system. Moreover, we report here the induction of expression of multiple transposases in a Streptococcus mitis biofilm when the periodontopathogen P. gingivalis is present. Based on these results, we hypothesize that P. gingivalis induces S. mitis cell death by an unknown mechanism, shaping the oral microbiome to its advantage. PMID:24866802

  14. Metabolome variations in the Porphyromonas gingivalis vimA mutant during hydrogen peroxide-induced oxidative stress

    PubMed Central

    McKenzie, R.M.E.; Aruni, W.; Johnson, N.A.; Robles, A.; Dou, Y.; Henry, L.; Boskovic, D.S.; Fletcher, H.M.

    2015-01-01

    SUMMARY The adaptability and survival of Porphyromonas gingivalis in the oxidative microenvironment of the periodontal pocket are indispensable for survival and virulence, and are modulated by multiple systems. Among the various genes involved in P. gingivalis oxidative stress resistance, vimA gene is a part of the 6.15-kb locus. To elucidate the role of a P. gingivalis vimA-defective mutant in oxidative stress resistance, we used a global approach to assess the transcriptional profile, to study the unique metabolome variations affecting survival and virulence in an environment typical of the periodontal pocket. A multilayered protection strategy against oxidative stress was noted in P. gingivalis FLL92 with upregulation of detoxifying genes. The duration of oxidative stress was shown to differentially modulate transcription with 94 (87%) genes upregulated twofold during 10 min and 55 (83.3%) in 15 min. Most of the up-regulated genes (55%), fell in the hypothetical/unknown/unassigned functional class. Metabolome variation showed reduction in fumarate and formaldehyde, hence resorting to alternative energy generation and maintenance of a reduced metabolic state. There was upregulation of transposases, genes encoding for the metal ion binding protein transport and secretion system. PMID:25055986

  15. 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

  16. Metabolome variations in the Porphyromonas gingivalis vimA mutant during hydrogen peroxide-induced oxidative stress.

    PubMed

    McKenzie, R M E; Aruni, W; Johnson, N A; Robles, A; Dou, Y; Henry, L; Boskovic, D S; Fletcher, H M

    2015-04-01

    The adaptability and survival of Porphyromonas gingivalis in the oxidative microenvironment of the periodontal pocket are indispensable for survival and virulence, and are modulated by multiple systems. Among the various genes involved in P. gingivalis oxidative stress resistance, vimA gene is a part of the 6.15-kb locus. To elucidate the role of a P. gingivalis vimA-defective mutant in oxidative stress resistance, we used a global approach to assess the transcriptional profile, to study the unique metabolome variations affecting survival and virulence in an environment typical of the periodontal pocket. A multilayered protection strategy against oxidative stress was noted in P. gingivalis FLL92 with upregulation of detoxifying genes. The duration of oxidative stress was shown to differentially modulate transcription with 94 (87%) genes upregulated twofold during 10 min and 55 (83.3%) in 15 min. Most of the upregulated genes (55%), fell in the hypothetical/unknown/unassigned functional class. Metabolome variation showed reduction in fumarate and formaldehyde, hence resorting to alternative energy generation and maintenance of a reduced metabolic state. There was upregulation of transposases, genes encoding for the metal ion binding protein transport and secretion system. PMID:25055986

  17. 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

  18. Active sites of salivary proline-rich protein for binding to Porphyromonas gingivalis fimbriae.

    PubMed Central

    Kataoka, K; Amano, A; Kuboniwa, M; Horie, H; Nagata, H; Shizukuishi, S

    1997-01-01

    Porphyromonas gingivalis fimbriae specifically bind salivary acidic proline-rich protein 1 (PRP1) through protein-protein interactions. The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were analyzed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 64:1631-1637, 1996). In this study, we investigated the sites of binding of the PRP1 molecules to the fimbriae. PRP1 (amino acid residues 1 to 150) was proteolysed to three fragments (residues 1 to 74 [fragment 1-74], 75 to 129, and 130 to 150). 125I-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments also inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) beads by 50 and 83%, respectively. However, the N-terminal fragment failed to show any effect. Analogous peptides corresponding to residues 75 to 89, 90 to 106, 107 to 120, 121 to 129, and 130 to 150 of PRP1 were synthesized. The fimbriae significantly bound peptide 130-150, immobilized on 96-well plates, and the peptide also inhibited binding of 125I-labeled fimbriae to PRP1-coated HAP beads by almost 100%. Peptides 75-89, 90-106, and 121-129, immobilized on plates, showed considerable ability to bind fimbriae. For further analysis of active sites in residues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGPPQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%. This amino acid sequence was shared in the alignment of residues 75 to 89, 90 to 106, and 107 to 120. Six synthetic peptides were prepared by serial deletions of individual residues from the N and C termini of peptide GRPQGPPQ. Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ. Further deletions of the dipeptide Pro-Gln from the N and C termini of peptide PQGPPQ resulted in significant loss of the inhibitory effect. These results strongly suggest that PQGPPQ

  19. Capsular Polysaccharide-Fimbrial Protein Conjugate Vaccine Protects against Porphyromonas gingivalis Infection in SCID Mice Reconstituted with Human Peripheral Blood Lymphocytes

    PubMed Central

    Choi, Jeom-Il; Schifferle, Robert E.; Yoshimura, Fuminobu; Kim, Byung-Woo

    1998-01-01

    The effect of immunization with either a Porphyromonas gingivalis fimbrial protein, a capsular polysaccharide, or a capsular polysaccharide-fimbrial protein conjugate vaccine were compared in hu-PBL-SCID mice. A significantly higher human immunoglobulin G antibody response and the highest degree of in vivo protection against bacterial challenge was observed in the group immunized with the conjugate vaccine. It was concluded that capsular polysaccharide-fimbrial protein conjugate from P. gingivalis could potentially be developed as a vaccine against periodontal infection by P. gingivalis. PMID:9423888

  20. 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

  1. Porphyromonas gingivalis Virulence Factor Gingipain RgpB Shows a Unique Zymogenic Mechanism for Cysteine Peptidases*

    PubMed Central

    de Diego, Iñaki; Veillard, Florian T.; Guevara, Tibisay; Potempa, Barbara; Sztukowska, Maryta; Potempa, Jan; Gomis-Rüth, F. Xavier

    2013-01-01

    Zymogenicity is a regulatory mechanism that prevents inadequate catalytic activity in the wrong context. It plays a central role in maintaining microbial virulence factors in an inactive form inside the pathogen until secretion. Among these virulence factors is the cysteine peptidase gingipain B (RgpB), which is the major virulence factor secreted by the periodontopathogen Porphyromonas gingivalis that attacks host vasculature and defense proteins. The structure of the complex between soluble mature RgpB, consisting of a catalytic domain and an immunoglobulin superfamily domain, and its 205-residue N-terminal prodomain, the largest structurally characterized to date for a cysteine peptidase, reveals a novel fold for the prodomain that is distantly related to sugar-binding lectins. It attaches laterally to the catalytic domain through a large concave surface. The main determinant for latency is a surface “inhibitory loop,” which approaches the active-site cleft of the enzyme on its non-primed side in a substrate-like manner. It inserts an arginine (Arg126) into the S1 pocket, thus matching the substrate specificity of the enzyme. Downstream of Arg126, the polypeptide leaves the cleft, thereby preventing cleavage. Moreover, the carbonyl group of Arg126 establishes a very strong hydrogen bond with the co-catalytic histidine, His440, pulling it away from the catalytic cysteine, Cys473, and toward Glu381, which probably plays a role in orienting the side chain of His440 during catalysis. The present results provide the structural determinants of zymogenic inhibition of RgpB by way of a novel inhibitory mechanism for peptidases in general and open the field for the design of novel inhibitory strategies in the treatment of human periodontal disease. PMID:23558682

  2. Porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system.

    PubMed

    Progulske-Fox, A; Kozarov, E; Dorn, B; Dunn, W; Burks, J; Wu, Y

    1999-10-01

    Our laboratory is interested in the genes and gene products involved in the interactions between Porphyromonas gingivalis (Pg) and the host. These interactions may occur in either the periodontal tissues or other non-oral host tissues such as those of the cardiovascular system. We have previously reported the cloning of several genes encoding hemagglutinins, surface proteins that interact with the host tissues, and are investigating their roles in the disease process. Primary among these is HagA, a very large protein with multiple functional groups that have significant sequence homology to protease genes of this species. Preliminary evidence indicates that an avirulent Salmonella typhimurium strain containing hagA is virulent in mice. These data indicate that HagA may be a key virulence factor of Pg. Additionally, we are investigating the invasion of primary human coronary artery endothelial cells (HCAEC) by Pg because of the recent epidemiological studies indicating a correlation between periodontal disease (PD) and coronary heart disease (CHD). We found that some, but not all, strains of Pg are able to invade these cells. Scanning electron microsopy of the infected HCAEC demonstrated that the invading organisms initially attached to the host cell surface as aggregates and by a "pedestal"-like structure. By transmission electronmicroscopy it could be seen that internalized bacteria were present within multimembranous compartments localized with rough endoplasmic reticulum. In addition, invasion of the HCAEC by Pg resulted in an increase in the degradation of long-lived cellular proteins. These data indicate that Pg are present within autophagosomes and may use components of the autophagic pathway as a means to survive intracellularly. However, Pg presence within autophagosomes in KB cells could not be observed or detected. It is therefore likely that Pg uses different invasive mechanisms for different host cells. This and the role of HagA in invasion is currently

  3. Regulon Controlled by the GppX Hybrid Two Component system in Porphyromonas gingivalis

    PubMed Central

    Hirano, Takanori; Beck, David A. C.; Wright, Chris J.; Demuth, Donald R.; Hackett, Murray; Lamont, Richard J.

    2012-01-01

    Summary The periodontal pathogen Porphyromonas gingivalis experiences a number of environmental conditions in the oral cavity and must monitor and respond to a variety of environmental cues. However the organism possesses only five full two-component systems, one of which is the hybrid system GppX. To investigate the regulon controlled by GppX we performed RNA-Seq on a ΔgppX mutant. Fifty three genes were up-regulated and 37 genes were down-regulated in the ΔgppX mutant. Pathway analyses revealed no systemic function for GppX under nutrient replete conditions; however, over 40% of the differentially abundant genes were annotated as encoding hypothetical proteins indicating a novel role for GppX. Abundance of small (s)RNA was, in general, not affected by the absence of GppX. To further define the role of GppX with respect to regulation of a hypothetical protein observed with the greatest significant relative abundance change relative to a wild-type control, PGN_0151, we constructed a series of strains in which a ΔgppX mutation was complemented with GppX protein containing specific domain and phosphotransfer mutations. The transmembrane domains, the DNA binding domain and the phosphotransfer residues were all required for regulation of PGN_0151. In addition, binding of GppX to the PGN_0151 promoter regions was confirmed by an electrophoretic mobility shift assay (EMSA). Both the ΔgppX mutant and a ΔPGN_0151 mutant were deficient in monospecies biofilm formation, suggesting a role for the GppX-PGN_0151 regulon in colonization and survival of the organism. PMID:23194602

  4. 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

  5. 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

  6. Human immunoglobulin G antibody response to iron-repressible and other membrane proteins of Porphyromonas (Bacteroides) gingivalis.

    PubMed Central

    Chen, C K; DeNardin, A; Dyer, D W; Genco, R J; Neiders, M E

    1991-01-01

    The human immunoglobulin G (IgG) immune response against Porphyromonas (Bacteroides) gingivalis A7A1-28 iron-repressible membrane proteins (IRMPs) and other membrane proteins was examined by immunoblot analysis. Thirty sera from patients with adult periodontitis and 30 sera from periodontally healthy subjects were included. Iron limitation of P. gingivalis was achieved by growing bacteria in brain heart infusion broth supplemented with protoporphyrin IX and 250 microM alpha, alpha'-dypyridyl, a ferrous iron chelator. Iron-sufficient growth was achieved by growing bacteria in the same medium without alpha, alpha'-dypyridyl. Human sera, in particular those from patients with periodontitis who exhibited high levels of IgG against whole cells of P. gingivalis A7A1-28 in serum in an enzyme-linked immunosorbent assay (ELISA), commonly reacted with five membrane proteins with apparent molecular masses of 80, 67.5, 51, 40.5, and 28 kDa and four IRMPs of 46, 43, 37.5, and 22 kDa. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with the 46-, 43-, and 37.5-kDa IRMPs, and 40% of these subjects expressed immunoreactivity against the 22-kDa IRMP. Sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects exhibited less immunoreactivity against IRMPs and the five membrane proteins of P. gingivalis. The present study indicates that P. gingivalis IRMPs are immunogenic and that these proteins are expressed in vivo. Images PMID:2050407

  7. Protective Role of the PG1036-PG1037-PG1038 Operon in Oxidative Stress in Porphyromonas gingivalis W83

    PubMed Central

    Henry, Leroy G.; Aruni, Wilson; Sandberg, Lawrence; Fletcher, Hansel M.

    2013-01-01

    As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis. PMID:23990885

  8. 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

  9. Porphyromonas gingivalis GroEL Induces Osteoclastogenesis of Periodontal Ligament Cells and Enhances Alveolar Bone Resorption in Rats

    PubMed Central

    Lin, Feng-Yen; Hsiao, Fung-Ping; Huang, Chun-Yao; Shih, Chun-Ming; Tsao, Nai-Wen; Tsai, Chien-Sung; Yang, Shue-Fen; Chang, Nen-Chung; Hung, Shan-Ling; Lin, Yi-Wen

    2014-01-01

    Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption. PMID:25058444

  10. Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonas gingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant

    PubMed Central

    Yagishita, Hisao; Kumagai, Yumi; Konishi, Kiyoshi; Takahashi, Yukihiro; Aoba, Takaaki; Yoshikawa, Masanosuke

    2001-01-01

    To elucidate the role of dipeptidyl aminopeptidase IV (DPPIV) in the virulence of Porphyromonas gingivalis, mice were infected with either a wild-type strain or a DPPIV-deficient mutant using an abscess model. Histopathological analysis of the resulting lesions indicated that DPPIV participates in virulence through the destruction of connective tissue and the less effective mobilization of inflammatory cells. PMID:11598093

  11. 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

  12. 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

  13. Cloning, expression, and sequencing of a protease gene (tpr) from Porphyromonas gingivalis W83 in Escherichia coli.

    PubMed Central

    Bourgeau, G; Lapointe, H; Péloquin, P; Mayrand, D

    1992-01-01

    Porphyromonas gingivalis is a highly proteolytic organism which metabolizes small peptides and amino acids. Indirect evidence suggests that the proteases produced by this microorganism constitute an important virulence factor. In this study, a gene bank of P. gingivalis W83 DNA was constructed by cloning 0.5- to 20-kb HindIII-cut DNA fragments into Escherichia coli DH5 alpha by using the plasmid vector pUC19. A clone expressing a protease from P. gingivalis was isolated on LB agar containing 1% skim milk. The clone contained a 3.0-kb insert that coded for a protease with an apparent molecular mass of 64 kDa. Sequencing part of the 3.0-kb DNA fragment revealed an open reading frame encoding a protein of 482 amino acids with a molecular mass of 62.5 kDa. Putative promoter and termination elements flanking the open reading frame were identified. The activity expressed in E. coli was extensively characterized by using various substrates and protease inhibitors, and the results suggest that it is possibly a thiol protease. Images PMID:1322368

  14. 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

  15. 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

  16. LuxS-Based Signaling in Streptococcus gordonii: Autoinducer 2 Controls Carbohydrate Metabolism and Biofilm Formation with Porphyromonas gingivalis

    PubMed Central

    McNab, Roderick; Ford, Suzannah K.; El-Sabaeny, Azza; Barbieri, Bruno; Cook, Guy S.; Lamont, Richard J.

    2003-01-01

    Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-β-d-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii. PMID:12486064

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Effect of Porphyromonas gingivalis and Lactobacillus acidophilus on secretion of IL1B, IL6, and IL8 by gingival epithelial cells.

    PubMed

    Zhao, Jun-jun; Feng, Xi-ping; Zhang, Xiu-li; Le, Ke-yi

    2012-08-01

    Porphyromonas gingivalis alters cytokine expression in gingival epithelial cells, stimulating inflammatory responses that may lead to periodontal disease. This study explored the effect of Lactobacillus acidophilus on the specific expressions of the interleukins (ILs) IL1B, IL6, and IL8 induced by the pathogen. Human gingival epithelial cells were co-cultured with P. gingivalis, L. acidophilus, or L. acidophilus + P. gingivalis; the control group consisted of the cells alone. Protein and gene expression levels of the ILs were detected using ELISA and qRT-PCR, respectively. The supernatant from the P. gingivalis group held significantly higher protein and mRNA levels of IL1B, IL6, and IL8, compared to the control group. In the mixed bacterial group (L. acidophilus + P. gingivalis), the levels of all three ILs decreased with increasing concentrations of L. acidophilus and were significantly different from the P. gingivalis group. This suggests that in gingival cells, L. acidophilus offsets the P. gingivalis-induced secretion of these ILs in a dose-dependent manner. PMID:22382516

  3. Determination of Active Phagocytosis of Unopsonized Porphyromonas gingivalis by Macrophages and Neutrophils Using the pH-Sensitive Fluorescent Dye pHrodo.

    PubMed

    Lenzo, Jason C; O'Brien-Simpson, Neil M; Cecil, Jessica; Holden, James A; Reynolds, Eric C

    2016-06-01

    Phagocytosis of pathogens is an important component of the innate immune system that is responsible for the removal and degradation of bacteria as well as their presentation via the major histocompatibility complexes to the adaptive immune system. The periodontal pathogen Porphyromonas gingivalis exhibits strain heterogeneity, which may affect a phagocyte's ability to recognize and phagocytose the bacterium. In addition, P. gingivalis is reported to avoid phagocytosis by antibody and complement degradation and by invading phagocytic cells. Previous studies examining phagocytosis have been confounded by both the techniques employed and the potential of the bacteria to invade the cells. In this study, we used a novel, pH-sensitive dye, pHrodo, to label live P. gingivalis strains and examine unopsonized phagocytosis by murine macrophages and neutrophils and human monocytic cells. All host cells examined were able to recognize and phagocytose unopsonized P. gingivalis strains. Macrophages had a preference to phagocytose P. gingivalis strain ATCC 33277 over other strains and clinical isolates in the study, whereas neutrophils favored P. gingivalis W50, ATCC 33277, and one clinical isolate over the other strains. This study revealed that all P. gingivalis strains were capable of being phagocytosed without prior opsonization with antibody or complement. PMID:27021243

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

    PubMed

    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

  5. 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

  6. 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

  7. ATP scavenging by the intracellular pathogen Porphyromonas gingivalis inhibits P2X7-mediated host-cell apoptosis

    PubMed Central

    Yilmaz, Özlem; Yao, Luyu; Maeda, Kazuhiko; Rose, Timothy M.; Lewis, Emma L.; Duman, Memed; Lamont, Richard J.; Ojcius, David M.

    2009-01-01

    Summary The purinergic receptor P2X7 is involved in cell death, inhibition of intracellular infection and secretion of inflammatory cytokines. The role of the P2X7 receptor in bacterial infection has been primarily established in macrophages. Here we show that primary gingival epithelial cells, an important component of the oral innate immune response, also express functional P2X7 and are sensitive to ATP-induced apoptosis. Porphyromonas gingivalis, an intracellular bacterium and successful colonizer of oral tissues, can inhibit gingival epithelial cell apoptosis induced by ATP ligation of P2X7 receptors. A P. gingivalis homologue of nucleoside diphosphate kinase (NDK), an ATP-consuming enzyme, is secreted extracellularly and is required for maximal suppression of apoptosis. An ndk-deficient mutant was unable to prevent ATP-induced host-cell death nor plasma membrane permeabilization in the epithelial cells. Treatment with purified recombinant NDK inhibited ATP-mediated host-cell plasma membrane permeabilization in a dose-dependent manner. Therefore, NDK promotes survival of host cells by hydrolysing extracellular ATP and preventing apoptosis-mediated through P2X7. PMID:18005240

  8. The bcp gene in the bcp-recA-vimA-vimE-vimF operon is important in oxidative stress resistance in Porphyromonas gingivalis W83.

    PubMed

    Johnson, N A; McKenzie, R M E; Fletcher, H M

    2011-02-01

    The ability of Porphyromonas gingivalis to overcome oxidative stress in the inflammatory environment of the periodontal pocket is critical for its survival. We have previously demonstrated that the recA locus, which carries the bacterioferritin co-migratory protein (bcp) gene and has a unique genetic architecture, plays a role in virulence regulation and oxidative stress resistance in P. gingivalis. To further characterize the bcp gene, which was confirmed to be part of the bcp-recA-vimA-vimE-vimF operon, we created a P. gingivalis bcp-defective isogenic mutant (FLL302) by allelic exchange. Compared with the wild-type, FLL302 had a similar growth rate, black pigmentation, β-hemolysis and UV sensitivity. Although there was no change in the distribution of gingipain activity, there was a 30% reduction in both Arg-X and Lys-X activities in the mutant strain compared with the wild-type. When exposed to 0.25 mm hydrogen peroxide, P. gingivalis FLL302 was more sensitive than the wild-type. In addition, the cloned P. gingivalis bcp gene increased resistance to 0.25 mm hydrogen peroxide in a bcp-defective Escherichia coli mutant. The mutant also demonstrated decreased aerotolerance when compared with the wild-type. Porphyromonas gingivalis FLL302 and the wild-type strain had similar virulence profiles in a mouse model of virulence. These observations suggest that the bcp gene may play a role in oxidative stress resistance but has a decreased functional significance in the pathogenic potential of P. gingivalis. PMID:21214873

  9. 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

  10. 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. PMID:25543295

  11. 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

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

    PubMed

    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

  13. 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

  14. Porphyromonas gingivalis Infection during Pregnancy Increases Maternal Tumor Necrosis Factor Alpha, Suppresses Maternal Interleukin-10, and Enhances Fetal Growth Restriction and Resorption in Mice

    PubMed Central

    Lin, Dongming; Smith, Mary Alice; Champagne, Catherine; Elter, John; Beck, James; Offenbacher, Steven

    2003-01-01

    Epidemiological studies have shown a potential association between maternal periodontitis and pregnancy complications. We used a pregnant murine model to study the effect of infection with the periodontal pathogen Porphyromonas gingivalis on pregnancy outcomes. Female BALB/c mice were inoculated with heat-killed P. gingivalis (109 CFU) in a subcutaneous chamber and mated 2 weeks later. At gestation day (GD) 7.5, mice were challenged with live P. gingivalis (107 CFU) (n = 20) or broth (control; n = 8) and sacrificed at GD 16.5. Fetal growth restriction (FGR, <0.46 g) was defined as fetuses with weights 2 standard deviations (SD) smaller than controls (0.56 ± 0.05 g [mean ± SD]). Among the 20 challenged mice, 8 had both normal-weight (0.51 ± 0.11 g) and FGR (0.34 ± 0.1 g) fetuses within the same litter. All other challenged dams had normal-weight fetuses (0.57 ± 0.04 g). Maternal liver, uterus, and spleen samples were examined for P. gingivalis DNA using a PCR technique. Of the eight challenged mice with FGR fetuses, three had PCR signals for P. gingivalis in liver and uterus, but not in the spleen. Liver, uterus, and spleen were negative for P. gingivalis DNA among all other challenged and control mice. In serum of dams with FGR fetuses, tumor necrosis factor alpha levels were elevated significantly, while interluekin-10 levels were significantly reduced compared to levels in dams with normal fetuses. P. gingivalis-specific serum immunoglobulin G levels were significantly elevated in dams with FGR fetuses compared to dams without any FGR fetuses. These data demonstrate that P. gingivalis-induced murine FGR is associated with systemic dissemination of the organism and activated maternal immune and inflammatory responses. PMID:12933859

  15. Deletion of a 77-Base-Pair Inverted Repeat Element Alters the Synthesis of Surface Polysaccharides in Porphyromonas gingivalis

    PubMed Central

    Bainbridge, Brian W.; Hirano, Takanori; Grieshaber, Nicole

    2015-01-01

    ABSTRACT Bacterial cell surface glycans, such as capsular polysaccharides and lipopolysaccharides (LPS), influence host recognition and are considered key virulence determinants. The periodontal pathogen Porphyromonas gingivalis is known to display at least three different types of surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown that PG0121 (in strain W83) encodes a DNABII histone-like protein and that this gene is transcriptionally linked to the K-antigen capsule synthesis genes, generating a large ∼19.4-kb transcript (PG0104-PG0121). Furthermore, production of capsule is deficient in a PG0121 mutant strain. In this study, we report on the identification of an antisense RNA (asRNA) molecule located within a 77-bp inverted repeat (77bpIR) element located near the 5′ end of the locus. We show that overexpression of this asRNA decreases the amount of capsule produced, indicating that this asRNA can impact capsule synthesis in trans. We also demonstrate that deletion of the 77bpIR element and thereby synthesis of the large 19.4-kb transcript also diminishes, but does not eliminate, capsule synthesis. Surprisingly, LPS structures were also altered by deletion of the 77bpIR element, and reactivity to monoclonal antibodies specific to both O-LPS and A-LPS was eliminated. Additionally, reduced reactivity to these antibodies was also observed in a PG0106 mutant, indicating that this putative glycosyltransferase, which is required for capsule synthesis, is also involved in LPS synthesis in strain W83. We discuss our finding in the context of how DNABII proteins, an antisense RNA molecule, and the 77bpIR element may modulate expression of surface polysaccharides in P. gingivalis. IMPORTANCE The periodontal pathogen Porphyromonas gingivalis displays at least three different types of cell surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown using Northern analysis that the K-antigen capsule locus encodes a large transcript (∼19.4 kb

  16. 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

  17. 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

  18. 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

  19. 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

  20. Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases

    PubMed Central

    Veillard, Florian; Sztukowska, Maryta; Mizgalska, Danuta; Ksiazek, Mirosław; Houston, John; Potempa, Barbara; Enghild, Jan J.; Thogersen, Ida B.; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

    2013-01-01

    Background Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments. Methods Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis. Results PDRgpB and PDRgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2 nM to 0.85 nM). In contrast, PDKgp showed no inhibitory activity. A conserved Arg-102 residue in PDRgpB and PDRgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PDRgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-L-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex. Conclusion Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains. General significance Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity. PMID:23583629

  1. Filifactor alocis Has Virulence Attributes That Can Enhance Its Persistence under Oxidative Stress Conditions and Mediate Invasion of Epithelial Cells by Porphyromonas gingivalis ▿ †

    PubMed Central

    Aruni, A. Wilson; Roy, Francis; Fletcher, H. M.

    2011-01-01

    Filifactor alocis, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis. In contrast to P. gingivalis, F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesicle-mediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease. PMID:21825062

  2. 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

  3. 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

    Bittner-Eddy, Peter D; Fischer, Lori A; Kaplan, Daniel H; Thieu, Kathleen; Costalonga, Massimo

    2016-08-15

    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

  4. Nucleotide sequence of the Porphyromonas gingivalis W83 recA homolog and construction of a recA-deficient mutant.

    PubMed Central

    Fletcher, H M; Morgan, R M; Macrina, F L

    1997-01-01

    Degenerate oligonucleotide primers were used in PCR to amplify a region of the recA homolog from Porphyromonas gingivalis W83. The resulting PCR fragment was used as a probe to identify a recombinant lambda DASH phage (L10) carrying the P. gingivalis recA homolog. The recA homolog was localized to a 2.1-kb BamHI fragment. The nucleotide sequence of this 2.1-kb fragment was determined, and a 1.02-kb open reading frame (341 amino acids) was detected. The predicted amino acid sequence was strikingly similar (90% identical residues) to the RecA protein from Bacteroides fragilis. No SOS box, characteristic of LexA-regulated promoters, was found in the 5' upstream region of the P. gingivalis recA homolog. In both methyl methanesulfonate and UV survival experiments the recA homolog from P. gingivalis complemented the recA mutation of Escherichia coli HB101. The cloned P. gingivalis recA gene was insertionally inactivated with the ermF-ermAM antibiotic resistance cassette to create a recA-deficient mutant (FLL33) by allelic exchange. The recA-deficient mutant was significantly more sensitive to UV irradiation than the wild-type strain, W83. W83 and FLL33 showed the same level of virulence in in vivo experiments using a mouse model. These results suggest that the recA gene in P. gingivalis W83 plays the expected role of repairing DNA damage caused by UV irradiation. However, inactivation of this gene did not alter the virulence of P. gingivalis in the mouse model. PMID:9353038

  5. Effect of Inactivation of the Arg- and/or Lys-Gingipain Gene on Selected Virulence and Physiological Properties of Porphyromonas gingivalis

    PubMed Central

    Grenier, Daniel; Roy, Sophie; Chandad, Fatiha; Plamondon, Pascale; Yoshioka, Masami; Nakayama, Koji; Mayrand, Denis

    2003-01-01

    Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects. PMID:12874356

  6. Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

    PubMed Central

    Tran, Simon Dangtuan; Rudney, Joel D.

    1999-01-01

    Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays. PMID

  7. 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

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

    2015-11-01

    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

  8. Baicalin Downregulates Porphyromonas gingivalis Lipopolysaccharide-Upregulated IL-6 and IL-8 Expression in Human Oral Keratinocytes by Negative Regulation of TLR Signaling

    PubMed Central

    Luo, Wei; Wang, Cun-Yu; Jin, Lijian

    2012-01-01

    Periodontal (gum) disease is one of the main global oral health burdens and severe periodontal disease (periodontitis) is a leading cause of tooth loss in adults globally. It also increases the risk of cardiovascular disease and diabetes mellitus. Porphyromonas gingivalis lipopolysaccharide (LPS) is a key virulent attribute that significantly contributes to periodontal pathogenesis. Baicalin is a flavonoid from Scutellaria radix, an herb commonly used in traditional Chinese medicine for treating inflammatory diseases. The present study examined the modulatory effect of baicalin on P. gingivalis LPS-induced expression of IL-6 and IL-8 in human oral keratinocytes (HOKs). Cells were pre-treated with baicalin (0–80 µM) for 24 h, and subsequently treated with P. gingivalis LPS at 10 µg/ml with or without baicalin for 3 h. IL-6 and IL-8 transcripts and proteins were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins was analyzed by western blot. A panel of genes related to toll-like receptor (TLR) signaling was examined by PCR array. We found that baicalin significantly downregulated P. gingivalis LPS-stimulated expression of IL-6 and IL-8, and inhibited P. gingivalis LPS-activated NF-κB, p38 MAPK and JNK. Furthermore, baicalin markedly downregulated P. gingivalis LPS-induced expression of genes associated with TLR signaling. In conclusion, the present study shows that baicalin may significantly downregulate P. gingivalis LPS-upregulated expression of IL-6 and IL-8 in HOKs via negative regulation of TLR signaling. PMID:23239998

  9. 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

  10. In-Vivo Effect of Andrographolide on Alveolar Bone Resorption Induced by Porphyromonas gingivalis and Its Relation with Antioxidant Enzymes

    PubMed Central

    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. PMID:24151590