33 results on '"Miroslaw Ksiazek"'
Search Results
2. Structure-based mechanism of cysteine-switch latency and of catalysis by pappalysin-family metallopeptidases
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Tibisay Guevara, Arturo Rodriguez-Banqueri, Miroslaw Ksiazek, Jan Potempa, and F. Xavier Gomis-Rüth
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pappalysin family ,metallopeptidases ,mirolysin ,peridontopathogens ,zymogens ,catalytic mechanisms ,Crystallography ,QD901-999 - Abstract
Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54–Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Å revealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1′. Moreover, residue Cys23 within a conserved cysteine–glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Å structure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1′ to S6′. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms.
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- 2020
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3. Development of a novel, high-affinity ssDNA trypsin inhibitor
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Stanislaw Malicki, Miroslaw Ksiazek, Pawel Majewski, Aleksandra Pecak, Piotr Mydel, Przemyslaw Grudnik, and Grzegorz Dubin
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aptamer ,ssdna ,trypsin ,protease inhibitor ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors.
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- 2019
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4. PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis , Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins
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Mariusz Madej, Zuzanna Nowakowska, Miroslaw Ksiazek, Anna M. Lasica, Danuta Mizgalska, Magdalena Nowak, Anna Jacula, Monika Bzowska, Carsten Scavenius, Jan J. Enghild, Joseph Aduse-Opoku, Michael A. Curtis, F. Xavier Gomis-Rüth, and Jan Potempa
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Microbiology ,QR1-502 - Abstract
Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis
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- 2021
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5. 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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Rielana Wichert, Anna Ermund, Stefanie Schmidt, Matthias Schweinlin, Miroslaw Ksiazek, Philipp Arnold, Katharina Knittler, Frederike Wilkens, Barbara Potempa, Björn Rabe, Marit Stirnberg, Ralph Lucius, Jörg W. Bartsch, Susanna Nikolaus, Maren Falk-Paulsen, Philip Rosenstiel, Marco Metzger, Stefan Rose-John, Jan Potempa, Gunnar C. Hansson, Peter J. Dempsey, and Christoph Becker-Pauly
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host-microbiome interaction ,mucus ,intestinal mucus barrier ,ectodomain shedding ,metalloprotease ,Biology (General) ,QH301-705.5 - 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.
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- 2017
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6. The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function
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Anna M. Lasica, Miroslaw Ksiazek, Mariusz Madej, and Jan Potempa
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secretion ,T9SS ,Porphyromonas gingivalis ,pathogenesis ,gliding motility ,proteins ,Microbiology ,QR1-502 - Abstract
Protein secretion systems are vital for prokaryotic life, as they enable bacteria to acquire nutrients, communicate with other species, defend against biological and chemical agents, and facilitate disease through the delivery of virulence factors. In this review, we will focus on the recently discovered type IX secretion system (T9SS), a complex translocon found only in some species of the Bacteroidetes phylum. T9SS plays two roles, depending on the lifestyle of the bacteria. It provides either a means of movement (called gliding motility) for peace-loving environmental bacteria or a weapon for pathogens. The best-studied members of these two groups are Flavobacterium johnsoniae, a commensal microorganism often found in water and soil, and Porphyromonas gingivalis, a human oral pathogen that is a major causative agent of periodontitis. In P. gingivalis and some other periodontopathogens, T9SS translocates proteins, especially virulence factors, across the outer membrane (OM). Proteins destined for secretion bear a conserved C-terminal domain (CTD) that directs the cargo to the OM translocon. At least 18 proteins are involved in this still enigmatic process, with some engaged in the post-translational modification of T9SS cargo proteins. Upon translocation across the OM, the CTD is removed by a protease with sortase-like activity and an anionic LPS is attached to the newly formed C-terminus. As a result, a cargo protein could be secreted into the extracellular milieu or covalently attached to the bacterial surface. T9SS is regulated by a two-component system; however, the precise environmental signal that triggers it has not been identified. Exploring unknown systems contributing to bacterial virulence is exciting, as it may eventually lead to new therapeutic strategies. During the past decade, the major components of T9SS were identified, as well as hints suggesting the possible mechanism of action. In addition, the list of characterized cargo proteins is constantly growing. The actual structure of the translocon, situated in the OM of bacteria, remains the least explored area; however, new technical approaches and increasing scientific attention have resulted in a growing body of data. Therefore, we present a compact up-to-date review of this topic.
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- 2017
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7. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.
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Peter Durand Skottrup, Grete Sørensen, Miroslaw Ksiazek, Jan Potempa, and Erik Riise
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Medicine ,Science - Abstract
Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.
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- 2012
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8. Response regulator PorX coordinates oligonucleotide signalling and gene expression to control the secretion of virulence factors
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Claus Schmitz, Mariusz Madej, Zuzanna Nowakowska, Anna Cuppari, Anna Jacula, Miroslaw Ksiazek, Katarzyna Mikruta, Jerzy Wisniewski, Natalia Pudelko-Malik, Anshu Saran, Natalie Zeytuni, Piotr Mlynarz, Richard J Lamont, Isabel Usón, Virginijus Siksnys, Jan Potempa, Maria Solà, National Science Centre (Poland), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Ministerio de Economía y Competitividad (España), Generalitat de Catalunya, National Institute of Dental and Craniofacial Research (US), Ministry of Science and Higher Education (Poland), and Wroclaw Center for Biotechnology
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Bacterial Proteins ,PorX ,PglZ ,virulence factor ,Virulence Factors ,Genetics ,Oligonucleotides ,Gene Expression ,Alkaline Phosphatase - Abstract
The PglZ family of proteins belongs to the alkaline phosphatase superfamily, which consists of metallohydrolases with limited sequence identity but similar metal-coordination architectures in otherwise divergent active sites. Proteins with a well-defined PglZ domain are ubiquitous among prokaryotes as essential components of BREX phage defence systems and two-component systems (TCSs). Whereas other members of the alkaline phosphatase superfamily are well characterized, the activity, structure and biological function of PglZ family proteins remain unclear. We therefore investigated the structure and function of PorX, an orphan response regulator of the Porphyromonas gingivalis TCS containing a putative PglZ effector domain. The crystal structure of PorX revealed a canonical receiver domain, a helical bundle, and an unprecedented PglZ domain, similar to the general organization of the phylogenetically related BREX-PglZ proteins. The PglZ domain of PorX features an active site cleft suitable for large substrates. An extensive search for substrates revealed that PorX is a phosphodiesterase that acts on cyclic and linear oligonucleotides, including signalling molecules such as cyclic oligoadenylates. These results, combined with mutagenesis, biophysical and enzymatic analysis, suggest that PorX coordinates oligonucleotide signalling pathways and indirectly regulates gene expression to control the secretion of virulence factors., National Science Centre, Poland [UMO-2015/19/N/NZ1/00322 to M.M., UMO-2016/23/N/NZ1/01513 to Z.N., UMO-2018/31/B/NZ1/03968 to J.P., UMO-2019/35/B/NZ1/03118 to M.K.]; Spanish Ministry of Science, Innovation and Universities [MCIN/AEI/ 10.13039/501100011033 ERDF ‘A way to make Europe’, BFU2015-70645-R, RTI2018-101015-B-100, PID2021-129038NB-I00, MDM-2014–0435 to M.S., BES-2013–063407 to C.S., PGC2018-101370-B-I00 and PID2021-128751NB-I00 (MICINN/AEI/FEDER/UE) to I.U.]; Generalitat de Catalunya [2014-SGR-99, 2017-SGR-1192 to M.S.]; European Social Fund [09.3.3-LMT-K-712-01-0126 to V.S.]; National Institute of Dental and Craniofacial Research (NIDCR) [DE012505, DE011111 to R.J.L.]; Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Science and Technology. Funding for open access charge: National Science Centre, Poland [UMO-2018/31/B/NZ1/03968].
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- 2022
9. Plasmin inhibition by bacterial serpin: Implications in gum disease
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Anna Straczek, Jan Potempa, Jan J. Enghild, Irena Waligorska, Miroslaw Ksiazek, Malgorzata Benedyk, Alicja Sochaj-Gregorczyk, Danuta Mizgalska, and Ida B. Thøgersen
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0301 basic medicine ,Plasmin ,medicine.medical_treatment ,Serpin ,Biochemistry ,Article ,03 medical and health sciences ,0302 clinical medicine ,Forsythia ,In vivo ,Catalytic Domain ,Fibrinolysis ,Genetics ,medicine ,Animals ,Humans ,Tannerella forsythia ,Protease Inhibitors ,Fibrinolysin ,periodontitis ,Molecular Biology ,Periodontal Diseases ,Serpins ,plasmin ,Bacteria ,biology ,Chemistry ,serpin ,Biofilm ,biology.organism_classification ,Antifibrinolytic Agents ,Mice, Inbred C57BL ,stomatognathic diseases ,030104 developmental biology ,Female ,fibrinolysis ,Bacterial outer membrane ,030217 neurology & neurosurgery ,Biotechnology ,medicine.drug - Abstract
Tannerella forsythia is a periodontopathogen that expresses miropin, a protease inhibitor in the serpin superfamily. In this study, we show that miropin is also a specific and efficient inhibitor of plasmin; thus it represents the first proteinaceous plasmin inhibitor of prokaryotic origin described to date. Miropin inhibits plasmin through the formation of a stable covalent complex triggered by cleavage of the Lys(368)-Thr(369) (P2-P1) reactive site bond with a stoichiometry of inhibition of 3.8 and an association rate constant (k(ass)) of 3.3×10(5) M(−1)s(-1). The inhibition of the fibrinolytic activity of plasmin was nearly as effective as that exerted by α(2)-antiplasmin. Miropin also acted in vivo by reducing blood loss in a mice tail bleeding assay. Importantly, intact T. forsythia cells or outer membrane vesicles, both of which carry surface-associated miropin, strongly inhibited plasmin. In intact bacterial cells, the antiplasmin activity of miropin protects envelope proteins from plasmin-mediated degradation. In summary, in the environment of periodontal pockets, which are bathed in gingival crevicular fluid consisting of 70% of blood plasma, an abundance of T. forsythia in the bacterial biofilm can cause local inhibition of fibrinolysis, which could have possible deleterious effects on the tooth-supporting structures of the periodontium.
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- 2019
10. Structural determinants of inhibition of Porphyromonas gingivalis gingipain K by KYT-36, a potent, selective, and bioavailable peptidase inhibitor
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Tibisay Guevara, Anna M. Lasica, Miroslaw Ksiazek, Barbara Potempa, F. Xavier Gomis-Rüth, Arturo Rodríguez-Banqueri, Jan Potempa, National Institutes of Health (US), Ministry of Science and Higher Education (Poland), Ministerio de Economía y Competitividad (España), Generalitat de Catalunya, Fundació La Marató de TV3, and Ministerio de Ciencia, Innovación y Universidades (España)
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0301 basic medicine ,Benzylamines ,Virulence Factors ,Virulence ,lcsh:Medicine ,Crystallography, X-Ray ,Article ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,Drug Development ,Protein Domains ,Catalytic Domain ,Hydrolase ,Bacteroidaceae Infections ,Structure–activity relationship ,Protease Inhibitors ,Periodontitis ,lcsh:Science ,Pathogen ,Porphyromonas gingivalis ,Gingipain K ,X-ray crystallography ,Bacterial structural biology ,Multidisciplinary ,biology ,Chemistry ,lcsh:R ,biology.organism_classification ,3. Good health ,Hydrazines ,030104 developmental biology ,Biochemistry ,Enzyme mechanisms ,Gingipain Cysteine Endopeptidases ,lcsh:Q ,Carbamates ,Salt bridge ,Hydrophobic and Hydrophilic Interactions ,030217 neurology & neurosurgery ,Cysteine - Abstract
© The Author(s) 2019., Porphyromonas gingivalis is a member of the dysbiotic oral microbiome and a “keystone pathogen” that causes severe periodontal disease, which is among the most prevalent infectious diseases. Part of the virulence factors secreted by P. gingivalis are the essential cysteine peptidases gingipain K (Kgp) and R (RgpA and RgpB), which account for 85% of the extracellular proteolytic activity of the pathogen and are thus prime targets for inhibition. We report the high-resolution (1.20 Å) complex structure of Kgp with KYT-36, a peptide-derived, potent, bioavailable and highly selective inhibitor, which is widely used for studies in vitro, in cells and in vivo. Sub-nanomolar inhibition of Kgp is achieved by tight binding to the active-site cleft, which is covered for its sub-sites S3 through S1’ under establishment of nine hydrophobic interactions, 14 hydrogen bonds and one salt bridge. In addition, an inhibitor carbonyl carbon that mimics the scissile carbonyl of substrates is pyramidalized and just 2.02 Å away from the catalytic nucleophile of Kgp, C477Sγ. Thus, the crystal structure emulates a reaction intermediate of the first nucleophilic attack during catalysis of cysteine peptidases. The present study sets the pace for the development of tailored next-generation drugs to tackle P. gingivalis., This study was supported in part by grants from US American (NIH/NIDCR R01 DE022597), Polish (National Science Center and Ministry of Science and Higher Education, Miniatura 2017/01/X/NZ1/01378, UMO-2015/199/N/NZ1/00322, UMO-2015/17/B/NZ1/00666, UMO-2016/21/B/NZ1/00292, and Mobility Plus 1306/MOB/IV/2015/0), Spanish (BFU2015-64487R and MDM-2014-0435), and Catalan (2017SGR3, and Fundació “La Marató de TV3” 201815) agencies. The Structural Biology Unit of IBMB is a “María de Maeztu” Unit of Excellence from the Spanish Ministry of Science, Innovation and Universities.
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- 2019
11. Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system
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Danuta Mizgalska, Arturo Rodríguez-Banqueri, Florian Veillard, Mirosław Książęk, Theodoros Goulas, Tibisay Guevara, Ulrich Eckhard, Jan Potempa, and F. Xavier Gomis-Rüth
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periodontal disease ,bacterial virulence factor ,infectious disease ,protein secretion ,X-ray crystal structure ,T9SS ,Biology (General) ,QH301-705.5 - Abstract
Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 β-sandwich (β1–β7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus (‘motif C-t.’) and the loop connecting strands β3 and β4 (‘motif Lβ3β4’) as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lβ3β4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.
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- 2024
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12. PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins
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Jan Potempa, F. Xavier Gomis-Rüth, Anna Jacula, Mariusz Madej, Zuzanna Nowakowska, Monika Bzowska, Carsten Scavenius, Miroslaw Ksiazek, Joseph Aduse-Opoku, Danuta Mizgalska, Anna M. Lasica, Magdalena Nowak, Michael A. Curtis, Jan J. Enghild, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Fundació La Marató de TV3, National Science Centre (Poland), Ministry of Science and Higher Education (Poland), National Institutes of Health (US), and Medical Research Council (UK)
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biology ,Effector ,Chemistry ,Lipopolysaccharide ,Gingipains ,biology.organism_classification ,Ligand (biochemistry) ,Microbiology ,QR1-502 ,Lipid A ,Biochemistry ,Sortase ,Virology ,T9SS ,Secretion ,Bacterial outer membrane ,Porphyromonas gingivalis ,Mannan - Abstract
© 2021 Madej et al., Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface., This study was supported in part by grants from Spanish and Catalan public and private bodies (PID2019-107725RG-I00, 2017SGR3, and Fundació “La Marató de TV3” 201815 to F.X.G.-R.); National Science Centre, Poland grants UMO-2015/19/N/NZ1/ 00322 to M.M., UMO-2012/04/A/NZ1/00051 to J.P., and UMO-2016/23/N/NZ1/01513 to Z.N.; Polish Ministry of Science and Higher Education grant 1306/MOB/IV/2015/0 to M. K.; U.S. National Institutes of Health, NIDCR, grants DE09761 and DE026280 to J.P.; and Medical Research Council grant MR/P012175/2 to M.A.C.
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- 2021
13. Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia
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Ida B. Thøgersen, Krzysztof M. Zak, Mark J Bostock, Jan Potempa, Irena Waligorska, Jan J. Enghild, Grzegorz M Popowicz, Przemyslaw Grudnik, and Miroslaw Ksiazek
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MECHANISM ,PERIODONTAL HEALTH ,Proteases ,proteolysis ,medicine.medical_treatment ,Proteolysis ,protease inhibitors ,RM1-950 ,medicine.disease_cause ,ACTIVATION ,nmr-based fragment screening ,Forsythia ,GINGIPAINS ,Zymogen ,PORPHYROMONAS-GINGIVALIS ,Drug Discovery ,medicine ,Tannerella forsythia ,tannerella forsythia ,periodontitis ,SUPPRESSION ,Pharmacology ,chemistry.chemical_classification ,Mutation ,Protease ,biology ,medicine.diagnostic_test ,General Medicine ,biology.organism_classification ,PREVALENCE ,NMR-based fragment screening ,RED COMPLEX ,stomatognathic diseases ,Enzyme ,chemistry ,Biochemistry ,NMR-SPECTROSCOPY ,Therapeutics. Pharmacology ,VIRULENCE FACTORS - Abstract
Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a “cysteine-switch” mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1′ subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.
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- 2021
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14. The essential Porphyromonas gingivalis type IX secretion system component PorZ delivers anionic-lipopolysaccharide to the PorU sortase for transpeptidase processing of cargos
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Joseph Aduse-Opoku, A. Jacula, N. Magdalena, Danuta Mizgalska, Jan Potempa, Mariusz Madej, Jan J. Enghild, Michael A. Curtis, Carsten Scavenius, Miroslaw Ksiazek, Anna M. Lasica, Zuzanna Nowakowska, and F.X. Gomis-Ruth
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Lipid A ,Biochemistry ,biology ,Effector ,Sortase ,Chemistry ,Secretion ,Ligand (biochemistry) ,Bacterial outer membrane ,biology.organism_classification ,Porphyromonas gingivalis ,Mannan - Abstract
Cargo proteins of the type IX secretion system (T9SS) in human pathogens from phylum Bacteroidetes invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of selected cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ, and found that it consists of two β-propeller moieties followed by a CTD. In this study, we performed structure-based modelling suggesting that PorZ is a carbohydrate-binding protein. We found that recombinant PorZ specifically binds A-LPS. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of the A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS–dependent manner. This interaction on the cell surface is crucial for the function of the attachment complex composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in post-translational modification and retention of T9SS cargos on the bacterial surface.Author summaryBacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis. Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS– bound PorZ interacts with sortase with significantly greater affinity, facilitating modification of cargo proteins by the cell-surface attachment complex of the T9SS.
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- 2020
15. Structure-based mechanism of cysteine-switch latency and of catalysis by pappalysin-family metallopeptidases
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Miroslaw Ksiazek, Arturo Rodríguez-Banqueri, Jan Potempa, F.X. Gomis-Ruth, Tibisay Guevara, Generalitat de Catalunya, National Institutes of Health (US), Polish Academy of Sciences, Ministerio de Economía y Competitividad (España), Fundació La Marató de TV3, and Ministry of Science and Higher Education (Poland)
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Peridontopathogens ,Metallopeptidase ,mirolysin ,catalytic mechanisms ,Cleavage (embryo) ,pappalysin family ,Biochemistry ,03 medical and health sciences ,zymogens ,Zymogens ,Zymogen ,Hydrolase ,Mirolysin ,Tannerella forsythia ,General Materials Science ,Methanosarcina acetivorans ,Metallopeptidases ,lcsh:Science ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,peridontopathogens ,Catalytic mechanisms ,030302 biochemistry & molecular biology ,General Chemistry ,Condensed Matter Physics ,biology.organism_classification ,Research Papers ,metallopeptidases ,Pappalysin family ,Enzyme ,chemistry ,metallopeptidases ,lcsh:Q ,Cysteine - Abstract
Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54–Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Å revealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1′. Moreover, residue Cys23 within a conserved cysteine–glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Å structure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1′ to S6′. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms., This study was supported in part by grants from Spanish, Catalan, US American (NIH/NIDR) and Polish (NCN) public agencies (BFU2015-64487R; MDM-2014-0435; Fundacio´ ‘La Marato´ de TV3’ 201815 and 2017SGR3, 2015/17/B/NZ1/ 00666, 2016/21/B/NZ1/00292, and R21DE026280). MK was recipient of a scholarship from the Polish Ministry of Science and Higher Education (1306/MOB/IV/2015/0, ‘Mobilnoc´ Plus’). The Structural Biology Unit of IBMB was a ‘Marı´a de Maeztu’ Unit of Excellence of the Spanish Ministry of Science, Innovation and Universities (2015–2019).
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- 2020
16. An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva
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Jan Potempa, Miroslaw Ksiazek, Vibeke Baelum, Peter Durand Skottrup, Rodrigo López, Jakub Zbigniew Kaczmarek, and Peter Højrup
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0301 basic medicine ,Male ,Saliva ,virulence factor ,0302 clinical medicine ,Antibody Specificity ,Immunology and Allergy ,Tannerella forsythia ,periodontitis ,biology ,medicine.diagnostic_test ,Virulence ,Antibodies, Bacterial ,Titer ,biomarker ,Female ,Antibody ,Adolescent ,Virulence Factors ,Immunology ,Immunoglobulins ,Enzyme-Linked Immunosorbent Assay ,Article ,Microbiology ,03 medical and health sciences ,Immune system ,Forsythia ,Bacterial Proteins ,Predictive Value of Tests ,IgY ,medicine ,Humans ,Periodontitis ,saliva ,business.industry ,karilysin ,Reproducibility of Results ,biology.organism_classification ,medicine.disease ,Matrix Metalloproteinases ,030104 developmental biology ,inflammation ,Immunoassay ,Case-Control Studies ,biology.protein ,business ,Gram-Negative Bacterial Infections ,030215 immunology - Abstract
Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p = .0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499–2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210–1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.
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- 2019
17. Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis
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Jan Potempa, Maryta Sztukowska, Ky-Anh Nguyen, Miroslaw Ksiazek, John A. Houston, Yonghua Guo, Lahari Koneru, Florian Veillard, and Barbara Potempa
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Proteases ,Clinical Biochemistry ,Virulence ,Biology ,virulence factor ,Biochemistry ,Chromatography, Affinity ,Article ,law.invention ,Affinity chromatography ,law ,protein secretion ,Bacteroidaceae Infections ,Humans ,cysteine protease ,Adhesins, Bacterial ,periodontitis ,Molecular Biology ,Porphyromonas gingivalis ,posttranslational modification ,biology.organism_classification ,Cysteine protease ,Recombinant Proteins ,Gingipain ,Cysteine Endopeptidases ,proteolytic processing ,Gingipain Cysteine Endopeptidases ,Recombinant DNA ,Bacterial outer membrane - 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.
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- 2015
18. Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia, Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop
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Miroslaw Ksiazek, Ida B. Thøgersen, Jan J. Enghild, Carsten Scavenius, Danuta Mizgalska, and Jan Potempa
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Cathepsin G ,serine protease ,periodontal disease ,Gene Expression ,Biochemistry ,Substrate Specificity ,Serine ,chemistry.chemical_compound ,Catalytic Domain ,Tannerella forsythia ,Trypsin ,Reactive center ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,serpin ,Recombinant Proteins ,embryonic structures ,Thermodynamics ,Proteases ,animal structures ,Serine Proteinase Inhibitors ,infectious disease ,Molecular Sequence Data ,Serpin ,Microbiology ,protease inhibitor ,03 medical and health sciences ,Bacterial Proteins ,Escherichia coli ,Humans ,Periodontal Pocket ,Amino Acid Sequence ,Molecular Biology ,Serpins ,030304 developmental biology ,Serine protease ,Bacteroidetes ,Subtilisin ,Cell Biology ,biology.organism_classification ,carbohydrates (lipids) ,Kinetics ,chemistry ,Enzymology ,biology.protein ,Leukocyte Elastase ,Sequence Alignment - Abstract
All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10(4) (cathepsin G) to 7.1 × 10(5) m(-1)s(-1) (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1' site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.
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- 2015
19. A structure-derived snap-trap mechanism of a multispecific serpin from the dysbiotic human oral microbiome
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Marcin Wasylewski, Alicja Sochaj-Gregorczyk, Irena Waligorska, Theodoros Goulas, F. Xavier Gomis-Rüth, Miroslaw Ksiazek, Irene Garcia-Ferrer, Jan Potempa, European Synchrotron Radiation Facility, and ALBA Synchrotron
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0301 basic medicine ,Molecular biology ,medicine.medical_treatment ,Gingiva ,periodontal disease ,Proteinase ,Biochemistry ,Protein Structure, Secondary ,Protein structure ,Tannerella forsythia ,molecular biology ,Inhibition mechanism ,medicine.diagnostic_test ,Microbiota ,peptidase ,3. Good health ,Cell biology ,inhibitor ,Protein Synthesis and Degradation ,Horizontal gene transfer ,inhibition mechanism ,proteinase ,Periodontal disease ,proteolysis ,Inhibitor ,Proteolysis ,Biology ,Serpin ,Cleavage (embryo) ,protease inhibitor ,03 medical and health sciences ,Bacterial Proteins ,Hydrolase ,medicine ,Humans ,Peptidase ,protein structure ,Serpins ,Protease ,protease ,Cell Biology ,biology.organism_classification ,030104 developmental biology ,Protease inhibitor ,Dysbiosis ,Peptide Hydrolases - Abstract
Enduring host-microbiome relationships are based on adaptive strategies within a particular ecological niche. Tannerella forsythia is a dysbiotic member of the human oral microbiome that inhabits periodontal pockets and contributes to chronic periodontitis. To counteract endopeptidases from the host or microbial competitors, T. forsythia possesses a serpin-type proteinase inhibitor called miropin. Although serpins from animals, plants, and viruses have been widely studied, those from prokaryotes have received only limited attention. Here we show that miropin uses the serpin-type suicidal mechanism. We found that, similar to a snap trap, the protein transits from a metastable native form to a relaxed triggered or induced form after cleavage of a reactive-site target bond in an exposed reactive-center loop. The prey peptidase becomes covalently attached to the inhibitor, is dragged 75 Å apart, and is irreversibly inhibited. This coincides with a large conformational rearrangement of miropin, which inserts the segment upstream of the cleavage site as an extra-strand in a central-sheet. Standard serpins possess a single target bond and inhibit selected endopeptidases of particular specificity and class. In contrast, miropin uniquely blocked many serine and cysteine endopeptidases of disparate architecture and substrate specificity owing to several potential target bonds within the reactive-center loop and to plasticity in accommodating extra -strands of variable length. Phylogenetic studies revealed a patchy distribution of bacterial serpins incompatible with a vertical descent model. This finding suggests that miropin was acquired from the host through horizontal gene transfer, perhaps facilitated by the long and intimate association of T. forsythia with the human gingiva, We further acknowledge the help provided by local contacts at the European Synchrotron Radiation Facility and ALBA synchrotrons.
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- 2017
20. '''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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Jan Potempa, Frederike Wilkens, Christoph Becker-Pauly, Jörg W. Bartsch, Philipp Arnold, Miroslaw Ksiazek, Susanna Nikolaus, Stefan Rose-John, Barbara Potempa, Marit Stirnberg, Anna Ermund, Peter J. Dempsey, Rielana Wichert, Matthias Schweinlin, Marco Metzger, Philip Rosenstiel, Björn Rabe, Gunnar C. Hansson, Ralph Lucius, Maren Falk-Paulsen, Katharina Knittler, Stefanie Schmidt, and Publica
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0301 basic medicine ,Male ,metalloprotease ,medicine.medical_treatment ,Cell ,Mice, Transgenic ,Mucin 2 ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,03 medical and health sciences ,mucus ,Organoid ,medicine ,intestinal mucus barrier ,Animals ,Humans ,Amino Acid Sequence ,Adhesins, Bacterial ,lcsh:QH301-705.5 ,Metalloproteinase ,Mucin-2 ,host-microbiome interaction ,Protease ,030102 biochemistry & molecular biology ,Protein ,Cell Membrane ,Metalloendopeptidases ,Epithelial Cells ,Mucus ,Cysteine protease ,Cell biology ,Cysteine Endopeptidases ,030104 developmental biology ,medicine.anatomical_structure ,HEK293 Cells ,lcsh:Biology (General) ,Cell culture ,Gingipain Cysteine Endopeptidases ,Metalloproteases ,Female ,ectodomain shedding ,Schleim - Abstract
Summary 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.
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- 2017
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21. Structure of the catalytic domain of theTannerella forsythiamatrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor
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Jan Potempa, Núria Cerdà-Costa, Peter Durand Skottrup, Tibisay Guevara, Iñaki de Diego, S. Trillo-Muyo, F. Xavier Gomis-Rüth, Miroslaw Ksiazek, and Erik Riise
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Phage display ,Metallopeptidase ,Stereochemistry ,Biophysics ,Peptide ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Biochemistry ,metalloproteinases ,Serine ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Structural Biology ,Catalytic Domain ,Genetics ,Consensus sequence ,Structural Communications ,Tannerella forsythia ,Inhibition ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Tetrapeptide ,Bacteroidetes ,030206 dentistry ,Condensed Matter Physics ,biology.organism_classification ,Metalloproteinases ,Matrix Metalloproteinases ,inhibition ,3. Good health ,chemistry ,peptides ,Peptides ,Oligopeptides ,Protein Binding - Abstract
5 páginas, 1 figura, 1 tabla.-- et al., Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule. © 2013., This study was supported in part by grants from European, American, Polish, Spanish, Danish and Catalan agencies (2012/04/A/NZ1/00051, 2011/03/N/NZ1/00586, 2137/7.PR-EU/2011/2, DE09761, FP7-HEALTH-F3-2009-223101 ‘AntiPathoGN’, FP7-HEALTH-2010-261460 ‘Gums&Joints’, FP7-PEOPLE-2011-ITN-290246 ‘RAPID’, BIO2009-10334, BFU2012-32862, CSD2006-00015, Lundbeck Foundation grant R54-A5291 and Fundació ‘La Marató de TV3’ grants 2009-100732 and 2009SGR1036). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University in Kraków (Poland) is a beneficiary of structural funds from the European Union (grant No POIG.02.01.00-12-064/08 ‘Molecular Biotechnology for Health’).
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- 2013
22. Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases
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Florian Veillard, Miroslaw Ksiazek, Jan Potempa, Barbara Potempa, F. Xavier Gomis-Rüth, Ky-Anh Nguyen, Ida B. Thøgersen, Danuta Mizgalska, John A. Houston, Jan J. Enghild, and Maryta Sztukowska
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Proteases ,Inhibitor ,Glycosylation ,proteolysis control ,Biophysics ,Virulence ,Cysteine Proteinase Inhibitors ,Biochemistry ,Article ,Microbiology ,03 medical and health sciences ,Enzyme activator ,stomatognathic system ,Secretion ,Periodontitis ,zymogen activation ,Adhesins, Bacterial ,periodontitis ,Molecular Biology ,Porphyromonas gingivalis ,Cellular compartment ,030304 developmental biology ,Zymogen activation ,0303 health sciences ,biology ,Pathogen ,030306 microbiology ,biology.organism_classification ,Peptide Fragments ,Recombinant Proteins ,Protein Structure, Tertiary ,3. Good health ,inhibitor ,Enzyme Activation ,Cysteine Endopeptidases ,stomatognathic diseases ,Proteolysis control ,Gingipain Cysteine Endopeptidases ,pathogen - Abstract
et al., [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]: PD RgpB and PDRgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2 nM to 0.85 nM). In contrast, PDKgp showed no inhibitory activity. A conserved Arg-102 residue in PDRgpB and PDRgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PD RgpB 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. © 2013 Elsevier B.V., This study was supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, 2011/03/N/NZ1/00586, 2137/7.PR-EU/2011/2, DE09761, FP7-HEALTH-F3-2009-223101 “AntiPathoGN”; FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; BIO2009-10334; BFU2012-32862; CSD2006-00015; Fundació “La Marató de TV3” grant 2009-100732; and 2009SGR1036).
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- 2013
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23. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain
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Danuta Mizgalska, B. Szmigielski, Magdalena Nowak, Zuzanna Nowakowska, F. Xavier Gomis-Rüth, Jan J. Enghild, Jan Potempa, Ida B. Thøgersen, Przemyslaw Golik, Miroslaw Ksiazek, Barbara Potempa, Jill Trewhella, Iñaki de Diego, Ky-Anh Nguyen, Grzegorz Dubin, Lahari Koneru, John A. Houston, Ann H. Kwan, Jinlong Gao, National Science Centre (Poland), Ministerio de Economía y Competitividad (España), Generalitat de Catalunya, and National Science Foundation (US)
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Models, Molecular ,0301 basic medicine ,030106 microbiology ,Immunoglobulins ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,Bacterial Proteins ,Sortase ,Scattering, Small Angle ,Secretion ,Amino Acid Sequence ,Structural motif ,Bacterial Secretion Systems ,Porphyromonas gingivalis ,Conserved Sequence ,Nuclear Export Signals ,Genetics ,Binding Sites ,Multidisciplinary ,biology ,biology.organism_classification ,3. Good health ,Cell biology ,Gingipain ,Protein Transport ,Secretory protein ,CTD ,Bacterial outer membrane - Abstract
Iñaki de Diego et al., In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway., This study was financially supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, UMO-2012/05/B/NZ6/00581, UMO-2013/08/W/NZ1/00696, UMO-2011/01/D/NZ1/01169, 2975/7.PR/13/2014/2, NIH NIDCR DE09761; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; BIO2013-49320-EXP; MDM-2014-0435; 1306/MOB/IV/2015/0 (“Mobilność Plus” MK) and 2014SGR9). The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. Funding for data collection was provided in part by ESRF
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- 2016
24. Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis
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Iñaki de Diego, Apoorv Goel, Maryta Sztukowska, Ky-Anh Nguyen, Yonghua Guo, Monika Bzowska, Anna M. Lasica, Arkadiusz W. Kulczyk, Mary Simonian, Magdalena Nowak, Theodoros Goulas, Xiaoyan Zhou, Danuta Mizgalska, Jan Potempa, Barbara Potempa, Magdalena Widziolek, Mariusz Madej, Jan J. Enghild, Tibisay Guevara, F. Xavier Gomis-Rüth, Miroslaw Ksiazek, Apurva T. Prabhakar, and Ida B. Thøgersen
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0301 basic medicine ,030106 microbiology ,Protein domain ,Biology ,Crystallography, X-Ray ,Protein Structure, Secondary ,Article ,03 medical and health sciences ,Protein structure ,Bacterial Proteins ,Protein Domains ,Sortase ,Escherichia coli ,Humans ,Secretion ,Amino Acid Sequence ,Adhesins, Bacterial ,Bacterial Secretion Systems ,Porphyromonas gingivalis ,X-ray crystallography ,Mouth ,Multidisciplinary ,Pigmentation ,Microbiota ,Cell Membrane ,Bacteriology ,biology.organism_classification ,3. Good health ,Cell biology ,Transport protein ,Cysteine Endopeptidases ,Phenotype ,030104 developmental biology ,Gingipain Cysteine Endopeptidases ,Protein-Arginine Deiminases ,CTD ,Pathogens ,Bacterial outer membrane ,Protein Processing, Post-Translational ,Gene Deletion ,Subcellular Fractions - Abstract
Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two ß-propeller domains and a C-terminal ß-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.
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- 2016
25. A Metalloproteinase Karilysin Present in the Majority of Tannerella forsythia Isolates Inhibits All Pathways of the Complement System
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Miroslaw Ksiazek, Sigrun Eick, Monika Jusko, Kristian Riesbeck, Anna M. Blom, Abdulkarim Yasin Karim, Peter Garred, and Jan Potempa
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0303 health sciences ,biology ,Immunology ,030206 dentistry ,Complement factor I ,biology.organism_classification ,Virulence factor ,3. Good health ,Complement system ,Microbiology ,stomatognathic diseases ,03 medical and health sciences ,0302 clinical medicine ,Forsythia ,Complement Inactivator Proteins ,Immunology and Allergy ,Tannerella forsythia ,Complement membrane attack complex ,Opsonin ,030304 developmental biology - Abstract
Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways, T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low-expressing strains. Furthermore, the low-expressing strain was significantly more opsonized with activated complement factor 3 and membrane attack complex from serum compared with the other strains. The high-expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited because of the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4 by karilysin, whereas inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in >90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion.
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- 2012
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26. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenologue of animal matrix metalloproteinases
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Jan Potempa, Miroslaw Ksiazek, Tibisay Guevara, Ky-Anh Nguyen, Núria Cerdà-Costa, Abdulkarim Yasin Karim, Joan L. Arolas, and F. Xavier Gomis-Rüth
- Subjects
0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Genetic transfer ,Sequence alignment ,Plasma protein binding ,biology.organism_classification ,Microbiology ,03 medical and health sciences ,Biochemistry ,Horizontal gene transfer ,Tannerella forsythia ,Binding site ,Molecular Biology ,Peptide sequence ,Gene ,030304 developmental biology - Abstract
Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52-kDa multidomain protein comprising a central 18-kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analyzed the 3D structure of Kly18 in the absence and presence of Mg 2+ or Ca 2+ , which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg 2+ or Ca 2+ binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally-characterized non-mammalian MMP is a xenolog co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs.
- Published
- 2010
27. Proteolytic Inactivation of LL-37 by Karilysin, a Novel Virulence Mechanism of Tannerella forsythia
- Author
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Jan Potempa, Aabdulkarim Y. Karim, Miroslaw Ksiazek, Joanna Koziel, Kornelia Przybyszewska, Maria Rapala-Kozik, and Ky-Anh Nguyen
- Subjects
Metalloproteinase ,Innate immune system ,biology ,medicine.medical_treatment ,Virulence ,biology.organism_classification ,Microbiology ,Cathelicidin ,stomatognathic diseases ,Forsythia ,medicine ,Immunology and Allergy ,Tannerella forsythia ,Bacteroides ,Bacteria - Abstract
Tannerella forsythia is a gram-negative bacterium strongly associated with the development and/or progression of periodontal disease. Here, we have shown that a newly characterized matrix metalloprotease-like enzyme, referred to as karilysin, efficiently cleaved the antimicrobial peptide LL-37, significantly reducing its bactericidal activity. This may contribute to the resistance of T. forsythia to the antibacterial activity of LL-37, since their vitality was found not to be affected by LL-37 at concentrations up to 2.2 µM. Furthermore, proteolysis of LL-37 by karilysin not only abolished its ability to bind lipopolysaccharide (LPS) to quench endotoxin-induced proinflammatory activity, but LL-37 cleavage also caused the release of active endotoxin from the LPS/LL-37 complex. Proteolytic inactivation of LL-37 bactericidal activity by karilysin may protect LL-37-sensitive species in the subgingival plaque and maintain the local inflammatory reaction driven by LPS from gram-negative bacteria. Consequently, the karilysin protease may directly contribute to periodontal tissue damage and the development and/or progression of chronic periodontitis.
- Published
- 2010
28. Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase*
- Author
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Aneta Sroka, Jan J. Enghild, Ida B. Thøgersen, Jan Potempa, Dominika Staniec, Matthew Bogyo, Magnus Abrahamson, Danuta Bryzek, and Miroslaw Ksiazek
- Subjects
Proteolysis ,Molecular Sequence Data ,periodontal disease ,virulence factor ,Biochemistry ,Microbiology ,Substrate Specificity ,calcium-dependent activity ,Bacterial Proteins ,Cathelicidins ,Zymogen ,Catalytic Domain ,Endopeptidases ,Enzyme Stability ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Porphyromonas gingivalis ,Peptide sequence ,calcium ,biology ,medicine.diagnostic_test ,Calpain ,Caseins ,Fibrinogen ,Cell Biology ,enzyme processing ,biology.organism_classification ,Recombinant Proteins ,Complement system ,Fibronectins ,Kinetics ,Genes, Bacterial ,biology.protein ,Mutagenesis, Site-Directed ,Calcium ,Cattle ,calpain ,Bacterial outer membrane ,Cysteine ,Antimicrobial Cationic Peptides - Abstract
Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease), which has sequence similarity to cysteine peptidases of the papain and calpain families. In this study, we biochemically characterize Tpr. We found that the 55 kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48 kDa, 37 kDa, and 33 kDa via sequential truncations at the N-terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane, where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations greater than 1 mM, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4 and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.
- Published
- 2015
29. KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure
- Author
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Danuta Mizgalska, Sigrum Eick, Miroslaw Ksiazek, Ida B. Thøgersen, Jan Potempa, and Jan J. Enghild
- Subjects
Microbiology (medical) ,Proteases ,infectious disease ,medicine.medical_treatment ,periodontal disease ,lcsh:QR1-502 ,Virulence ,Infectious Disease ,610 Medicine & health ,Microbiology ,lcsh:Microbiology ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Forsythia ,law ,medicine ,Tannerella forsythia ,Zymography ,Gene ,Original Research ,030304 developmental biology ,0303 health sciences ,Protease ,biology ,030206 dentistry ,biology.organism_classification ,virulence ,stomatognathic diseases ,Recombinant DNA ,proteases ,Public Health ,Periodontal disease - Abstract
Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.
- Published
- 2015
30. A novel mechanism of latency in matrix metalloproteinases
- Author
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F. Xavier Gomis-Rüth, Joan L. Arolas, Mar López-Pelegrín, Abdulkarim Yasin Karim, Tibisay Guevara, Jan Potempa, Miroslaw Ksiazek, and European Commission
- Subjects
Signal peptide ,Protein Folding ,Architecture domain ,Metallopeptidase ,Gene Transfer, Horizontal ,Bacteroidaceae ,Matrix metalloproteinase ,Biology ,X - ray crystal structure ,Biochemistry ,digestive system ,Conserved sequence ,Evolution, Molecular ,Periodontal Disease ,X-ray Crystallography ,Bacterial Proteins ,Protein Evolution ,matrixin ,Humans ,Peptidase ,Cysteine ,Protein precursor ,Periodontitis ,Molecular Biology ,Matrix Metalloproteinase (MMP) ,MMP ,food and beverages ,Cell Biology ,pro - domain ,Matrix Metalloproteinases ,Cell biology ,Protein Structure, Tertiary ,Protease ,Enzymology ,Protein folding ,microbial infection ,Astacin - Abstract
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Background: Animal and plant matrix metalloproteinases (MMPs) are kept zymogenic through large prodomains and a cysteine-switch mechanism., This work was supported in part by European, United States American, Polish, Spanish, and Catalan Grants UMO-2012/04/A/NZ1/00051, UMO-2013/08/T/NZ1/00315, 2137/7.PR-EU/2011/2, 2975/7.PR/13/2014/2, DE09761, DE022597, FP7-HEALTH-2010-261460 “Gums&Joints,” FP7-PEOPLE-2011-ITN-290246 “RAPID,” FP7-HEALTH-2012-306029-2 “TRIGGER,” BFU2012-32862, BIO2013-49320-EXP, CSD2006-00015, and 2014SGR9)
- Published
- 2015
31. A pathogenic trace of Tannerella forsythia – shedding of soluble fully active tumor necrosis factor alpha from the macrophages surface by karilysin
- Author
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Joanna Koziel, Danuta Bryzek, Miroslaw Ksiazek, Ewa Bielecka, Jan Potempa, Abdulkarim Yasin Karim, Barbara Potempa, and Dominika Staniec
- Subjects
Microbiology (medical) ,Proteases ,Virulence Factors ,medicine.medical_treatment ,Immunology ,Apoptosis ,Matrix metalloproteinase ,ADAM17 Protein ,Real-Time Polymerase Chain Reaction ,Microbiology ,Virulence factor ,Article ,Cell Line ,03 medical and health sciences ,Bacterial Proteins ,medicine ,Tannerella forsythia ,Humans ,Secretion ,Periodontitis ,General Dentistry ,periodontitis ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Bacteroidetes ,Protein Stability ,Tumor Necrosis Factor-alpha ,Macrophages ,karilysin ,Sheddase ,biology.organism_classification ,Matrix Metalloproteinases ,macrophages ,ADAM Proteins ,Cytokine ,Cytokines ,Tumor necrosis factor alpha ,tumor necrosis factor-α - Abstract
Summary Tannerella forsythia is implicated as a pathogen causing chronic and aggressive periodontitis. However, its virulence factors, including numerous putative proteases, are mostly uncharacterized. Karilysin is a newly described matrix metalloprotease-like enzyme of T. forsythia. Since pathogen-derived proteases may affect the host defense system via modulation of the cytokine network, the aim of this study was to determine the influence of karilysin on tumor necrosis factor-α (TNF-α). The results showed that karilysin cleaved the membrane form of TNF-α on the surface of macrophages, and that this led to an increased concentration of soluble TNF-α in the conditioned medium. Importantly, despite partial degradation of soluble TNF-α by karilysin, the released cytokine retained its biological activity, inducing apoptosis and stimulating autocrine pathway of pro-inflammatory gene expression. Notably, the observed effect required proteolytic activity by karilysin, since a catalytically inactive mutant of the enzyme did not affect TNF-α secretion. The shedding was independent of the activity of ADAM17, a major endogenous TNF-α converting enzyme. Karilysin-dependent TNF-α release from the cell surface is likely to occur in vivo because human plasma, the main constituent of gingival crevicular fluid, only slightly affected the sheddase activity of karilysin. Taken together, these results indicate that karilysin modulates the host immune response through regulation of TNF-α secretion, and should therefore be considered as a new virulence factor of T. forsythia.
- Published
- 2014
32. A metalloproteinase mirolysin of Tannerella forsythia inhibits all pathways of the complement system
- Author
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Kristian Riesbeck, Monika Jusko, Peter Garred, Miroslaw Ksiazek, Anna M. Blom, Jan Potempa, and Sigrun Eick
- Subjects
Metalloproteinase ,biology ,Chemistry ,Immunology ,Tannerella forsythia ,biology.organism_classification ,Molecular Biology ,Complement system ,Microbiology - Published
- 2013
33. A Phage Display Selected 7-mer Peptide Inhibitor of the Tannerella forsythia Metalloprotease-Like Enzyme Karilysin can be Truncated to Ser-Trp-Phe-Pro
- Author
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Erik Riise, Jan Potempa, Miroslaw Ksiazek, Peter Durand Skottrup, and Grete Sørensen
- Subjects
Bacterial Diseases ,Models, Molecular ,Phage display ,Protein Conformation ,Amino Acid Motifs ,lcsh:Medicine ,Peptide ,Biochemistry ,Serine ,chemistry.chemical_compound ,Maltose-binding protein ,0302 clinical medicine ,Catalytic Domain ,Drug Discovery ,Peptide synthesis ,Gram Negative ,Tannerella forsythia ,Enzyme Inhibitors ,lcsh:Science ,Peptide sequence ,chemistry.chemical_classification ,Escherichia Coli ,0303 health sciences ,Multidisciplinary ,biology ,Bacterial Pathogens ,Kinesis ,Host-Pathogen Interaction ,Infectious Diseases ,Prokaryotic Models ,Medicine ,Periodontal Abscesses ,Research Article ,Protein Binding ,Drugs and Devices ,Drug Research and Development ,Recombinant Fusion Proteins ,Microbiology ,03 medical and health sciences ,Model Organisms ,Bacterial Proteins ,Peptide Library ,Amino Acid Sequence ,Peptide library ,Biology ,030304 developmental biology ,Bacteroidetes ,lcsh:R ,030206 dentistry ,biology.organism_classification ,Molecular biology ,Matrix Metalloproteinases ,Solubility ,chemistry ,Proteolysis ,biology.protein ,lcsh:Q ,Peptides - Abstract
Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.
- Published
- 2012
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