51 results on '"Potempa, Jan"'
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2. Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.
- Author
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Bryzek D, Ciaston I, Dobosz E, Gasiorek A, Makarska A, Sarna M, Eick S, Puklo M, Lech M, Potempa B, Potempa J, and Koziel J
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- Adhesins, Bacterial metabolism, Animals, Bacteroidaceae Infections metabolism, Bacteroidaceae Infections microbiology, Bacteroidaceae Infections pathology, Cells, Cultured, Cysteine Endopeptidases metabolism, Extracellular Traps microbiology, Female, Gingipain Cysteine Endopeptidases, Humans, Mice, Mice, Inbred C57BL, Neutrophils microbiology, Neutrophils pathology, Peritonitis metabolism, Peritonitis microbiology, Receptor, PAR-2 immunology, Signal Transduction, Adhesins, Bacterial immunology, Bacteroidaceae Infections immunology, Cysteine Endopeptidases immunology, Extracellular Traps immunology, Neutrophils immunology, Peritonitis immunology, Porphyromonas gingivalis immunology, Porphyromonas gingivalis pathogenicity, Receptor, PAR-2 metabolism
- Abstract
Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment., Competing Interests: The authors have declared that no competing interests exist.
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- 2019
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3. Adhesive protein-mediated cross-talk between Candida albicans and Porphyromonas gingivalis in dual species biofilm protects the anaerobic bacterium in unfavorable oxic environment.
- Author
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Bartnicka D, Karkowska-Kuleta J, Zawrotniak M, Satała D, Michalik K, Zielinska G, Bochenska O, Kozik A, Ciaston I, Koziel J, Dutton LC, Nobbs AH, Potempa B, Baster Z, Rajfur Z, Potempa J, and Rapala-Kozik M
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- Adhesins, Bacterial genetics, Bacterial Adhesion, Fungal Proteins genetics, Fungal Proteins metabolism, Humans, Microbial Viability, Proteomics methods, Virulence, Adhesins, Bacterial metabolism, Bacteria, Anaerobic physiology, Biofilms, Candida albicans physiology, Microbial Interactions, Porphyromonas gingivalis physiology
- Abstract
The oral cavity contains different types of microbial species that colonize human host via extensive cell-to-cell interactions and biofilm formation. Candida albicans-a yeast-like fungus that inhabits mucosal surfaces-is also a significant colonizer of subgingival sites in patients with chronic periodontitis. It is notable however that one of the main infectious agents that causes periodontal disease is an anaerobic bacterium-Porphyromonas gingivalis. In our study, we evaluated the different strategies of both pathogens in the mutual colonization of an artificial surface and confirmed that a protective environment existed for P. gingivalis within developed fungal biofilm formed under oxic conditions where fungal cells grow mainly in their filamentous form i.e. hyphae. A direct physical contact between fungi and P. gingivalis was initiated via a modulation of gene expression for the major fungal cell surface adhesin Als3 and the aspartic proteases Sap6 and Sap9. Proteomic identification of the fungal surfaceome suggested also an involvement of the Mp65 adhesin and a "moonlighting" protein, enolase, as partners for the interaction with P. gingivalis. Using mutant strains of these bacteria that are defective in the production of the gingipains-the proteolytic enzymes that also harbor hemagglutinin domains-significant roles of these proteins in the formation of bacteria-protecting biofilm were clearly demonstrated.
- Published
- 2019
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4. Host cell-surface proteins as substrates of gingipains, the main proteases of Porphyromonas gingivalis.
- Author
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Hočevar K, Potempa J, and Turk B
- Subjects
- Gingipain Cysteine Endopeptidases, Substrate Specificity, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis enzymology
- Abstract
Gingipains are extracellular cysteine proteases of the oral pathogen Porphyromonas gingivalis and are its most potent virulence factors. They can degrade a great variety of host proteins, thereby helping the bacterium to evade the host immune response, deregulate signaling pathways, trigger anoikis and, finally, cause tissue destruction. Host cell-surface proteins targeted by gingipains are the main focus of this review and span three groups of substrates: immune-regulatory proteins, signaling pathways regulators and adhesion molecules. The analysis of published data revealed that gingipains predominantly inactivate their substrates by cleaving them at one or more sites, or through complete degradation. Sometimes, gingipains were even found to initially shed their membrane substrates, but this was mostly just the first step in the degradation of cell-surface proteins.
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- 2018
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5. Proteolytic effects of gingipains on trefoil factor family peptides.
- Author
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Chaiyarit P, Jaresitthikunchai J, Phaonakrop N, Roytrakul S, Potempa B, and Potempa J
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- Chromatography, Liquid, Gingipain Cysteine Endopeptidases, Humans, Recombinant Proteins pharmacology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Adhesins, Bacterial drug effects, Cysteine Endopeptidases drug effects, Proteolysis, Trefoil Factors pharmacology
- Abstract
Objectives: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis., Materials and Methods: Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides., Results: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin., Conclusions: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides., Clinical Relevance: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.
- Published
- 2018
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6. Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB.
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Wichert R, Ermund A, Schmidt S, Schweinlin M, Ksiazek M, Arnold P, Knittler K, Wilkens F, Potempa B, Rabe B, Stirnberg M, Lucius R, Bartsch JW, Nikolaus S, Falk-Paulsen M, Rosenstiel P, Metzger M, Rose-John S, Potempa J, Hansson GC, Dempsey PJ, and Becker-Pauly C
- Subjects
- Amino Acid Sequence genetics, Animals, Cell Membrane metabolism, Epithelial Cells metabolism, Female, Gingipain Cysteine Endopeptidases, HEK293 Cells, Humans, Male, Metalloendopeptidases genetics, Mice, Transgenic, Mucin-2 genetics, Mucin-2 metabolism, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Metalloendopeptidases metabolism, Metalloproteases metabolism
- Abstract
The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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7. Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner.
- Author
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Glowczyk I, Wong A, Potempa B, Babyak O, Lech M, Lamont RJ, Potempa J, and Koziel J
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- Bacterial Infections drug therapy, Bone Resorption, CD4-Positive T-Lymphocytes drug effects, Cell Differentiation, Cell Proliferation drug effects, Cytokines genetics, Cytokines metabolism, Dendritic Cells immunology, Fimbriae, Bacterial immunology, Gene Expression Regulation, Gingipain Cysteine Endopeptidases, Gingiva immunology, Gingiva microbiology, Humans, Inflammation, Interleukin-23 metabolism, Interleukin-6 genetics, Interleukins metabolism, Periodontitis immunology, Periodontitis microbiology, Phenotype, Porphyromonas gingivalis genetics, Porphyromonas gingivalis pathogenicity, Signal Transduction, Virulence Factors, Adhesins, Bacterial metabolism, Adhesins, Bacterial pharmacology, Cysteine Endopeptidases metabolism, Cysteine Endopeptidases pharmacology, Interleukin-6 metabolism, Porphyromonas gingivalis metabolism, Th17 Cells drug effects, Th17 Cells immunology
- 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.
- Published
- 2017
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8. Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis , a causative agent of gum disease from the human oral microbiome.
- Author
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Pomowski A, Usón I, Nowakowska Z, Veillard F, Sztukowska MN, Guevara T, Goulas T, Mizgalska D, Nowak M, Potempa B, Huntington JA, Potempa J, and Gomis-Rüth FX
- Subjects
- Adhesins, Bacterial genetics, Adhesins, Bacterial metabolism, Bacteroidaceae Infections enzymology, Bacteroidaceae Infections genetics, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Enzyme Precursors genetics, Enzyme Precursors metabolism, Gingipain Cysteine Endopeptidases, Gingivitis enzymology, Gingivitis genetics, Humans, Microbiota, Mouth microbiology, Porphyromonas gingivalis genetics, Protein Domains, Protein Multimerization, Structure-Activity Relationship, Virulence Factors metabolism, Adhesins, Bacterial chemistry, Cysteine Endopeptidases chemistry, Enzyme Precursors chemistry, Porphyromonas gingivalis enzymology, Porphyromonas gingivalis pathogenicity, Virulence Factors chemistry
- Abstract
Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis , the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-β protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys
129 ) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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9. Zebrafish as a new model to study effects of periodontal pathogens on cardiovascular diseases.
- Author
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Widziolek M, Prajsnar TK, Tazzyman S, Stafford GP, Potempa J, and Murdoch C
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- Adhesins, Bacterial genetics, Animals, Cardiovascular Diseases pathology, Cysteine Endopeptidases genetics, Gingipain Cysteine Endopeptidases, Larva microbiology, Porphyromonas gingivalis genetics, Zebrafish embryology, Adhesins, Bacterial metabolism, Cardiovascular Diseases microbiology, Cysteine Endopeptidases metabolism, Endothelium, Vascular microbiology, Periodontitis microbiology, Porphyromonas gingivalis pathogenicity
- Abstract
Porphyromonas gingivalis (Pg) is a keystone pathogen in the aetiology of chronic periodontitis. However, recent evidence suggests that the bacterium is also able to enter the bloodstream, interact with host cells and tissues, and ultimately contribute to the pathogenesis of cardiovascular disease (CVD). Here we established a novel zebrafish larvae systemic infection model showing that Pg rapidly adheres to and penetrates the zebrafish vascular endothelium causing a dose- and time-dependent mortality with associated development of pericardial oedemas and cardiac damage. The in vivo model was then used to probe the role of Pg expressed gingipain proteases using systemically delivered gingipain-deficient Pg mutants, which displayed significantly reduced zebrafish morbidity and mortality compared to wild-type bacteria. In addition, we used the zebrafish model to show efficacy of a gingipain inhibitor (KYT) on Pg-mediated systemic disease, suggesting its potential use therapeutically. Our data reveal the first real-time in vivo evidence of intracellular Pg within the endothelium of an infection model and establishes that gingipains are crucially linked to systemic disease and potentially contribute to CVD.
- Published
- 2016
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10. Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins.
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Plaza K, Kalinska M, Bochenska O, Meyer-Hoffert U, Wu Z, Fischer J, Falkowski K, Sasiadek L, Bielecka E, Potempa B, Kozik A, Potempa J, and Kantyka T
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- Adhesins, Bacterial chemistry, Cysteine Endopeptidases chemistry, Gingipain Cysteine Endopeptidases, Humans, Kallikreins antagonists & inhibitors, Kallikreins chemistry, Kallikreins metabolism, Protein Stability, Proteinase Inhibitory Proteins, Secretory chemistry, Saliva chemistry, Salivary Proteins and Peptides antagonists & inhibitors, Salivary Proteins and Peptides chemistry, Serine Peptidase Inhibitors, Kazal Type, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis enzymology, Proteinase Inhibitory Proteins, Secretory metabolism, Saliva metabolism, Salivary Proteins and Peptides metabolism
- Abstract
Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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11. Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis.
- Author
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Benedyk M, Mydel PM, Delaleu N, Płaza K, Gawron K, Milewska A, Maresz K, Koziel J, Pyrc K, and Potempa J
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- Adhesins, Bacterial genetics, Animals, Bacteroidaceae Infections genetics, Bacteroidaceae Infections microbiology, Bacteroidaceae Infections pathology, Blood Platelets immunology, Cysteine Endopeptidases genetics, Cytokines immunology, Female, Gingipain Cysteine Endopeptidases, Hemorrhage genetics, Hemorrhage immunology, Hemorrhage microbiology, Hemorrhage pathology, Mice, Mice, Inbred BALB C, Necrosis, Neutrophil Infiltration immunology, Neutrophils immunology, Neutrophils pathology, Platelet Activation immunology, Platelet Count, Pneumonia, Aspiration genetics, Pneumonia, Aspiration pathology, Porphyromonas gingivalis genetics, Adhesins, Bacterial immunology, Bacteroidaceae Infections immunology, Cysteine Endopeptidases immunology, Pneumonia, Aspiration immunology, Porphyromonas gingivalis immunology
- 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., (© 2015 S. Karger AG, Basel.)
- Published
- 2016
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12. Noncanonical activation of β-catenin by Porphyromonas gingivalis.
- Author
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Zhou Y, Sztukowska M, Wang Q, Inaba H, Potempa J, Scott DA, Wang H, and Lamont RJ
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- Adhesins, Bacterial genetics, Bacteroidaceae Infections enzymology, Bacteroidaceae Infections genetics, Bacteroidaceae Infections microbiology, Cell Line, Cell Nucleus genetics, Cell Nucleus metabolism, Cysteine Endopeptidases genetics, Epithelial Cells enzymology, Epithelial Cells metabolism, Epithelial Cells microbiology, Gingipain Cysteine Endopeptidases, Gingiva enzymology, Gingiva metabolism, Gingiva microbiology, Glycogen Synthase Kinase 3 genetics, Glycogen Synthase Kinase 3 metabolism, Glycogen Synthase Kinase 3 beta, Humans, Porphyromonas gingivalis genetics, Protein Processing, Post-Translational, Protein Transport, beta Catenin metabolism, Adhesins, Bacterial metabolism, Bacteroidaceae Infections metabolism, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis enzymology
- Abstract
Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on β-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of β-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for β-catenin processing. The β-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3β were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that β-catenin fragments were translocated to the nucleus. The accumulation of β-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the β-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis-induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of β-catenin and disassociation of the β-catenin destruction complex by gingipain-dependent proteolytic processing. β-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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13. Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis.
- Author
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Veillard F, Potempa B, Guo Y, Ksiazek M, Sztukowska MN, Houston JA, Koneru L, Nguyen KA, and Potempa J
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- Adhesins, Bacterial chemistry, Bacteroidaceae Infections microbiology, Chromatography, Affinity, Cysteine Endopeptidases chemistry, Gingipain Cysteine Endopeptidases, Humans, Porphyromonas gingivalis chemistry, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Adhesins, Bacterial isolation & purification, Adhesins, Bacterial metabolism, Cysteine Endopeptidases isolation & purification, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis metabolism
- 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 that can easily be purified by nickel-chelating affinity chromatography. The final product obtained high yielding high purity is biochemically indistinguishable from the native RgpB enzyme.
- Published
- 2015
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14. Cleavage of host cytokeratin-6 by lysine-specific gingipain induces gingival inflammation in periodontitis patients.
- Author
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Tancharoen S, Matsuyama T, Kawahara K, Tanaka K, Lee LJ, Machigashira M, Noguchi K, Ito T, Imamura T, Potempa J, Kikuchi K, and Maruyama I
- Subjects
- Animals, Cells, Cultured, Cytokines metabolism, Epithelial Cells drug effects, Epithelial Cells metabolism, Epithelial Cells pathology, Gingipain Cysteine Endopeptidases, Gingiva drug effects, Gingiva pathology, Gingival Crevicular Fluid drug effects, Humans, Inflammation metabolism, Inflammation pathology, Periodontitis pathology, Porphyromonas gingivalis, Rats, Signal Transduction drug effects, Adhesins, Bacterial pharmacology, Cysteine Endopeptidases pharmacology, Gingiva metabolism, Gingival Crevicular Fluid metabolism, Keratin-6 metabolism, Periodontitis metabolism
- Abstract
Background/purpose: Lysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion., Methods: K6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay., Results: We identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359-378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt., Conclusion: Kgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.
- Published
- 2015
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15. Structure and mechanism of cysteine peptidase gingipain K (Kgp), a major virulence factor of Porphyromonas gingivalis in periodontitis.
- Author
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de Diego I, Veillard F, Sztukowska MN, Guevara T, Potempa B, Pomowski A, Huntington JA, Potempa J, and Gomis-Rüth FX
- Subjects
- Amino Acid Sequence, Catalysis, Catalytic Domain, Crystallography, X-Ray, Gingipain Cysteine Endopeptidases, Humans, Immunoglobulins chemistry, Lysine chemistry, Models, Molecular, Molecular Sequence Data, Porphyromonas gingivalis pathogenicity, Sequence Homology, Amino Acid, Solvents chemistry, Virulence Factors, Adhesins, Bacterial chemistry, Cysteine Endopeptidases chemistry, Periodontitis enzymology, Periodontitis microbiology, Porphyromonas gingivalis enzymology
- Abstract
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, Cys(477)-His(444)-Asp(388), 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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2014
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16. Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1β but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.
- Author
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Akiyama T, Miyamoto Y, Yoshimura K, Yamada A, Takami M, Suzawa T, Hoshino M, Imamura T, Akiyama C, Yasuhara R, Mishima K, Maruyama T, Kohda C, Tanaka K, Potempa J, Yasuda H, Baba K, and Kamijo R
- Subjects
- Amino Acid Sequence, Animals, Animals, Outbred Strains, Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Cell Differentiation physiology, Cells, Cultured, Gingipain Cysteine Endopeptidases, Humans, Interleukin-17 metabolism, Interleukin-1beta metabolism, Mice, Molecular Sequence Data, Osteoblasts cytology, Osteoblasts metabolism, Osteoclasts metabolism, RANK Ligand metabolism, Receptor Activator of Nuclear Factor-kappa B metabolism, Tumor Necrosis Factor-alpha metabolism, Adhesins, Bacterial metabolism, Bacteroidaceae Infections metabolism, Cysteine Endopeptidases metabolism, Osteoclasts cytology, Osteoprotegerin metabolism, Periodontitis metabolism, Porphyromonas gingivalis enzymology
- Abstract
Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
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- 2014
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17. Sequence-independent processing site of the C-terminal domain (CTD) influences maturation of the RgpB protease from Porphyromonas gingivalis.
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Zhou XY, Gao JL, Hunter N, Potempa J, and Nguyen KA
- Subjects
- Adhesins, Bacterial genetics, Amino Acid Sequence, Cysteine Endopeptidases genetics, DNA Mutational Analysis, Gingipain Cysteine Endopeptidases, Glycosylation, Molecular Sequence Data, Porphyromonas gingivalis genetics, Porphyromonas gingivalis metabolism, Protein Structure, Tertiary, Protein Transport, Sequence Alignment, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis enzymology, Protein Processing, Post-Translational
- Abstract
The Gram-negative periodontal pathogen Porphyromonas gingivalis produces a family of outer membrane-anchored proteases, the gingipains, shown to play an essential role in virulence of the organism. The C-terminal domain (CTD) of gingipains and other secreted proteins is known to be the targeting signal for maturation and translocation of the protein through the outer membrane. The CTD is subsequently cleaved during the secretion process. Multiple alignment of various CTDs failed to define a consensus sequence at the putative CTD processing site. Using mutagenesis, we were able to show that cleavage at the site is not dependent on a specific residue and that recognition of the site is independent of local sequence. Interestingly, length of the junction between the CTD and adjacent Ig-like subdomain has a critical influence on post-translational glycan modification of the protein, whereby insertion of additional residues immediately N-terminal to the cleavage site results in failure of glycan modification and release of soluble protease into the culture medium. Various hypotheses are presented to explain these phenomena. Knowledge of the role CTDs play in maturation of gingipains has broader application for understanding maturation of CTD homologues expressed by bacteria of the Bacteriodetes phylum., (© 2013 John Wiley & Sons Ltd.)
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- 2013
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18. Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases.
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Veillard F, Sztukowska M, Mizgalska D, Ksiazek M, Houston J, Potempa B, Enghild JJ, Thogersen IB, Gomis-Rüth FX, Nguyen KA, and Potempa J
- Subjects
- Adhesins, Bacterial drug effects, Adhesins, Bacterial metabolism, Cysteine Endopeptidases drug effects, Cysteine Endopeptidases metabolism, Enzyme Activation, Gingipain Cysteine Endopeptidases, Glycosylation, Protein Structure, Tertiary, Recombinant Proteins pharmacology, Adhesins, Bacterial chemistry, Cysteine Endopeptidases chemistry, Cysteine Proteinase Inhibitors pharmacology, Peptide Fragments pharmacology, Porphyromonas gingivalis pathogenicity
- Abstract
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 6.2nM to 0.85nM). 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., (Copyright © 2013 Elsevier B.V. All rights reserved.)
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- 2013
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19. Site-specific O-glycosylation on the MUC2 mucin protein inhibits cleavage by the Porphyromonas gingivalis secreted cysteine protease (RgpB).
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van der Post S, Subramani DB, Bäckström M, Johansson MEV, Vester-Christensen MB, Mandel U, Bennett EP, Clausen H, Dahlén G, Sroka A, Potempa J, and Hansson GC
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- Amino Acid Sequence, Animals, CHO Cells, Chromatography, Colitis microbiology, Colon metabolism, Cricetinae, Epithelium metabolism, Gingipain Cysteine Endopeptidases, Glycosylation, Humans, Mass Spectrometry, Molecular Sequence Data, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Mucin-2 metabolism, Porphyromonas gingivalis enzymology
- Abstract
The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.
- Published
- 2013
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20. Porphyromonas gingivalis virulence factor gingipain RgpB shows a unique zymogenic mechanism for cysteine peptidases.
- Author
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de Diego I, Veillard FT, Guevara T, Potempa B, Sztukowska M, Potempa J, and Gomis-Rüth FX
- Subjects
- Arginine metabolism, Catalytic Domain, Crystallography, X-Ray methods, Enzyme Precursors metabolism, Escherichia coli metabolism, Gingipain Cysteine Endopeptidases, Models, Molecular, Molecular Conformation, Protein Folding, Protein Interaction Domains and Motifs, Adhesins, Bacterial metabolism, Cysteine metabolism, Cysteine Endopeptidases metabolism, Gene Expression Regulation, Bacterial, Porphyromonas gingivalis metabolism, Virulence Factors metabolism
- Abstract
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 (Arg(126)) into the S1 pocket, thus matching the substrate specificity of the enzyme. Downstream of Arg(126), the polypeptide leaves the cleft, thereby preventing cleavage. Moreover, the carbonyl group of Arg(126) establishes a very strong hydrogen bond with the co-catalytic histidine, His(440), pulling it away from the catalytic cysteine, Cys(473), and toward Glu(381), which probably plays a role in orienting the side chain of His(440) 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.
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- 2013
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21. Cleavage of extracellular matrix in periodontitis: gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C.
- Author
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Ruggiero S, Cosgarea R, Potempa J, Potempa B, Eick S, and Chiquet M
- Subjects
- Alternative Splicing genetics, Animals, Apoptosis, Arginine metabolism, Cell Adhesion, Cells, Cultured, Extracellular Matrix metabolism, Extracellular Matrix pathology, Fibroblasts, Gingipain Cysteine Endopeptidases, Humans, Lysine metabolism, Mice, Porphyromonas gingivalis enzymology, Porphyromonas gingivalis pathogenicity, Protein Binding, Adhesins, Bacterial genetics, Adhesins, Bacterial metabolism, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Fibronectins genetics, Fibronectins metabolism, Periodontitis genetics, Periodontitis metabolism, Tenascin genetics, Tenascin metabolism
- Abstract
Gingipains are cysteine proteases that represent major virulence factors of the periodontopathogenic bacterium Porphyromonas gingivalis. Gingipains are reported to degrade extracellular matrix (ECM) of periodontal tissues, leading to tissue destruction and apoptosis. The exact mechanism is not known, however. Fibronectin and tenascin-C are pericellular ECM glycoproteins present in periodontal tissues. Whereas fibronectin mediates fibroblast adhesion, tenascin-C binds to fibronectin and inhibits its cell-spreading activity. Using purified proteins in vitro, we asked whether fibronectin and tenascin-C are cleaved by gingipains at clinically relevant concentrations, and how fragmentation by the bacterial proteases affects their biological activity in cell adhesion. Fibronectin was cleaved into distinct fragments by all three gingipains; however, only arginine-specific HRgpA and RgpB but not lysine-specific Kgp destroyed its cell-spreading activity. This result was confirmed with recombinant cell-binding domain of fibronectin. Of the two major tenascin-C splice variants, the large but not the small was a substrate for gingipains, indicating that cleavage occurred primarily in the alternatively spliced domain. Surprisingly, cleavage of large tenascin-C variant by all three gingipains generated fragments with increased anti-adhesive activity towards intact fibronectin. Fibronectin and tenascin-C fragments were detected in gingival crevicular fluid of a subset of periodontitis patients. We conclude that cleavage by gingipains directly affects the biological activity of both fibronectin and tenascin-C in a manner that might lead to increased cell detachment and loss during periodontal disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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22. Gingipain aminopeptidase activities in Porphyromonas gingivalis.
- Author
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Veillard F, Potempa B, Poreba M, Drag M, and Potempa J
- Subjects
- Adhesins, Bacterial genetics, Bacteroidaceae Infections drug therapy, Bacteroidaceae Infections microbiology, Cysteine Endopeptidases genetics, Gene Deletion, Gingipain Cysteine Endopeptidases, Humans, Leucine pharmacology, Porphyromonas gingivalis genetics, Substrate Specificity, Adhesins, Bacterial metabolism, Aminopeptidases antagonists & inhibitors, Cysteine Endopeptidases metabolism, Leucine analogs & derivatives, Porphyromonas gingivalis drug effects, Porphyromonas gingivalis enzymology
- Abstract
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. The 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 the degradation of proteins and peptides in P. gingivalis.
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- 2012
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23. Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes.
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Sztukowska M, Veillard F, Potempa B, Bogyo M, Enghild JJ, Thogersen IB, Nguyen KA, and Potempa J
- Subjects
- Adhesins, Bacterial genetics, Amino Acid Sequence, Base Sequence, Cysteine Endopeptidases genetics, Gingipain Cysteine Endopeptidases, Molecular Sequence Data, Adhesins, Bacterial chemistry, Adhesins, Bacterial metabolism, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism
- Abstract
RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.
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- 2012
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24. Porphyromonas gingivalis enzymes enhance infection with human metapneumovirus in vitro.
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Pyrc K, Strzyz P, Milewska A, Golda A, Schildgen O, and Potempa J
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- Cell Line, Gingipain Cysteine Endopeptidases, Humans, Leukocyte Elastase metabolism, Staphylococcus aureus enzymology, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Metapneumovirus pathogenicity, Microbial Interactions, Porphyromonas gingivalis enzymology
- Abstract
Relatively recently discovered, human metapneumovirus (HMPV) is a human pathogen with worldwide prevalence, accounting for a substantial percentage of respiratory tract diseases. Concurrent viral and bacterial infections enable intricate mechanisms of cooperation between pathogens, which complicate the symptoms and outcome of the disease. Such bilateral interactions are based on the modulation of bacterial growth on epithelium pathologically altered during viral illness and the modulation of immune responses, as well as the enhancement of virus replication by bacterial virulence factors. This study showed that proteases produced by Porphyromonas gingivalis, a Gram-negative bacterium implicated in the development of periodontitis, named gingipains, facilitated HMPV replication in LLC-MK2 cells and may contribute to HMPV pathogenicity in patients with periodontitis. Gingipains at low nanomolar concentrations enabled HMPV replication and allowed virus propagation in vitro. In contrast to previously published data for influenza virus, however, Staphylococcus aureus proteases and human neutrophil elastase did not affect virus replication.
- Published
- 2011
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25. Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis.
- Author
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Vincents B, Guentsch A, Kostolowska D, von Pawel-Rammingen U, Eick S, Potempa J, and Abrahamson M
- Subjects
- Adhesins, Bacterial metabolism, Amino Acid Sequence, Cysteine Endopeptidases metabolism, Gingipain Cysteine Endopeptidases, Host-Pathogen Interactions, Humans, Molecular Sequence Data, Periodontitis microbiology, Adhesins, Bacterial pharmacology, Cysteine Endopeptidases pharmacology, Immunoglobulin G metabolism, Periodontitis pathology, Porphyromonas gingivalis metabolism
- Abstract
Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.
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- 2011
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26. Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis.
- Author
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Skottrup PD, Leonard P, Kaczmarek JZ, Veillard F, Enghild JJ, O'Kennedy R, Sroka A, Clausen RP, Potempa J, and Riise E
- Subjects
- Adhesins, Bacterial genetics, Adhesins, Bacterial immunology, Amino Acid Sequence, Bacteroidaceae Infections microbiology, Biomarkers metabolism, Cysteine Endopeptidases genetics, Cysteine Endopeptidases immunology, Gingipain Cysteine Endopeptidases, Humans, Molecular Sequence Data, Peptide Library, Porphyromonas gingivalis enzymology, Protein Binding, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Saliva microbiology, Adhesins, Bacterial analysis, Antibodies, Bacterial immunology, Bacteroidaceae Infections diagnosis, Cysteine Endopeptidases analysis, Porphyromonas gingivalis isolation & purification, Single-Chain Antibodies immunology
- Abstract
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., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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27. HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis.
- Author
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Smalley JW, Byrne DP, Birss AJ, Wojtowicz H, Sroka A, Potempa J, and Olczak T
- Subjects
- Adhesins, Bacterial chemistry, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Electrophoresis, Electrophysiological Phenomena, Gingipain Cysteine Endopeptidases, Heme chemistry, Hemoglobins metabolism, Multiprotein Complexes chemistry, Multiprotein Complexes metabolism, Multiprotein Complexes physiology, Oxyhemoglobins metabolism, Peptide Hydrolases chemistry, Peptide Hydrolases physiology, Porphyromonas gingivalis chemistry, Protein Binding drug effects, Protoporphyrins metabolism, Serum Albumin pharmacology, Adhesins, Bacterial physiology, Cysteine Endopeptidases physiology, Heme metabolism, Peptide Hydrolases metabolism, Porphyromonas gingivalis enzymology, Porphyromonas gingivalis metabolism
- Abstract
Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.
- Published
- 2011
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28. Adsorption of components of the plasma kinin-forming system on the surface of Porphyromonas gingivalis involves gingipains as the major docking platforms.
- Author
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Rapala-Kozik M, Bras G, Chruscicka B, Karkowska-Kuleta J, Sroka A, Herwald H, Nguyen KA, Eick S, Potempa J, and Kozik A
- Subjects
- Adhesins, Bacterial genetics, Adsorption, Biotinylation, Cell Membrane, Cysteine Endopeptidases genetics, Gene Expression Regulation, Bacterial physiology, Gingipain Cysteine Endopeptidases, Kininogens genetics, Kininogens metabolism, Kinins genetics, Membrane Proteins genetics, Porphyromonas gingivalis genetics, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Kinins metabolism, Membrane Proteins metabolism, Porphyromonas gingivalis metabolism
- Abstract
Enhanced production of proinflammatory bradykinin-related peptides, the kinins, has been suggested to contribute to the pathogenesis of periodontitis, a common inflammatory disease of human gingival tissues. In this report, we describe a plausible mechanism of activation of the kinin-generating system, also known as the contact system or kininogen-kallikrein-kinin system, by the adsorption of its plasma-derived components such as high-molecular-mass kininogen (HK), prekallikrein (PK), and Hageman factor (FXII) to the cell surface of periodontal pathogen Porphyromonas gingivalis. The adsorption characteristics of mutant strains deficient in selected proteins of the cell envelope suggested that the surface-associated cysteine proteinases, gingipains, bearing hemagglutinin/adhesin domains (RgpA and Kgp) serve as the major platforms for HK and FXII adhesion. These interactions were confirmed by direct binding tests using microplate-immobilized gingipains and biotinylated contact factors. Other bacterial cell surface components such as fimbriae and lipopolysaccharide were also found to contribute to the binding of contact factors, particularly PK. Analysis of kinin release in plasma upon contact with P. gingivalis showed that the bacterial surface-dependent mechanism is complementary to the previously described kinin generation system dependent on HK and PK proteolytic activation by the gingipains. We also found that several P. gingivalis clinical isolates differed in the relative significance of these two mechanisms of kinin production. Taken together, these data show the importance of this specific type of bacterial surface-host homeostatic system interaction in periodontal infections.
- Published
- 2011
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29. The lysine-specific gingipain of Porphyromonas gingivalis : importance to pathogenicity and potential strategies for inhibition.
- Author
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Yongqing T, Potempa J, Pike RN, and Wijeyewickrema LC
- Subjects
- Adhesins, Bacterial chemistry, Bacterial Proteins antagonists & inhibitors, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Biocatalysis drug effects, Cysteine Endopeptidases chemistry, Gingipain Cysteine Endopeptidases, Periodontal Diseases microbiology, Porphyromonas gingivalis drug effects, Protease Inhibitors pharmacology, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis enzymology, Porphyromonas gingivalis pathogenicity
- Abstract
Periodontitis is a disease affecting the supporting structures of the teeth. The most severe forms of the disease result in tooth loss and have recently been strongly associated with systemic diseases, including cardiovascular and lung diseases and cancer. The disease is caused by biofilms of predominantly anaerobic bacteria. A major pathogen associated with severe, adult forms of the disease is Porphyromonas gingivalis. This organism produces potent cysteine proteases known as gingipains, which have specificity for cleavage after arginine or lysine residues. The lysine-specific gingipain, Kgp, appears to be the major virulence factor of this organism and here we describe its structure and function. We also discuss the inhibitors of the enzyme produced to date and the potential pathways to newer versions of such molecules that will be required to combat periodontitis.
- Published
- 2011
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30. Dichotomy of gingipains action as virulence factors: from cleaving substrates with the precision of a surgeon's knife to a meat chopper-like brutal degradation of proteins.
- Author
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Guo Y, Nguyen KA, and Potempa J
- Subjects
- Animals, Antimicrobial Cationic Peptides metabolism, Bacterial Adhesion physiology, Chronic Periodontitis immunology, Chronic Periodontitis microbiology, Complement Activation, Gingipain Cysteine Endopeptidases, Gingiva microbiology, Heme metabolism, Humans, Immune Evasion physiology, Immunity, Innate physiology, Inflammation microbiology, Protein Denaturation, Receptors, Proteinase-Activated metabolism, Signal Transduction, Substrate Specificity, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Porphyromonas gingivalis enzymology, Virulence Factors
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- 2010
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31. Elafin is specifically inactivated by RgpB from Porphyromonas gingivalis by distinct proteolytic cleavage.
- Author
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Kantyka T, Latendorf T, Wiedow O, Bartels J, Gläser R, Dubin G, Schröder JM, Potempa J, and Meyer-Hoffert U
- Subjects
- Adhesins, Bacterial chemistry, Amino Acid Sequence, Cysteine Endopeptidases chemistry, Cysteine Proteases metabolism, Elafin chemistry, Gingipain Cysteine Endopeptidases, Humans, Models, Molecular, Molecular Sequence Data, Protein Structure, Quaternary, Serine Proteases metabolism, Staphylococcus aureus enzymology, Substrate Specificity, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Elafin metabolism, Porphyromonas gingivalis enzymology
- Abstract
Abstract Porphyromonas gingivalis, the major causative bacterium of periodontitis, contributes significantly to elevated proteolytic activity at periodontal pockets owing to the presence of both bacteria and host, predominantly neutrophil-derived, serine proteases. Normally the activity of the latter enzymes is tightly regulated by endogenous proteins, including elafin, a potent neutrophil elastase and proteinase 3 inhibitor released from epithelial cells at sites of inflammation. Here, we report that all three gingipains (HRgpA, RgpB, and Kgp) have the ability to degrade elafin, with RgpB being far more efficient than other gingipains. RgpB efficiently inactivates the inhibitory activity of elafin at subnanomolar concentrations through proteolysis limited to the Arg22-Cys23 peptide bond within the surface loop harboring the inhibitor active site. Notably, elafin resists inactivation by several Staphylococcus aureus-derived serine and cysteine proteases, confirming the high stability of this protein against proteolytic degradation. Therefore, we conclude that elafin inactivation by RgpB represents a specific pathogenic adaptation of P. gingivalis to disturb the protease-protease inhibitor balance in the infected gingival tissue. This contributes to enhanced degradation of host proteins and generation of a pool of peptides serving as nutrients for this asaccharolytic pathogen.
- Published
- 2009
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32. Kinin danger signals proteolytically released by gingipain induce Fimbriae-specific IFN-gamma- and IL-17-producing T cells in mice infected intramucosally with Porphyromonas gingivalis.
- Author
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Monteiro AC, Scovino A, Raposo S, Gaze VM, Cruz C, Svensjö E, Narciso MS, Colombo AP, Pesquero JB, Feres-Filho E, Nguyen KA, Sroka A, Potempa J, and Scharfstein J
- Subjects
- Animals, Gingipain Cysteine Endopeptidases, Immunity, Inflammation, Mice, Mouth Mucosa microbiology, Mouth Mucosa pathology, Peptide Hydrolases, Signal Transduction, T-Lymphocytes immunology, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Fimbriae, Bacterial immunology, Interferon-gamma biosynthesis, Interleukin-17 biosynthesis, Kinins metabolism, Porphyromonas gingivalis immunology, Receptor, Bradykinin B2 metabolism, T-Lymphocytes metabolism, Toll-Like Receptor 2 metabolism
- Abstract
Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, we studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B(2) receptors (B(2)R). Our data show that P. gingivalis W83 (wild type), but not gingipain-deficient mutant or wild-type bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in BALB/c or C57BL/6 mice. Studies in TLR2(-/-), B(2)R(-/-), and neutrophil-depleted C57BL/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a readout to verify whether activation of the TLR2-->PMN-->B(2)R axis (where PMN is polymorphonuclear neutrophil) at early stages of mucosal infection had impact on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B(2)R-dependent generation of IFN-gamma-producing Fim T cells in submandibular draining lymph nodes of BALB/c and C57BL/6 mice, whereas IL-17-producing Fim T cells were generated only in BALB/c mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forge a trans-cellular cross-talk between TLR2 and B(2)R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify whether kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis.
- Published
- 2009
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33. Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism.
- Author
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Stathopoulou PG, Galicia JC, Benakanakere MR, Garcia CA, Potempa J, and Kinane DF
- Subjects
- Caspase 3 metabolism, Cells, Cultured, DNA Fragmentation, Epithelial Cells microbiology, Gene Expression Profiling, Gingipain Cysteine Endopeptidases, Gingiva microbiology, Humans, Adhesins, Bacterial metabolism, Apoptosis, Cysteine Endopeptidases metabolism, Epithelial Cells pathology, Gingiva cytology, Porphyromonas gingivalis enzymology
- Abstract
Background: The oral pathogen Porphyromonas gingivalis has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear., Results: We demonstrate that primary human gingival epithelial cells (HGECs) challenged with live P. gingivalis for 24 hours exhibit apoptosis, and we characterize this by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Live bacteria strongly upregulated intrinsic and extrinsic apoptotic pathways. Pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax were upregulated after 24 hours. The anti-apoptotic Bcl-2 was also upregulated, but this was not sufficient to ensure cell survival. The main P. gingivalis proteases arginine and lysine gingipains are necessary and sufficient to induce host cell apoptosis. Thus, live P. gingivalis can invoke gingival epithelial cell apoptosis in a time and dose dependent manner with significant apoptosis occurring between 12 and 24 hours of challenge via a gingipain-dependent mechanism., Conclusion: The present study provides evidence that live, but not heat-killed, P. gingivalis can induce apoptosis after 24 hours of challenge in primary human gingival epithelial cells. Either arginine or lysine gingipains are necessary and sufficient factors in P. gingivalis elicited apoptosis.
- Published
- 2009
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34. Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin.
- Author
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Yasuhara R, Miyamoto Y, Takami M, Imamura T, Potempa J, Yoshimura K, and Kamijo R
- Subjects
- Animals, Cell Differentiation drug effects, Cells, Cultured, Gingipain Cysteine Endopeptidases, Mice, Mice, Mutant Strains, Osteoclasts cytology, Protein Stability drug effects, Adhesins, Bacterial pharmacology, Cholecalciferol pharmacology, Cysteine Endopeptidases pharmacology, Lipopolysaccharides pharmacology, Osteoclasts drug effects, Osteoclasts metabolism, Osteoprotegerin metabolism
- Abstract
Porphyromonas gingivalis is one of the major pathogens of periodontitis, a condition characterized by excessive alveolar bone resorption by osteoclasts. The bacterium produces cysteine proteases called gingipains, which are classified according to their cleavage-site specificity into Kgps (lysine-specific gingipains) and Rgps (arginine-specific gingipains). In the present study we examined the effects of gingipains on osteoclast differentiation. In co-cultures of mouse bone-marrow cells and osteoblasts, formation of multinucleated osteoclasts induced by 1alpha,25(OH)(2)D(3) (1alpha,25-dihydroxyvitamin D(3)) was augmented by Kgp but not by RgpB. A physiological concentration (0.1 nM) of 1alpha,25(OH)(2)D(3) induced the osteoclast formation in the presence of 100 nM Kgp to an extent comparable with that induced by 10 nM 1alpha,25(OH)(2)D(3). Kgp also enhanced osteoclastogenesis induced by various microbial components, including lipopolysaccharide. Combined use of Kgp and 1alpha,25(OH)(2)D(3) or lipopolysaccharide also increased the number of resorption pits developed on dentin slices, indicating that the osteoclasts formed in the presence of Kgp possess bone-resorbing activity. The enhanced osteoclastogenesis by Kgp was correlated with a depletion of osteoprotegerin in co-culture medium and was proteolytic-activity-dependent, since benzyloxycarbonyl-L-phenylalanyl-L-lysylacycloxyketone, an inhibitor of Kgp, completely abolished osteoclastogenesis induced by Kgp. Kgp digested osteoprotegerin, since its recombinant protein was susceptible to degradation by Kgp in the presence of serum. As a result, Kgp did not augment osteoclastogenesis in co-cultures of osteoprotegerin-deficient osteoblasts and bone-marrow cells. In addition, enhanced osteoclastogenesis by Kgp was abolished by an excess amount of recombinant osteoprotegerin. These findings suggest that degradation of osteoprotegerin is one of the mechanisms underlying promotion of osteoclastogenesis by Kgp.
- Published
- 2009
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35. Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.
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Potempa M, Potempa J, Kantyka T, Nguyen KA, Wawrzonek K, Manandhar SP, Popadiak K, Riesbeck K, Eick S, and Blom AM
- Subjects
- Blood Bactericidal Activity immunology, Complement Activation, Complement C1q immunology, Complement C4 immunology, Complement System Proteins immunology, Cysteine Proteinase Inhibitors metabolism, Drug Synergism, Gingiva metabolism, Humans, Adhesins, Bacterial physiology, Bacterial Proteins physiology, Complement C3 immunology, Cysteine Endopeptidases physiology, Prevotella intermedia enzymology
- Abstract
Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.
- Published
- 2009
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36. High molecular weight gingipains from Porphyromonas gingivalis induce cytokine responses from human macrophage-like cells via a nonproteolytic mechanism.
- Author
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Fitzpatrick RE, Aprico A, Wijeyewickrema LC, Pagel CN, Wong DM, Potempa J, Mackie EJ, and Pike RN
- Subjects
- Bacteroidaceae Infections microbiology, Cell Line, Gingipain Cysteine Endopeptidases, Granulocyte-Macrophage Colony-Stimulating Factor biosynthesis, Humans, Immunity, Innate, Interferon-gamma biosynthesis, Interleukin-1beta biosynthesis, Protein Subunits physiology, Up-Regulation, Adhesins, Bacterial physiology, Bacteroidaceae Infections immunology, Cysteine Endopeptidases physiology, Cytokines biosynthesis, Macrophages immunology, Porphyromonas gingivalis enzymology
- Abstract
Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. Porphyromonas gingivalis, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1beta, granulocyte-macrophage colony stimulatory factor and interferon-gamma were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1beta and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages., (Copyright 2008 S. Karger AG, Basel.)
- Published
- 2009
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37. Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis.
- Author
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Potempa M, Potempa J, Okroj M, Popadiak K, Eick S, Nguyen KA, Riesbeck K, and Blom AM
- Subjects
- Adhesins, Bacterial genetics, Adhesins, Bacterial metabolism, Bacteroidaceae Infections enzymology, Bacteroidaceae Infections genetics, Blood Bactericidal Activity genetics, Complement Activation genetics, Complement Activation immunology, Complement C4b-Binding Protein genetics, Complement C4b-Binding Protein metabolism, Complement C9 genetics, Complement C9 immunology, Complement C9 metabolism, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Gingipain Cysteine Endopeptidases, Humans, Ligands, Mutation, Porphyromonas gingivalis enzymology, Porphyromonas gingivalis pathogenicity, Protein Binding genetics, Protein Binding immunology, Protein Structure, Secondary genetics, Protein Structure, Tertiary genetics, Virulence Factors genetics, Virulence Factors metabolism, Adhesins, Bacterial immunology, Bacteroidaceae Infections immunology, Blood Bactericidal Activity immunology, Complement C4b-Binding Protein immunology, Cysteine Endopeptidases immunology, Porphyromonas gingivalis immunology, Virulence Factors immunology
- Abstract
The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.
- Published
- 2008
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38. Mechanism of methaemoglobin breakdown by the lysine-specific gingipain of the periodontal pathogen Porphyromonas gingivalis.
- Author
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Smalley JW, Birss AJ, Szmigielski B, and Potempa J
- Subjects
- Animals, Gingipain Cysteine Endopeptidases, Horses, Ligands, Methemoglobin analogs & derivatives, Adhesins, Bacterial metabolism, Cysteine Endopeptidases metabolism, Methemoglobin metabolism, Porphyromonas gingivalis enzymology
- Abstract
Abstract The R- and K-gingipain proteases of Porphyromonas gingivalis are involved in proteolysis of haemoglobin from which the defensive dimeric haem pigment is formed. Whilst oxyhaemoglobin is refractory towards K-gingipain, methaemoglobin is rapidly degraded. Ligation of methaemoglobin with N3-, which effectively blocks haem dissociation from the protein, prevented haemoglobin breakdown. Haem-free globin was rapidly degraded by K-gingipain. These data emphasise the need for haemoglobin oxidation which encourages haem dissociation and makes the haem-free globin susceptible to proteolytic attack.
- Published
- 2008
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39. Dual regulation of interleukin-8 production in human oral epithelial cells upon stimulation with gingipains from Porphyromonas gingivalis.
- Author
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Uehara A, Naito M, Imamura T, Potempa J, Travis J, Nakayama K, and Takada H
- Subjects
- Adhesins, Bacterial metabolism, Adhesins, Bacterial pharmacology, Cell Line, Tumor, Cysteine Endopeptidases metabolism, Cysteine Endopeptidases pharmacology, Down-Regulation, Epithelial Cells microbiology, Gingipain Cysteine Endopeptidases, Humans, Mouth Mucosa cytology, Porphyromonas gingivalis immunology, Porphyromonas gingivalis metabolism, Up-Regulation, Adhesins, Bacterial physiology, Cysteine Endopeptidases physiology, Epithelial Cells immunology, Gene Expression Regulation, Interleukin-8 biosynthesis, Mouth Mucosa immunology, Porphyromonas gingivalis enzymology
- Abstract
Cysteine proteinases from Porphyromonas gingivalis, or gingipains, are considered to be key virulence factors of the bacterium in relation to periodontal diseases. Incubation of human oral epithelial cells with lysine-specific gingipain (Kgp) and high-molecular-mass arginine-specific gingipain (HRgpA) resulted in a decrease in the production of interleukin (IL)-8, but not in the production of other pro-inflammatory cytokines. In contrast, arginine-specific gingipain 2 (RgpB) increased IL-8 production. RNA interference assays demonstrated that Kgp- and HRgpA-mediated downregulation and RgpB-mediated upregulation occurred through protease-activated receptor (PAR)-1 and PAR-2 signalling. Although the RgpB-mediated upregulation of IL-8 production occurred through nuclear factor-kappa B (NF-kappaB), the Kgp- and HRgpA-mediated downregulation was not negated in NF-kappaB-silenced cells. Both the haemagglutinin and the enzymic domains are required for Kgp and HRgpA to downregulate the production of IL-8 in human oral epithelial cells, and the two domains are thought to co-exist. These results suggest that gingipains preferentially suppress IL-8, resulting in attenuation of the cellular recognition of bacteria, and as a consequence, sustain chronic inflammation.
- Published
- 2008
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40. Friendly fire against neutrophils: proteolytic enzymes confuse the recognition of apoptotic cells by macrophages.
- Author
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Guzik K and Potempa J
- Subjects
- Bacteroidaceae Infections immunology, Bacteroidaceae Infections microbiology, Gingipain Cysteine Endopeptidases, Humans, Immunity, Innate, Neutrophils cytology, Periodontitis immunology, Periodontitis microbiology, Phagocytosis, Phosphatidylserines physiology, Porphyromonas gingivalis enzymology, Adhesins, Bacterial metabolism, Apoptosis, Cysteine Endopeptidases metabolism, Macrophages immunology, Neutrophils immunology
- Abstract
Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and 'eat me' signals. The complexity of the system is reflected by the term: 'engulfment synapse', used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent "eat me" signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy.
- Published
- 2008
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41. Sequential action of R- and K-specific gingipains of Porphyromonas gingivalis in the generation of the haem-containing pigment from oxyhaemoglobin.
- Author
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Smalley JW, Birss AJ, Szmigielski B, and Potempa J
- Subjects
- Gingipain Cysteine Endopeptidases, Kinetics, Adhesins, Bacterial chemistry, Cysteine Endopeptidases chemistry, Heme chemistry, Hemeproteins chemistry, Oxyhemoglobins chemistry, Pigments, Biological chemistry, Plant Extracts chemistry, Porphyromonas gingivalis chemistry
- Abstract
The arginine- and lysine-specific gingipains of Porphyromonas gingivalis have been implicated in the degradation of haemoglobin from which the black mu-oxo haem dimer-containing pigment is generated. Here, we examined interactions of oxyhaemoglobin (oxyHb) with the Arg-(R)-specific (HRgpA) and Lys-(K)-specific (Kgp) gingipains. Incubation of oxyHb with HRgpA resulted in formation of methaemoglobin (metHb), which could be prevented by the R-gingipain specific inhibitor leupeptin. oxyHb-Kgp interactions resulted in formation of a haemoglobin haemichrome. This was inhibited by the lysine-specific protease inhibitor Z-Phe-Lys-acyloxymethylketone (Z-FKck). metHb, formed by treatment of oxyHb with either NaNO(2) or by pre-incubation with HRgpA, was rapidly degraded by Kgp compared to oxyHb. metHb degradation by Kgp was also inhibited Z-FKck. Together these data show that R-gingipain activity is crucial for converting oxyHb into the metHb form which is rendered more susceptible to Kgp degradation for the eventual release of iron(III) protoporphyrin IX and production of the mu-oxo haem dimer. This explains previous observations [J.W. Smalley, M.F. Thomas, A.J. Birss, R. Withnall, J. Silver, Biochem. J. 379 (2004) 833-840.] of the requirement for a combination of both R- and K-gingipains for pigment production from oxyhaemoglobin by P. gingivalis.
- Published
- 2007
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42. Purification and characterization of gingipains.
- Author
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Potempa J and Nguyen KA
- Subjects
- Chromatography, Liquid methods, Gingipain Cysteine Endopeptidases, Porphyromonas gingivalis growth & development, Adhesins, Bacterial chemistry, Adhesins, Bacterial isolation & purification, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases isolation & purification, Porphyromonas gingivalis enzymology
- Abstract
Gingipains are cysteine proteases produced in large quantities by Porphyromonas gingivalis which together constitute important virulence factors in the pathogenesis of periodontal disease by that organism. Described is this unit is an efficient procedure for the purification of gingipains from the growth medium of P. gingivalis strain HG66, along with detailed protocols for growth of the organism and basic characterization of the purified proteases. The purification procedure consists of acetone precipitation followed by gel filtration to separate high-molecular-mass gingipains (Kgp and HRgpA) from RgpB. Kgp and HRgpA are further separated on Arg-Sepharose by the virtue of differential elution from the affinity matrix with lysine (Kgp) and arginine (HRgpA) eluant. Obtained from these procedures, the gingipains are stable and can be stored at -80 degrees C for years without loss of activity., ((c) 2007 by John Wiley & Sons, Inc.)
- Published
- 2007
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43. Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system.
- Author
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Popadiak K, Potempa J, Riesbeck K, and Blom AM
- Subjects
- Anti-Bacterial Agents pharmacology, Blood Bactericidal Activity immunology, Complement C1q metabolism, Complement C3 antagonists & inhibitors, Complement C4 antagonists & inhibitors, Complement C5 antagonists & inhibitors, Complement Pathway, Alternative immunology, Complement Pathway, Classical immunology, Complement Pathway, Mannose-Binding Lectin immunology, Gingipain Cysteine Endopeptidases, Humans, Porphyromonas gingivalis growth & development, Protein Subunits antagonists & inhibitors, Adhesins, Bacterial physiology, Complement Inactivator Proteins physiology, Cysteine Endopeptidases physiology, Porphyromonas gingivalis enzymology, Porphyromonas gingivalis immunology
- Abstract
Periodontitis is an inflammatory disease of the supporting structures of the teeth and is caused by, among other agents, Porphyromonas gingivalis. P. gingivalis is very resistant to killing by human complement, which is present in a gingival fluid at 70% of the serum concentration. We found that the incubation of human serum with purified cysteine proteases of P. gingivalis (gingipains) or P. gingivalis wild-type strains W83 and W50 resulted in a drastic decrease of the bactericidal activity of the serum. In contrast, serum treated with P. gingivalis mutants lacking gingipains (particularly strains without HRgpA) maintained significant bactericidal activity. To understand in detail the mechanism by which gingipains destroy the serum bactericidal activity, we investigated the effects of gingipains on the human complement system. We found that all three proteases degraded multiple complement components, with arginine-specific gingipains (HRgpA and RgpB) being more efficient than lysine-specific gingipain (Kgp). Interestingly, all three proteases at certain concentrations were able to activate the C1 complex in serum, which resulted in the deposition of C1q on inert surfaces and on bacteria themselves. It is therefore plausible that P. gingivalis activates complement when present at low numbers, resulting in a local inflammatory reaction and providing the bacteria with a colonization opportunity and nutrients. At later stages of infection the concentration of proteases is high enough to destroy complement factors and thus render the bacteria resistant to the bactericidal activity of complement.
- Published
- 2007
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44. Does the importance of the C-terminal residues in the maturation of RgpB from Porphyromonas gingivalis reveal a novel mechanism for protein export in a subgroup of Gram-Negative bacteria?
- Author
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Nguyen KA, Travis J, and Potempa J
- Subjects
- Alanine genetics, Alanine metabolism, Amino Acid Sequence, Bacterial Outer Membrane Proteins genetics, Bacterial Outer Membrane Proteins metabolism, Blotting, Western, Endopeptidases genetics, Endopeptidases metabolism, Gingipain Cysteine Endopeptidases, Glycosylation, Gram-Negative Bacteria genetics, Immunoprecipitation, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Porphyromonas gingivalis genetics, Protein Processing, Post-Translational, Protein Transport, Sequence Alignment, Adhesins, Bacterial genetics, Adhesins, Bacterial metabolism, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Gram-Negative Bacteria metabolism, Porphyromonas gingivalis metabolism
- Abstract
The mature 507-residue RgpB protein belongs to an important class of extracellular outer membrane-associated proteases, the gingipains, from the oral pathogen Porphyromonas gingivalis that has been shown to play a central role in the virulence of the organism. The C termini of these gingipains along with other outer membrane proteins from the organism share homologous sequences and have been suggested to function in attachment of these proteins to the outer membrane. In this report, we have created a series of truncated and site-directed mutants of the C terminus from a representative member of this class, the RgpB protease, to investigate its role in the maturation of these proteins. Truncation of the last two residues (valyl-lysine) from the C terminus is sufficient to create an inactive version of the protein that lacks the posttranslational glycosylation seen in the wild type, and the protein remains trapped behind the outer membrane. Alanine scanning of the last five residues revealed the importance of the C-terminal motif in mediating correct posttranslational modification of the protein. This result may have a wider implication in a novel secretory pathway in distinct members of the Cytophaga-Flavobacterium-Bacteroidetes phylum.
- Published
- 2007
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45. Gingipains from Porphyromonas gingivalis W83 synergistically disrupt endothelial cell adhesion and can induce caspase-independent apoptosis.
- Author
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Sheets SM, Potempa J, Travis J, Fletcher HM, and Casiano CA
- Subjects
- Adhesins, Bacterial drug effects, Adhesins, Bacterial isolation & purification, Amino Acid Chloromethyl Ketones pharmacology, Animals, Caspase Inhibitors, Caspases chemistry, Caspases metabolism, Cattle, Cells, Cultured, Cysteine Endopeptidases drug effects, Cysteine Endopeptidases isolation & purification, Cysteine Proteinase Inhibitors pharmacology, Endothelial Cells enzymology, Gingipain Cysteine Endopeptidases, Porphyromonas gingivalis pathogenicity, Adhesins, Bacterial pharmacology, Apoptosis, Cell Adhesion drug effects, Cell Adhesion Molecules metabolism, Cysteine Endopeptidases pharmacology, Endothelial Cells drug effects, Porphyromonas gingivalis enzymology
- Abstract
We have shown previously that gingipains from Porphyromonas gingivalis W83 can induce cell detachment, cell adhesion molecule (CAM) cleavage, and apoptosis in endothelial cells; however, the specific roles of the individual gingipains are unclear. Using purified gingipains, we determined that each of the gingipains can cleave CAMs to varying degrees with differing kinetics. Kgp and HRgpA work together to quickly detach endothelial cells. Interestingly, in the absence of active caspases, both gingipain-active W83 extracts and purified HRgpA and RgpB induce apoptotic morphology, suggesting that the gingipains can induce both caspase-dependent and caspase-independent apoptosis. Using z-VAD-FMK to inhibit Kgp activity and leupeptin to inhibit Rgp activity in gingipain-active W83 extracts, we investigated the relative significance of the synergistic role of the gingipains. z-VAD-FMK or leupeptin delayed, but did not inhibit, cell detachment induced by gingipain-active W83 extracts or purified gingipains. There was partial cleavage of N-cadherin and cleavage of VE-cadherin was not inhibited. Degradation of integrin beta1 was inhibited only in the presence of z-VAD-FMK. These results further clarify the role P. gingivalis plays in tissue destruction occurring in the periodontal pocket.
- Published
- 2006
- Full Text
- View/download PDF
46. Exploring the Sn binding pockets in gingipains by newly developed inhibitors: structure-based design, chemistry, and activity.
- Author
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Białas A, Grembecka J, Krowarsch D, Otlewski J, Potempa J, and Mucha A
- Subjects
- Amino Acid Sequence, Binding Sites, Crystallography, X-Ray, Dipeptides chemistry, Gingipain Cysteine Endopeptidases, Molecular Sequence Data, Protein Conformation, Sequence Homology, Amino Acid, Structure-Activity Relationship, Substrate Specificity, Adhesins, Bacterial chemistry, Amino Acid Chloromethyl Ketones chemistry, Cysteine Endopeptidases chemistry, Cysteine Proteinase Inhibitors chemistry, Models, Molecular
- Abstract
Arg-gingipains (Rgps) and Lys-gingipain (Kgp) are cysteine proteinases secreted by Porphyromonas gingivalis, the major pathogen implicated in periodontal disease. Gingipains are essential to the bacterium for its virulence and survival, and development of inhibitors targeting these proteins provides an approach to treat periodontal diseases. Here, we present the first example of structure-based design of gingipains inhibitors, with the use of the crystal structure of RgpB and the homology model of Kgp. Chloromethyl ketones were selected as suitable compounds to explore the specificity of the Sn binding region of both enzymes. Three series of inhibitors bearing Arg or Lys at P1 and different substituents at P2 and P3 were designed, synthesized, and tested. High potency (k(obs)/[I] approximately 10(7) M(-1) s(-1)) was achieved for small ligands, such as the dipeptide analogues. The detailed analysis of Sn binding pockets revealed the molecular basis of inhibitory affinity and provided insight into the structure-activity relationship.
- Published
- 2006
- Full Text
- View/download PDF
47. Arginine-specific gingipains from Porphyromonas gingivalis stimulate production of hepatocyte growth factor (scatter factor) through protease-activated receptors in human gingival fibroblasts in culture.
- Author
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Uehara A, Muramoto K, Imamura T, Nakayama K, Potempa J, Travis J, Sugawara S, and Takada H
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Cells, Cultured, Fibroblasts metabolism, Gingipain Cysteine Endopeptidases, Gingiva cytology, Gingiva metabolism, Humans, NF-kappa B physiology, Periodontitis etiology, Type C Phospholipases physiology, Adhesins, Bacterial pharmacology, Cysteine Endopeptidases pharmacology, Gingiva drug effects, Hepatocyte Growth Factor biosynthesis, Receptor, PAR-1 physiology, Receptor, PAR-2 physiology
- Abstract
Cystein proteinases (gingipains) from Porphyromonas gingivalis cleave a broad range of in-host proteins and are considered to be key virulence factors in the onset and development of adult periodontitis and host defense evasion. In periodontitis, an inflammatory disease triggered by bacterial infection, the production of hepatocyte growth factor (HGF) is induced not only by various factors derived from the host, such as inflammatory cytokines, but also by bacterial components. In this study we examined the possible enhanced production of HGF produced by human gingival fibroblasts upon stimulation with gingipains. Arginine-specific gingipain (Rgp) caused a marked production of HGF into the supernatant, the induction of HGF expression on the cell surface, and the up-regulation of HGF mRNA expression in a dose-dependent and an enzymatic activity-dependent manner. Because it has been reported that Rgp activated protease-activated receptors (PARs), we examined whether the induction of HGF triggered by Rgps on human gingival fibroblasts occurred through PARs. An RNA interference assay targeted to PAR-1 and PAR-2 mRNA revealed that gingipains-induced secretion of HGF was significantly inhibited by RNA interference targeted to PAR-1 and PAR-2. In addition, the Rgps-mediated HGF induction was completely inhibited by the inhibition of phospholipase C and was clearly inhibited by RNA interference targeted to p65, which is an NF-kappaB component. These results suggest that Rgps activated human gingival fibroblasts to secrete HGF in the inflamed sites and the mechanism(s) involved may actively participate in both inflammatory and reparative processes in periodontal diseases.
- Published
- 2005
- Full Text
- View/download PDF
48. Gingipains : critical factors in the development of aspiration pneumonia caused by Porphyromonas gingivalis
- Author
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Benedyk, Małgorzata, Mydel, Piotr, Delaleu, Nicolas, Płaza, Karolina, Gawron, Katarzyna, Milewska, Aleksandra, Maresz, Katarzyna, Koziel, Joanna, Pyrc, Krzysztof, and Potempa, Jan
- Subjects
Blood Platelets ,Mice, Inbred BALB C ,Neutrophils ,Platelet Count ,Hemorrhage ,Platelet Activation ,Pneumonia, Aspiration ,Article ,stomatognathic diseases ,Cysteine Endopeptidases ,Mice ,Necrosis ,stomatognathic system ,Neutrophil Infiltration ,Bacteroidaceae Infections ,Gingipain Cysteine Endopeptidases ,Animals ,Cytokines ,Female ,Adhesins, Bacterial ,Porphyromonas gingivalis - 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.
- Published
- 2016
49. Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis1
- Author
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Potempa, Michal, Potempa, Jan, Okroj, Marcin, Popadiak, Katarzyna, Eick, Sigrun, Nguyen, Ky-Anh, Riesbeck, Kristian, and Blom, Anna M.
- Subjects
Blood Bactericidal Activity ,Virulence Factors ,Complement C4b-Binding Protein ,Complement C9 ,Ligands ,Article ,Protein Structure, Secondary ,Protein Structure, Tertiary ,Cysteine Endopeptidases ,Mutation ,Bacteroidaceae Infections ,Gingipain Cysteine Endopeptidases ,Humans ,Adhesins, Bacterial ,Complement Activation ,Porphyromonas gingivalis ,Protein Binding - Abstract
The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.
- Published
- 2008
50. Erratum to: Smoking, Porphyromonas gingivalis and the immune response to citrullinated autoantigens before the clinical onset of rheumatoid arthritis in a Southern European nested case-control study
- Author
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Benjamin A. Fisher, Alison J. Cartwright, Anne-Marie Quirke, Paola de Pablo, Dora Romaguera, Salvatore Panico, Amalia Mattiello, Diana Gavrila, Carmen Navarro, Carlotta Sacerdote, Paolo Vineis, Rosario Tumino, David F. Lappin, Danae Apatzidou, Shauna Culshaw, Jan Potempa, Dominique S. Michaud, Elio Riboli, Patrick J. Venables, Fisher, Benjamin A, Cartwright, Alison J, Quirke, Anne Marie, de Pablo, Paola, Romaguera, Dora, Panico, Salvatore, Mattiello, Amalia, Gavrila, Diana, Navarro, Carmen, Sacerdote, Carlotta, Vineis, Paolo, Tumino, Rosario, Lappin, David F, Apatzidou, Danae, Culshaw, Shauna, Potempa, Jan, Michaud, Dominique S, Riboli, Elio, and Venables, Patrick J.
- Subjects
Adult ,Male ,Hydrolases ,Autoantigens ,Peptides, Cyclic ,Arthritis, Rheumatoid ,Rheumatology ,Humans ,Orthopedics and Sports Medicine ,Prospective Studies ,Adhesins, Bacterial ,Periodontitis ,Science & Technology ,Smoking ,1103 Clinical Sciences ,Middle Aged ,Europe ,Cysteine Endopeptidases ,Orthopedics ,Case-Control Studies ,Gingipain Cysteine Endopeptidases ,Protein-Arginine Deiminases ,Female ,Erratum ,Porphyromonas gingivalis ,Life Sciences & Biomedicine - Abstract
Antibodies to citrullinated proteins (ACPA) occur years before RA diagnosis. Porphyromonas gingivalis expresses its own peptidylarginine deiminase (PPAD), and is a proposed aetiological factor for the ACPA response. Smoking is a risk factor for both ACPA-positive RA and periodontitis. We aimed to study the relation of these factors to the risk of RA in a prospective cohort.We performed a nested case-control study by identifying pre-RA cases in four populations from the European Prospective Investigation into Cancer and nutrition, matched with three controls. Data on smoking and other covariates were obtained from baseline questionnaires. Antibodies to CCP2 and citrullinated peptides from α-enolase, fibrinogen, vimentin and PPAD were measured. Antibodies to arginine gingipain (RgpB) were used as a marker for P.gingivalis infection and validated in a separate cohort of healthy controls and subjects with periodontitis.We studied 103 pre-RA cases. RA development was associated with several ACPA specificities, but not with antibodies to citrullinated PPAD peptides. Antibody levels to RgpB and PPAD peptides were higher in smokers but were not associated with risk of RA or with pre-RA autoimmunity. Former but not current smoking was associated with antibodies to α-enolase (OR 4.06; 95 % CI 1.02, 16.2 versus 0.54; 0.09-3.73) and fibrinogen peptides (OR 4.24; 95 % CI 1.2-14.96 versus 0.58; 0.13-2.70), and later development of RA (OR 2.48; 95 % CI 1.27-4.84 versus 1.57; 0.85-2.93), independent of smoking intensity.Smoking remains a risk factor for RA well before the clinical onset of disease. In this cohort, P.gingivalis is not associated with pre-RA autoimmunity or risk of RA in an early phase before disease-onset. Antibodies to PPAD peptides are not an early feature of ACPA ontogeny.
- Published
- 2016
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