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Jan Potempa - One of the best experts on this subject based on the ideXlab platform.

  • Gingipains impair attachment of epithelial cell to dental titanium abutment surfaces
    Journal of Biomedical Materials Research Part B, 2019
    Co-Authors: Sigrun Eick, Jan Potempa, Naida Gadzo, Manuel Tacchi, Anton Sculean, Andreas Stavropoulos
    Abstract:

    : The study investigated in vitro the effect of Porphyromonas gingivalis and its cysteine proteases (Gingipains) on epithelial cell adhesion to titanium-zirconium alloy surfaces. Titanium-zirconium discs with a standard machined (M) or chemically modified hydrophilic surface (modM) were coated with lamin-5 and incubated with telomerase-inactivated gingival keratinocytes (TIGK). Three P. gingivalis strains or Gingipains were either added simultaneously with TIGK or after TIGK cells were already attached to the disks. Adhered TIGK cells were counted at 24 h. All P. gingivalis strains clearly inhibited adhesion of TIGK cells to M and modM surfaces. Compared with bacteria/Gingipain-free TIGK cell cultures, the number of attached TIGK cells was reduced by about 80% and 60% when P. gingivalis was added simultaneously or after TIGK cells were already attached to the disks (each p < 0.01), respectively. Counts of attached cells were similarly reduced when only Gingipains were used. Adhesion molecules of TIGK cells, in particular E-cadherin, were cleaved by P. gingivalis. In conclusion, P. gingivalis and Gingipains interfere with the adhesion of epithelial cells to titanium-zirconium alloy surfaces by cleaving adhesion molecules, while a chemically modified hydrophilic titanium-zirconium alloy surface did not yield any protection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2549-2556, 2019.

  • inhibition of Gingipains and porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids
    Journal of Periodontal Research, 2017
    Co-Authors: Toru Kariu, Jan Potempa, R Nakao, Tsuyoshi Ikeda, Keisuke Nakashima, Takahisa Imamura
    Abstract:

    Background and Objective Porphyromonas gingivalis is considered a major pathogen of chronic periodontitis, which also may be implicated with systemic diseases such as atherosclerosis. Secreted cysteine proteases, Gingipains Rgp and Kgp, are essential for P. gingivalis virulence. Some polyphenols and flavonoids are known to inhibit Gingipain activity and interfere with biofilm formation by P. gingivalis. Many bioactive compounds have been isolated from Epimedium species, but availability of these compounds on Gingipains and P. gingivalis is still unclear. Therefore, the aim of this study was to evaluate natural products from medical plants to develop a new therapeutic agent against periodontal disease. Material and Methods Prenylated flavonoids were isolated from Epimedium species plant using column chromatographies. The inhibitory effect of the prenylated flavonoids against protease activity of Gingipains were examined using purified Gingipains and fluorogenic substrates. Anti-P. gingivalis activity was evaluated to analyze planktonic growth and biofilm formation in brain heart infusion medium in the presence of the prenylated flavonoids. Results We isolated 17 prenylated flavonoids (Limonianin, Epimedokoreanin B, etc.) from Epimedium species. We found that some prenylated flavonoids inhibited Gingipain activity in a non-competitive manner with Ki values at μm order. The prenylated flavonoids also hindered growth and biofilm formation of P. gingivalis, in a manner independent of Gingipain inhibition by the compounds. Conclusion The results indicated an inhibitory effect of the prenylated flavonoids against P. gingivalis and would provide useful information for future development of periodontitis treatment that suppresses Gingipains, P. gingivalis growth and biofilm formation.

  • Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner
    Frontiers Media S.A., 2017
    Co-Authors: Jan Potempa, Barbara Potempa, Joanna Koziel, Izabela Glowczyk, Alicia Wong, Olena Babyak, Maciej Lech, Richard J. Lamont
    Abstract:

    Gingipain cysteine proteases are considered key virulence factors of Porphyromonas gingivalis. They significantly influence antibacterial and homeostatic functions of macrophages, neutrophils, the complement system, and cytokine networks. Recent data indicate the role of P. gingivalis in T cell differentiation; however, the involvement of Gingipains in this process remains elusive. Therefore, the aim of this study was to investigate the contribution of danger signals triggered by the Gingipains on the generation of Th17 cells, which play a key role in protection against bacterial diseases but may cause chronic inflammation and bone resorption. To this end we compared the effects of the wild-type strain of P. gingivalis (W83) with its isogenic mutant devoid of Gingipain activity (ΔKΔRAB), and bacterial cells pretreated with a highly-specific inhibitor of Gingipains activity (KYTs). Antigen presenting cells (APCs), both professional (dendritic cells), and non-professional (gingival keratinocytes), exposed to viable bacteria expressed high amounts of cytokines (IL-6, IL-21, IL-23). These cytokines are reported to either stimulate or balance the Th17-dependent immune response. Surprisingly, cells infected with P. gingivalis devoid of Gingipain activity showed increased levels of all tested cytokines compared to bacteria with fully active enzymes. The effect was dependent on both the reduction of cytokine proteolysis and the lack of cross-talk with other bacterial virulence factors, including LPS and fimbriae that induce de novo synthesis of cytokines. The profile of lymphocyte T differentiation from naive T cells showed enhanced generation of Th17 in response to bacteria with inactive Gingipains. Moreover, we found that Gingipain-dependent induction of Th17 cells was highly specific, since other T cell-subsets remained unchanged. Finally, inhibition of IL-6 signaling in dendritic cells led to a significant depletion of the Th17 population. Cumulatively, this study revealed a previously undisclosed role of Gingipain activity in the process of Th17 differentiation reliant on blocking signaling through IL-6. Since inactivation of Gingipains accelerates the skewing of T cells toward Th17 cells, which are detrimental in periodontitis, IL-6 signaling may serve as an attractive target for treatment of the disease

  • Gingipains critical factors in the development of aspiration pneumonia caused by porphyromonas gingivalis
    Journal of Innate Immunity, 2016
    Co-Authors: Malgorzata Benedyk, Piotr Mydel, Nicolas Delaleu, Karolina Plaza, Katarzyna Gawron, Aleksandra Milewska, Katarzyna Maresz, Joanna Koziel, Krzysztof Pyrc, Jan Potempa
    Abstract:

    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.

  • purification and characterisation of recombinant his tagged rgpb Gingipain from porphymonas gingivalis
    Biological Chemistry, 2015
    Co-Authors: Florian Veillard, Barbara Potempa, Yonghua Guo, Kyanh Nguyen, Maryta Sztukowska, Miroslaw Ksiazek, John A Houston, Lahari Koneru, Jan Potempa
    Abstract:

    Gingipain proteases are important virulence factors from the periodontal pathogen Porphyromonas gingivalis and are the target of many in vitro studies. Due to their close biochemical properties, purification of individual Gingipains is difficult and requires multiple chromatographic steps. In this study, we demonstrate that insertion of a hexahistidine affinity tag upstream of a C-terminal outer membrane translocation signal in RgpB Gingipain leads to the secretion of a soluble, mature form of RgpB bearing the affinity tag which can easily be purified by nickel-chelating affinity chromatography. The final product obtained in high yielding and high purity is biochemically indistinguishable from the native RgpB enzyme.

James Travis - One of the best experts on this subject based on the ideXlab platform.

  • Gingipain enzymes from porphyromonas gingivalis preferentially bind immobilized extracellular proteins a mechanism favouring colonization
    Journal of Periodontal Research, 2009
    Co-Authors: Adrian Dale Mcalister, Jan Potempa, James Travis, Aneta Sroka, Rebecca E Fitzpatrick, Noelene Sheila Quinsey, Robert N Pike
    Abstract:

    Background and Objective: Porphyromonas gingivalis, an anaerobic bacterium associated with adult periodontal disease, employs a number of pathogenic mechanisms, including protease/adhesin complexes (Gingipains), fimbriae and hemagglutinins, to maintain attachment within colonized hosts. Here we examined the binding of Gingipains and whole, live P. gingivalis cells to immobilized extracellular matrix proteins in the presence of soluble forms of the same proteins, to investigate whether this may constitute a colonization mechanism in the oral environment. Material and Methods:  Binding of purified Gingipain molecules and whole bacterial cells to immobilized matrix proteins was examined in the presence and absence of soluble competitors using enzyme-linked immunosorbent assays. Results:  Purified Gingipains or whole, live bacteria preferentially bound immobilized forms of matrix proteins, even in the presence of soluble forms of the same proteins. Fimbriae appeared to be redundant for adhesion to immobilized proteins in the presence of the Gingipains, indicating that the protease/adhesins and hemagglutinins may be more important for adhesion under these conditions. Conclusion:  The data presented here provide evidence for a model of adhesion for P. gingivalis within the fluid environment of the oral cavity, where preferential binding of matrix-located proteins over soluble forms facilitates colonization of the host.

  • a peptide domain on Gingipain r which confers immunity against porphyromonas gingivalis infection in mice
    Infection and Immunity, 1998
    Co-Authors: Caroline A Genco, Jan Potempa, B M Odusanya, Jowita Mikolajczykpawlinska, James Travis
    Abstract:

    The cysteine proteinases referred to as Gingipains R (Gingipain R1 and Gingipain R2) and Gingipain K produced by Porphyromonas gingivalis are virulence factors of this periodontal pathogen which likely act by interrupting host defense mechanisms and by participating in the penetration and destruction of host connective tissue. To examine the effect of immunization with Gingipains R on the ability of P. gingivalis to colonize and invade in the mouse chamber model, BALB/c mice were immunized intraperitoneally with the 95-kDa Gingipain R1, the 50-kDa Gingipain R2, or multiple antigenic peptide (MAP)-conjugated Gingipain R-derived peptides and then challenged with P. gingivalis. Immunization of mice with the 95-kDa Gingipain R1, the 50-kDa Gingipain R2, or a peptide derived from the N-terminal sequence of the catalytic domain of Gingipains R (peptide A) followed by challenge with P. gingivalis A7436 resulted in protection from P. gingivalis invasion. In contrast, immunization with peptides corresponding to either a sequence encompassing the catalytic cysteine residue of Gingipains R (peptide B) or an identical sequence within the catalytic domains of Gingipain R1 and Gingipain K (peptide C), followed by challenge with P. gingivalis, did not protect animals, nor did immunization with a peptide corresponding to sequences within the adhesion/hemagglutinin domain of Gingipain R1 (peptide D) which have been shown to be directly involved in the hemagglutinin activity of Gingipain R1. However, the immunoglobulin G (IgG) titer obtained following immunization with peptide D was comparable to that obtained following immunization with the N-terminal peptide (peptide A). Competitive enzyme-linked immunosorbent assays, using either the 95-kDa Gingipain R1 or Gingipain K as the competing soluble antigen, indicated that 42 and 53% of the antibodies induced by immunization with heat-killed bacteria recognize Gingipain R1 and Gingipain K, respectively; however, even at very high concentrations, the 50-kDa Gingipain R2 did not hinder IgG binding to P. gingivalis. These results indicate that antibodies directed to the amino-terminal region of the catalytic domain of Gingipains R are capable of inducing a protective immune response against P. gingivalis infection in the mouse chamber model.

  • inactivation of tumor necrosis factor α by proteinases Gingipains from the periodontal pathogen porphyromonas gingivalis implications of immune evasion
    Journal of Biological Chemistry, 1998
    Co-Authors: Catharine C Calkins, Jan Potempa, Kimberley Platt, James Travis
    Abstract:

    Porphyromonas gingivalis is one of the major pathogens associated with adult periodontitis, a major chronic inflammatory disease. Potent proteinases elaborated by these bacteria aid directly and indirectly in both the development of the pathophysiology of the disease and in host defense evasion. For these reasons they are considered key virulence factors. To investigate whether possible immune evasion mechanisms involve the dysregulation of the host cytokine network, we examined the ability of P. gingivalis cysteine proteinases, including Arg-specific Gingipains HRGP and RGP2 and Lys-specific KGP, to degrade the proinflammatory cytokine tumor necrosis factor-α (TNF-α). All three Gingipains rapidly degraded TNF-α as exhibited by immunoblot analysis. Moreover, all biological activity was significantly reduced over extended incubation periods with the proteinases tested, whereas the host neutrophil proteinases were ineffective. These results indicate that the Gingipain proteinases elaborated by P. gingivalis are capable of disrupting the cytokine network at the site of infection through the degradation of the proinflammatory cytokine TNF-α, suggesting the removal of one of several mediators important to the function of polymorphonuclear leukocytes. Such a mechanism is likely to be utilized by other infective organisms not only for survival but also for growth and proliferation.

  • activation of blood coagulation factor x by arginine specific cysteine proteinases Gingipain rs from porphyromonas gingivalis
    Journal of Biological Chemistry, 1997
    Co-Authors: Takahisa Imamura, Jan Potempa, Sumio Tanase, James Travis
    Abstract:

    The effect of two arginine-specific cysteine proteinases (Gingipain Rs) from Porphyromonas gingivalis, a causative bacterium of adult periodontitis, on human blood coagulation was investigated. Activated partial thromboplastin time and prothrombin time were shortened by these proteinases, with a 95-kDa Gingipain R containing adhesin domains being 5-fold more efficient in comparison to a 50-kDa Gingipain R containing the catalytic domain alone. The 50-kDa enzyme reduced each coagulation time in several plasmas deficient in various coagulation factors, while it was ineffective in factor X-deficient plasma unless reconstituted with this protein. Each proteinase activated factor X in a dose- and time-dependent manner, with Michaelis constants (K m) being found to be lower than the normal plasma factor X concentration, strongly suggesting that factor X activation by Gingipain Rs, especially the 95-kDa form which is strongly activated by phospholipids, could occur in plasma. This is the first report of factor X activation by bacterial proteinases and indicates that the Gingipain Rs could be responsible for the production of thrombin and, indirectly, with the generation of prostaglandins, interleukin-1, etc., which have been found to be associated with the development of periodontitis induced by P. gingivalis infections. Furthermore, the data support the hypothesis that induction of blood coagulation by bacterial proteinases may be a causative agent in the pathogenesis of disseminated intravascular coagulation in sepsis.

  • molecular cloning and characterization of porphyromonas gingivalis lysine specific Gingipain a new member of an emerging family of pathogenic bacterial cysteine proteinases
    Journal of Biological Chemistry, 1997
    Co-Authors: Nadine Pavloff, Robert N Pike, Jan Potempa, James Travis, Philip A Pemberton, Wan Ching Audrey Chen, Vaclav Prochazka, Michael C Kiefer, Philip J Barr
    Abstract:

    The proteinases of Porphyromonas gingivalis are key virulence factors in the etiology and progression of periodontal disease. Previous work in our laboratories resulted in the purification of arginine- and lysine-specific cysteine proteinases, designated Gingipains, that consist of several tightly associated protein subunits. Recent characterization of arginine-specific Gingipain-1 (Gingipain R1; RGP-1) revealed that the sequence is unique and that the protein subunits are initially translated as a polyprotein encoding a proteinase domain and multiple adhesin domains (Pavloff, N., Potempa, J., Pike, R. N., Prochazka, V., Kiefer, M. C., Travis, J., and Barr, P. J. (1995) J. Biol. Chem. 270, 1007-1010). We now show that the lysine-specific Gingipain (Gingipain K; KGP) is also biosynthesized as a polyprotein precursor that contains a proteinase domain that is 22% homologous to the proteinase domain of RGP-1 and multiple adhesin domains. This precursor is similarly processed at distinct sites to yield active KGP. The key catalytic residues in the proteinase domain of KGP are identical to those found in RGP-1, but there are significant differences elsewhere within this domain that likely contribute to the altered substrate specificity of KGP. Independent expression of the proteinase domain in insect cells has shown that KGP does not require the presence of the adhesin domains for correct folding to confer proteolytic activity.

Koji Nakayama - One of the best experts on this subject based on the ideXlab platform.

  • porm a core component of bacterial type ix secretion system forms a dimer with a unique kinked rod shape
    Biochemical and Biophysical Research Communications, 2020
    Co-Authors: Keiko Sato, Koji Nakayama, Kodai Okada, Katsumi Imada
    Abstract:

    Abstract Porphyromonas gingivalis, which is a major pathogen of the periodontal disease, secrets virulence factors such as Gingipain proteases via the type IX secretion system (T9SS). T9SS consists of a trans-periplasmic core complex, the outer membrane translocon complex and the cell-surface complex attached on the outer membrane. PorM is a major component of the trans-periplasmic core complex and is believed to connect the outer membrane component with the inner membrane component. Recent structural studies have revealed that the periplasmic region of GldM, a PorM homolog of a gliding bacterium, consist of four domains and forms a dimer with a straight rod shape. However, only fragment structures are known for PorM. Moreover, one of the PorM fragment structure shows a kink. Here we show the structure of the entire structure of the periplasmic region of PorM (PorMp) at 3.7 A resolution. PorMp is made up of four domains and forms a unique dimeric structure with an asymmetric, kinked-rod shape. The structure and the following mutational analysis revealed that R204 stabilizes the kink between the D1 and D2 domains and is essential for Gingipains secretion, suggesting that the kinked structure of PorM is important for the functional T9SS formation.

  • attenuation of the phosphatidylinositol 3 kinase akt signaling pathway by porphyromonas gingivalis Gingipains rgpa rgpb and kgp
    Journal of Biological Chemistry, 2015
    Co-Authors: Masaaki Nakayama, Koji Nakayama, Mariko Naito, Tetsuyoshi Inoue, Naoya Ohara
    Abstract:

    Porphyromonas gingivalis is a major pathogen of periodontal diseases, including periodontitis. We have investigated the effect of P. gingivalis infection on the PI3K/Akt (protein kinase B) signaling pathway in gingival epithelial cells. Here, we found that live P. gingivalis, but not heat-killed P. gingivalis, reduced Akt phosphorylation at both Thr-308 and Ser-473, which implies a decrease in Akt activity. Actually, PI3K, which is upstream of Akt, was also inactivated by P. gingivalis. Furthermore, glycogen synthase kinase 3α/β, mammalian target of rapamycin, and Bad, which are downstream proteins in the PI3K/Akt cascade, were also dephosphorylated, a phenomenon consistent with Akt inactivation by P. gingivalis. However, these events did not require direct interaction between bacteria and host cells and were independent of P. gingivalis invasion into the cells. The use of Gingipain-specific inhibitors and a Gingipain-deficient P. gingivalis mutant KDP136 revealed that the Gingipains and their protease activities were essential for the inactivation of PI3K and Akt. The associations between the PI3K regulatory subunit p85α and membrane proteins were disrupted by wild-type P. gingivalis. Moreover, PDK1 translocation to the plasma membrane was reduced by wild-type P. gingivalis, but not KDP136, indicating little production of phosphatidylinositol 3,4,5-triphosphate by PI3K. Therefore, it is likely that PI3K failed to transmit homeostatic extracellular stimuli to intracellular signaling pathways by Gingipains. Taken together, our findings indicate that P. gingivalis attenuates the PI3K/Akt signaling pathway via the proteolytic effects of Gingipains, resulting in the dysregulation of PI3K/Akt-dependent cellular functions and the destruction of epithelial barriers.

  • porphyromonas gingivalis and related bacteria from colonial pigmentation to the type ix secretion system and gliding motility
    Journal of Periodontal Research, 2015
    Co-Authors: Koji Nakayama
    Abstract:

    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.

  • A protein secretion system linked to bacteroidete gliding motility and pathogenesis
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Keiko Sato, Mariko Naito, Hideharu Yukitake, Hideki Hirakawa, Mikio Shoji, Ryan G. Rhodes, Koji Nakayama
    Abstract:

    Porphyromonas gingivalis secretes strong proteases called Gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to Gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in Gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.

  • identification of a Gingipain sensitive surface ligand of porphyromonas gingivalis that induces toll like receptor 2 and 4 independent nf kappab activation in cho cells
    Infection and Immunity, 2009
    Co-Authors: Koki Haruyama, Mariko Naito, Mikio Shoji, Atsutoshi Yoshimura, Mami Kishimoto, Yoshimitsu Abiko, Yoshitaka Hara, Koji Nakayama
    Abstract:

    Porphyromonas gingivalis is a major periodontal pathogen that has the pathogenic proteinases Arg-specific Gingipain and Lys-specific Gingipain. We previously found that a cell surface component on P. gingivalis is able to induce Toll-like receptor 2 (TLR2)- and TLR4-independent signaling in 7.19 cells and that this component can be degraded by Gingipains. In this study, we purified this component from the P. gingivalis Gingipain-null mutant KDP136 and obtained two candidate proteins. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis showed that the proteins, with molecular masses of 123 and 43 kDa, were encoded by PGN_0748 and PGN_0728 (pgm6), respectively, in the P. gingivalis ATCC 33277 genome sequence. The PGN_0748-encoded protein, which we refer to as Gingipain-sensitive ligand A (GslA), reacted with antiserum that could effectively inhibit the activity of KDP136 to induce NF-kappaB activation in 7.19 cells, but Pgm6 did not. To further determine what protein is responsible for the NF-kappaB activation, we constructed gslA, pgm6, and pgm6 pgm7 deletion mutants from KDP136. When 7.19 cells were exposed to those mutants, the gslA deletion mutant did not induce NF-kappaB activation, whereas the pgm6 and pgm6 pgm7 deletion mutants did. Furthermore, NF-kappaB activation in 7.19 cells induced by KDP136 was partially inhibited by antiserum against a recombinant protein expressed from the 5'-terminal third of gslA. These results indicate that GslA is one of the factors that induce NF-kappaB activation in 7.19 cells. Interestingly, the gslA gene was present in four of seven P. gingivalis strains tested. This restricted distribution might be associated with the virulence potential of each strain.

Hansel M Fletcher - One of the best experts on this subject based on the ideXlab platform.

  • role of acetyltransferase pg1842 in Gingipain biogenesis in porphyromonas gingivalis
    Journal of Bacteriology, 2018
    Co-Authors: Arunima Mishra, Hansel M Fletcher, Francis Roy, Yuetan Dou, Kangling Zhang, Hui Tang
    Abstract:

    ABSTRACT Porphyromonas gingivalis, the major etiologic agent in adult periodontitis, produces large amounts of proteases that are important for its survival and pathogenesis. The activation/maturation of Gingipains, the major proteases, in P. gingivalis involves a complex network of processes which are not yet fully understood. VimA, a putative acetyltransferase and virulence-modulating protein in P. gingivalis, is known to be involved in Gingipain biogenesis. P. gingivalis FLL92, a vimA-defective isogenic mutant (vimA::ermF-ermAM) showed late-onset Gingipain activity at stationary phase, indicating the likelihood of a complementary functional VimA homolog in that growth phase. This study aimed to identify a functional homolog(s) that may activate the Gingipains in the absence of VimA at stationary phase. A bioinformatics analysis showed five putative GCN5-related N-acetyltransferases (GNAT) encoded in the P. gingivalis genome that are structurally related to VimA. Allelic exchange mutagenesis was used to make deletion mutants for these acetyltransferases in the P. gingivalisvimA-defective mutant FLL102 (ΔvimA::ermF) genetic background. One of the mutants, designated P. gingivalis FLL126 (ΔvimA-ΔPG1842), did not show any late-onset Gingipain activity at stationary phase compared to that of the parent strain P. gingivalis FLL102. A Western blot analysis of stationary-phase extracellular fractions with antiGingipain antibodies showed immunoreactive bands that were similar in size to those for the proGingipain species present only in the ΔvimA-ΔPG1842 isogenic mutant. Both recombinant VimA and PG1842 proteins acetylated Y230, K247, and K248 residues in the pro-RgpB substrate. Collectively, these findings indicate that PG1842 may play a significant role in the activation/maturation of Gingipains in P. gingivalis. IMPORTANCE Gingipain proteases are key virulence factors secreted by Porphyromonas gingivalis that cause periodontal tissue damage and the degradation of the host immune system proteins. Gingipains are translated as an inactive zymogen to restrict intracellular proteolytic activity before secretion. Posttranslational processing converts the inactive proenzyme to a catalytically active protease. Gingipain biogenesis, including its secretion and activation, is a complex process which is still not fully understood. One recent study identified acetylated lysine residues in the three Gingipains RgpA, RgpB, and Kgp, thus indicating a role for acetylation in Gingipain biogenesis. Here, we show that the acetyltransferases VimA and PG1842 can acetylate the pro-RgpB Gingipain species. These findings further indicate that acetylation is a potential mechanism in the Gingipain activation/maturation pathway in P. gingivalis.

  • the roles of rgpb and kgp in late onset Gingipain activity in the vima defective mutant of porphyromonas gingivalis w83
    Molecular Oral Microbiology, 2015
    Co-Authors: Yuetan Dou, Hansel M Fletcher, Francis Roy, A Robles, A W Aruni, Lawrence B Sandberg, E Nothnagel
    Abstract:

    Previous studies have shown that VimA, an acetyltransferase, can modulate Gingipain biogenesis in Porphyromonas gingivalis. Inactivation of the vimA gene resulted in isogenic mutants that showed a late onset of Gingipain activity that only occurred during the stationary growth phase. To further elucidate the role and contribution of the Gingipains in this VimA-dependent process, isogenic mutants defective in the Gingipain genes in the vimA-deficient genetic background were evaluated. In contrast with the wild-type strain, RgpB and Kgp Gingipain activities were absent in exponential phase in the ∆rgpA::tetQ-vimA::ermF mutant. However, these activities increased to 31 and 53%, respectively, of that of the wild-type during stationary phase. In the ∆rgpA::cat-∆kgp::tetQ-vimA::ermF mutant, the RgpB protein was observed in the extracellular fraction but no activity was present even at the stationary growth phase. There was no Gingipain activity observed in the ∆rgpB::cat-∆kgp::tetQ-vimA::ermF mutant whereas Kgp activity in ∆rgpA::cat-∆rgpB::tetQ-vimA::ermF mutant was 24% of the wild-type at late stationary phase. In contrast to RgpA, the glycosylation profile of the RgpB catalytic domain from both W83 and P. gingivalis FLL92 (vimA::ermF) showed similarity. Taken together, the results suggest multiple Gingipain activation pathways in P. gingivalis. Whereas the maturation pathways for RgpA and RgpB are different, the late-onset Gingipain activity in the vimA-defective mutant was due to activation/maturation of RgpB and Kgp. Moreover, unlike RgpA, which is VimA-dependent, the maturation/activation pathways for RgpB and Kgp are interdependent in the absence VimA.

  • involvement of extracytoplasmic function sigma factors in virulence regulation in porphyromonas gingivalis w83
    Fems Microbiology Letters, 2010
    Co-Authors: Devon Osbourne, Rachelle M E Mckenzie, Hansel M Fletcher
    Abstract:

    Extracytoplasmic function (ECF) sigma factors are known to play an important role in the bacterial response to various environmental stresses and can significantly modulate their pathogenic potential. In the genome of Porphyromonas gingivalis W83, six putative ECF sigma factors were identified. To further evaluate their role in this organism, a PCR-based linear transformation method was used to inactivate five ECF sigma factor genes ( PG0162 , PG0214 , PG0985 , PG1660 , and PG1827 ) by allelic exchange mutagenesis. All five isogenic mutants formed black-pigmented colonies on blood agar. Mutants defective in PG0985 , PG1660 , and PG1827 genes were more sensitive to 0.25 mM of hydrogen peroxide compared with the wild-type strain. Isogenic mutants of PG0162 and PG1660 showed a 50% decrease in Gingipain activity. Reverse transcription-PCR analysis showed that there was no alteration in the expression of rgpA , rgpB , and kgp Gingipain genes in these mutants. Hemolytic and hemagglutination activities were decreased by more than 50% in the PG0162 mutant compared with the wild type. Taken together, these findings suggest that ECF sigma factors can modulate important virulence factors in P. gingivalis . ECF sigma factors encoded by the PG0162 and PG1660 genes might also be involved in the post-transcriptional regulation of the Gingipains.

  • regt can modulate Gingipain activity and response to oxidative stress in porphyromonas gingivalis
    Microbiology, 2010
    Co-Authors: E Vanterpool, Francis Roy, Wilson A Aruni, Hansel M Fletcher
    Abstract:

    Recombinant VimA protein can interact with the Gingipains and several other proteins that may play a role in its biogenesis in Porphyromonas gingivalis. In silico analysis of PG2096, a hypothetical protein that was shown to interact with VimA, suggests that it may have environmental stress resistance properties. To further evaluate the role(s) of PG2096, the predicted open reading frame was PCR amplified from P. gingivalis W83 and insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette. One randomly chosen PG2096-defective mutant created by allelic exchange and designated FLL205 was further characterized. Under normal growth conditions at 37 °C, Arg-X and Lys-X Gingipain activities in FLL205 were reduced by approximately 35 % and 21 %, respectively, compared to the wild-type strain. However, during prolonged growth at an elevated temperature of 42 °C, Arg-X activity was increased by more than 40 % in FLL205 in comparison to the wild-type strain. In addition, the PG2096-defective mutant was more resistant to oxidative stress when treated with 0.25 mM hydrogen peroxide. Taken together these results suggest that the PG2096 gene, designated regT (regulator of Gingipain activity at elevated temperatures), may be involved in regulating Gingipain activity at elevated temperatures and be important in oxidative stress resistance in P. gingivalis.

  • Gingipain dependent interactions with the host are important for survival of porphyromonas gingivalis
    Frontiers in Bioscience, 2008
    Co-Authors: Shaun M Sheets, Rachelle M E Mckenzie, Antonette G Roblesprice, Carlos A Casiano, Hansel M Fletcher
    Abstract:

    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.

Kenji Yamamoto - One of the best experts on this subject based on the ideXlab platform.

  • a novel potent dual inhibitor of arg Gingipains and lys Gingipain as a promising agent for periodontal disease therapy
    The FASEB Journal, 2014
    Co-Authors: Shinsuke Kataoka, Tomoko Kadowaki, Atsuyo Baba, Yoshimitsu Suda, Ryosuke Takii, Munetaka Hashimoto, Tomoyo Kawakubo, Tetsuji Asao, Kenji Yamamoto
    Abstract:

    The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed Gingipains that comprises Arg-Gingipain (Rgp) and Lys-Gingipain (Kgp). Growing evidence indicates that these 2 types of Gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both Gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD.

  • a functional virulence complex composed of Gingipains adhesins and lipopolysaccharide shows high affinity to host cells and matrix proteins and escapes recognition by host immune systems
    Infection and Immunity, 2005
    Co-Authors: Ryosuke Takii, Tomoko Kadowaki, Atsuyo Baba, Takayuki Tsukuba, Kenji Yamamoto
    Abstract:

    Arg-Gingipain (Rgp) and Lys-Gingipain (Kgp) are Porphyromonas gingivalis cysteine proteinases implicated as major virulence factors in pathologies of periodontitis. We purified a 660-kDa cell-associated Gingipain complex existing as a homodimer of two catalytically active monomers which comprises their catalytic and adhesin domains. Electron microscopy revealed that the complex was composed of a globular particle with a 10-nm external diameter possessing one or two electron-dense hole-like structures. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide (LPS) with the catalytic domains and a hemagglutinin domain, Hgp44, of Rgp and Kgp in the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endotherial cells with an efficiency which was higher than that of the monomeric Gingipains. The native complex produced only a small amount of nitrogen dioxide, tumor necrosis factor alpha, and interleukin-6 by macrophages, whereas the heat-denatured complex resulted in increased production. Inhibition of the proteolytic activities of the Gingipain complex did not up-regulate the cytokine production, indicating that the functional domains in LPS are structurally masked by the complex proteins. These results indicate the importance of the complex in evasion of host defense mechanisms as well as in host tissue breakdown.

  • construction and characterization of arginine specific cysteine proteinase arg Gingipain deficient mutants of porphyromonas gingivalis evidence for significant contribution of arg Gingipain to virulence
    Journal of Biological Chemistry, 1995
    Co-Authors: Koji Nakayama, Tomoko Kadowaki, Kuniaki Okamoto, Kenji Yamamoto
    Abstract:

    Abstract Arginine-specific cysteine proteinase (Arg-Gingipain; formerly, arGingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arg-Gingipain is important for periodontopathogenicity of the organism, Arg-Gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-Gingipain-encoding genes designated rgpA and rgpB existed on 12.5- and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-Gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpB mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arg-Gingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-Gingipain makes a significant contribution to the virulence of P. gingivalis.