Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100263
ISSN1083-351X
AutoresNadine Taudte, Miriam Linnert, Jens‐Ulrich Rahfeld, Anke Piechotta, Daniel Ramsbeck, Mirko Buchholz, Petr Kolenko, C. Parthier, John A. Houston, Florian Veillard, Sigrun Eick, Jan Potempa, Stephan Schilling, Hans‐Ulrich Demuth, Milton T. Stubbs,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases. The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases. 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Currently, periodontitis is treated by nonsurgical therapy such as debridement and the application of adjunctive antimicrobials, for example, chlorhexidine and the antibiotics minocycline, doxycycline, amoxicillin, or metronidazole (19Smiley C.J. Tracy S.L. Abt E. Michalowicz B.S. John M.T. Gunsolley J. Cobb C.M. Rossmann J. Harrel S.K. Forrest J.L. Hujoel P.P. Noraian K.W. Greenwell H. Frantsve-Hawley J. Estrich C. et al.Systematic review and meta-analysis on the nonsurgical treatment of chronic periodontitis by means of scaling and root planing with or without adjuncts.J. Am. Dent. Assoc. 2015; 146: 508-524.e5Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Matthews D. Local antimicrobials in addition to scaling and root planing provide statistically significant but not clinically important benefit.Evid. Based Dent. 2013; 14: 87-88Crossref PubMed Scopus (5) Google Scholar). As general antibiotic therapies can involve the risk of development of antibiotic-resistance bacteria and destruction of the host microbiome (leading in turn to a loss of metabolic support, immune modulation, and enabling recolonization by potential pathogens), an alternative therapy would be desirable, such as the selective small-molecule inhibition of a physiologically relevant bacterial enzyme. The glutaminyl cyclase (QC) from P. gingivalis (PgQC), identified recently using sophisticated proteomic analyses (21Bochtler M. Mizgalska D. Veillard F. Nowak M.L. Houston J. Veith P. Reynolds E.C. Potempa J. The Bacteroidetes Q-rule: Pyroglutamate in signal peptidase I substrates.Front. Microbiol. 2018; 9: 230Crossref PubMed Scopus (9) Google Scholar), represents such an attractive target. QCs belong to the family of aminoacyltransferases and catalyze the cyclization of N-terminal glutamine/glutamate residues of peptides and proteins with concomitant release of ammonia/water (Fig. 1A). They are of widespread distribution and can be found in mammals (22Busby W.H. Quackenbush G.E. Humm J. Youngblood W.W. Kizer J.S. An enzyme(s) that converts glutaminyl-peptides into pyroglutamyl-peptides. Presence in pituitary, brain, adrenal medulla, and lymphocytes.J. Biol. Chem. 1987; 262: 8532-8536Abstract Full Text PDF PubMed Google Scholar, 23Pohl T. Zimmer M. Mugele K. Spiess J. Primary structure and functional expression of a glutaminyl cyclase.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10059-10063Crossref PubMed Scopus (82) Google Scholar), bacteria (24Huang W.L. Wang Y.R. Ko T.P. Chia C.Y. Huang K.F. Wang A.H. Crystal structure and functional analysis of the glutaminyl cyclase from Xanthomonas campestris.J. Mol. Biol. 2010; 401: 374-388Crossref PubMed Scopus (17) Google Scholar, 25Carrillo D.R. Parthier C. Jänckel N. Grandke J. Stelter M. Schilling S. Boehme M. Neumann P. Wolf R. Demuth H.U. Stubbs M.T. Rahfeld J.U. 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The physiological role and targets of type I QCs are not fully known, whereas the type II human QC (HsQC) catalyzes the N-terminal pyroglutamate formation of the chemokines CX3CL1 (fractalkine) and CCL2 (MCP-1, monocyte chemoattractant protein-1), of hormones such as thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), neurotensin, and gastrin and of collagen or fibronectin. In humans, introduction of this posttranslational modification has been shown to be essential for stability against N-terminal degradation and for modulation of receptor binding (23Pohl T. Zimmer M. Mugele K. Spiess J. Primary structure and functional expression of a glutaminyl cyclase.Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10059-10063Crossref PubMed Scopus (82) Google Scholar, 30Schilling S. Wasternack C. Demuth H.U. Glutaminyl cyclases from animals and plants: A case of functionally convergent protein evolution.Biol. 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Graubner S. et al.Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β.Nature. 2012; 485: 651-655Crossref PubMed Scopus (286) Google Scholar). In addition to these physiological substrates, HsQC also cyclizes truncated Glu3-Aβ peptide, generating pGlu3-Aβ, a significant component of Aβ plaques in AD brains (34Schilling S. Zeitschel U. Hoffmann T. Heiser U. Francke M. Kehlen A. Holzer M. Hutter-Paier B. Prokesch M. Windisch M. Jagla W. Schlenzig D. Lindner C. Rudolph T. Reuter G. et al.Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology.Nat. Med. 2008; 14: 1106-1111Crossref PubMed Scopus (268) Google Scholar). The cyclization of the N-terminal residue increases the stability, hydrophobicity, aggregation potential, and thereby toxicity of the peptide (35Nussbaum J.M. Schilling S. Cynis H. Silva A. Swanson E. Wangsanut T. Tayler K. Wiltgen B. Hatami A. Rönicke R. Reymann K. Hutter-Paier B. Alexandru A. Jagla W. 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Surprisingly, putative QC open reading frames (ORFs) from the order Bacteroidales, which includes the families Porphyromonadaceae, Bacteroidaceae and Prevotellaceae and the oral pathogens P. gingivalis, T. forsythia, and Prevotella intermedia, would appear to belong to type II ("animal"-type) QCs (21Bochtler M. Mizgalska D. Veillard F. Nowak M.L. Houston J. Veith P. Reynolds E.C. Potempa J. The Bacteroidetes Q-rule: Pyroglutamate in signal peptidase I substrates.Front. Microbiol. 2018; 9: 230Crossref PubMed Scopus (9) Google Scholar). Primary sequences of these gene products exhibit 25% (PgQC) or 23% (TfQC and PiQC) sequence identity to human glutaminyl cyclase (HsQC), including residues corresponding to the highly conserved metal binding motif in mammalian QCs (Asp159, Glu202, and His330 in HsQC) (32Huang K.F. Liu Y.L. Cheng W.J. Ko T.P. Wang A.H. Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13117-13122Crossref PubMed Scopus (78) Google Scholar); the zinc ligand HsGlu202 is replaced by a conserved Asp residue (PgAsp183) in the bacteroidal sequences. In addition, the latter possess counterparts to residues assumed to be involved in catalysis (HsGlu201 and HsAsp248) and to line the active site (HsPhe325 and HsTrp329). Further analyses indicated the presence of an N-terminal lipid anchor (37Kovacs-Simon A. Titball R.W. Michell S.L. Lipoproteins of bacterial pathogens.Infect. Immun. 2011; 79: 548-561Crossref PubMed Scopus (263) Google Scholar), consistent with the authors' demonstration that PgQC localizes to the inner periplasmatic membrane, which in turn is supported by freeze-fracture replica immunolabeling (FRIL) electron microscopy (38Bender P. Egger A. Westermann M. Taudte N. Sculean A. Potempa J. Möller B. Buchholz M. Eick S. Expression of human and Porphyromonas gingivalis glutaminyl cyclases in periodontitis and rheumatoid arthritis-A pilot study.Arch. Oral Biol. 2019; 97: 223-230Crossref PubMed Scopus (12) Google Scholar). It is thought that the enzyme catalyzes the cyclization of N-terminal glutamine residues of periplasmic, outer membrane integrated, and extracellular proteins after their translocation into the periplasm and subsequent removal of the signal peptides by the SP I signal peptidase (21Bochtler M. Mizgalska D. Veillard F. Nowak M.L. Houston J. Veith P. Reynolds E.C. Potempa J. The Bacteroidetes Q-rule: Pyroglutamate in signal peptidase I substrates.Front. Microbiol. 2018; 9: 230Crossref PubMed Scopus (9) Google Scholar, 39Veith P.D. Nor Muhammad N.A. Dashper S.G. Likić V.A. Gorasia D.G. Chen D. Byrne S.J. Catmull D.V. Reynolds E.C. Protein substrates of a novel secretion system are numerous in the Bacteroidetes phylum and have in common a cleavable C-terminal secretion signal, extensive post-translational modification, and cell-surface attachment.J. Proteome Res. 2013; 12: 4449-4461Crossref PubMed Scopus (74) Google Scholar, 40Gorasia D.G. Veith P.D. Chen D. Seers C.A. Mitchell H.A. Chen Y.Y. Glew M.D. Dashper S.G. Reynolds E.C. Porphyromonas gingivalis type IX secretion substrates are cleaved and modified by a sortase-like mechanism.PLoS Pathog. 2015; 11e1005152Crossref PubMed Scopus (41) Google Scholar), which could stabilize substrate proteins by protecting them against proteolytic degradation by periplasmatic and host cell aminopeptidases. As such protein modifications could be important for the survival of P. gingivalis (by ensuring nutrient acquisition from the host, facilitating response to environmental changes, and/or delivering virulence factors), the catalytic action of QC may be beneficial for the overall physiological fitness of P. gingivalis. This is supported by saturation mariner transposon insertion sequencing of the genome of P. gingivalis ATCC 33277 by two independent groups, which identified PgQC (PGN_0202) as one of 281 candidate essential genes (from ∼117.000 TA sites distributed randomly over 2155 genes in the whole genome) (41Hutcherson J.A. Gogeneni H. Yoder-Himes D. Hendrickson E.L. Hackett M. Whiteley M. Lamont R.J. Scott D.A. Comparison of inherently essential genes of Porphyromonas gingivalis identified in two transposon-sequencing libraries.Mol. Oral Microbiol. 2016; 31: 354-364Crossref PubMed Scopus (17) Google Scholar, 42Klein B.A. Tenorio E.L. Lazinski D.W. Camilli A. Duncan M.J. Hu L.T. Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis.BMC Genomics. 2012; 13: 578Crossref PubMed Scopus (100) Google Scholar). Thus, the QC of P. gingivalis and other oral pathogens represent attractive targets for small-molecule inhibitor development for the treatment or prevention of chronic periodontitis. In this study, we characterize and compare enzymatic properties of three different bacteroidal QCs: from P. gingivalis (PgQC), T. forsythia (TfQC), and P. intermedia (PiQC), all bacteria that are strongly associated with periodontal disease (2Socransky S.S. Haffajee A.D. Cugini M.A. Smith C. Kent R.L. Microbial complexes in subgingival plaque.J. Clin. Periodontol. 1998; 25: 134-144Crossref PubMed Scopus (2831) Google Scholar). The crystal structures of PgQC and TfQC at 2.8 and 2.1 Å resolution clearly define them as type II QCs, with notable differences to their animal counterparts. These differences allow for the development of specific inhibitors of the bacteroidal enzymes, and we demonstrate with one such PgQC inhibitor the successful inhibition of bacterial growth in a dose-dependent manner. Together, these data provide an excellent starting point for structure-based development of selectiv
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