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Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum


Autoři: Zoltán Attila Nagy aff001;  Dávid Szakács aff001;  Eszter Boros aff001;  Dávid Héja aff001;  Eszter Vígh aff001;  Noémi Sándor aff003;  Mihály Józsi aff003;  Gábor Oroszlán aff004;  József Dobó aff004;  Péter Gál aff004;  Gábor Pál aff001
Působiště autorů: Department of Biochemistry, ELTE, Eötvös Loránd University, Budapest, Hungary aff001;  Department of Nephrology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America aff002;  Department of Immunology, ELTE, Eötvös Loránd University, Budapest, Hungary aff003;  Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary aff004
Vyšlo v časopise: Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum. PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1008232
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008232

Souhrn

Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.

Klíčová slova:

Bacterial pathogens – Complement system – Enzyme inhibitors – Flow cytometry – Proteases – Pseudomonas aeruginosa – Complement inhibitors – Yersinia pestis


Zdroje

1. Chung C. H., Ives H. E., Almeda S. & Goldberg A. L. Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. J. Biol. Chem. 258, 11032–11038 (1983). 6411724

2. Schechter I. & Berger A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157–162 (1967). doi: 10.1016/s0006-291x(67)80055-x 6035483

3. McGrath M. E., Erpel T., Bystroff C. & Fletterick R. J. Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex. EMBO J. 13, 1502–1507 (1994). 8156987

4. Yang S. Q., Wang C. I., Gillmor S. A., Fletterick R. J. & Craik C. S. Ecotin: a serine protease inhibitor with two distinct and interacting binding sites. J. Mol. Biol. 279, 945–957 (1998). doi: 10.1006/jmbi.1998.1748 9642073

5. Eggers C. T., Wang S. X., Fletterick R. J. & Craik C. S. The role of ecotin dimerization in protease inhibition. J. Mol. Biol. 308, 975–991 (2001). doi: 10.1006/jmbi.2001.4754 11352586

6. Seymour J. L. et al. Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. Biochemistry 33, 3949–3958 (1994). doi: 10.1021/bi00179a022 8142399

7. Ulmer J. S., Lindquist R. N., Dennis M. S. & Lazarus R. A. Ecotin is a potent inhibitor of the contact system proteases factor XIIa and plasma kallikrein. FEBS Lett. 365, 159–163 (1995). doi: 10.1016/0014-5793(95)00466-m 7781771

8. Clark E. A. et al. Molecular Recognition of Chymotrypsin by the Serine Protease Inhibitor Ecotin from Yersinia pestis. J. Biol. Chem. 286, 24015–24022 (2011). doi: 10.1074/jbc.M111.225730 21531711

9. Ireland P. M., Marshall L., Norville I. & Sarkar-Tyson M. The serine protease inhibitor Ecotin is required for full virulence of Burkholderia pseudomallei. Microb. Pathog. 67–68, 55–58 (2014). doi: 10.1016/j.micpath.2014.01.001 24462575

10. Eschenlauer S. C. P. et al. Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major. Cell. Microbiol. 11, 106–120 (2009). doi: 10.1111/j.1462-5822.2008.01243.x 19016791

11. Eggers C. T., Murray I. A., Delmar V. A., Day A. G. & Craik C. S. The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase. Biochem. J. 379, 107–118 (2004). doi: 10.1042/BJ20031790 14705961

12. Ricklin D., Hajishengallis G., Yang K. & Lambris J. D. Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785–797 (2010). doi: 10.1038/ni.1923 20720586

13. Héja D. et al. Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2. Proc. Natl. Acad. Sci. U. S. A. 109, 10498–10503 (2012). doi: 10.1073/pnas.1202588109 22691502

14. Dobó J. et al. MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked. Sci. Rep. 6, 31877 (2016). doi: 10.1038/srep31877 27535802

15. Merle N. S., Noe R., Halbwachs-Mecarelli L., Fremeaux-Bacchi V. & Roumenina L. T. Complement System Part II—Role in Immunity. Front. Immunol. 6, 257 (2015). doi: 10.3389/fimmu.2015.00257 26074922

16. Merle N. S., Church S. E., Fremeaux-Bacchi V. & Roumenina L. T. Complement System Part I—Molecular Mechanisms of Activation and Regulation. Front. Immunol. 6, 262 (2015). doi: 10.3389/fimmu.2015.00262 26082779

17. Pál G., Sprengel G., Patthy A. & Gráf L. Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis. FEBS Lett. 342, 57–60 (1994). doi: 10.1016/0014-5793(94)80584-9 8143850

18. Pál G., Szilágyi L. & Gráf L. Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis. FEBS Lett. 385, 165–170 (1996). doi: 10.1016/0014-5793(96)00376-6 8647243

19. Paréj K. et al. Cutting Edge: A New Player in the Alternative Complement Pathway, MASP-1 Is Essential for LPS-Induced, but Not for Zymosan-Induced, Alternative Pathway Activation. J. Immunol. 200, 2247–2252 (2018). doi: 10.4049/jimmunol.1701421 29475986

20. Szakács D., Kocsis A., Szász R., Gál P. & Pál G. Novel MASP-2 inhibitors developed via directed evolution of human TFPI1 are potent lectin pathway inhibitors. J. Biol. Chem. 294, 8227–8237 (2019). doi: 10.1074/jbc.RA119.008315 30952698

21. Héja D. et al. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 Are essential for lectin pathway activation and reveal structural plasticity of MASP-2. J. Biol. Chem. 287, 20290–20300 (2012). doi: 10.1074/jbc.M112.354332 22511776

22. Oroszlán G. et al. MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors. J. Immunol. 196, 857–865 (2016). doi: 10.4049/jimmunol.1501717 26673137

23. Gaboriaud C. et al. The Serine Protease Domain of MASP-3: Enzymatic Properties and Crystal Structure in Complex with Ecotin. PLoS ONE 8, (2013).

24. Lynch N. J. et al. Composition of the lectin pathway of complement in Gallus gallus: absence of mannan-binding lectin-associated serine protease-1 in birds. J. Immunol. Baltim. Md 1950 174, 4998–5006 (2005).

25. Nakao M. et al. Lectin pathway of bony fish complement: identification of two homologs of the mannose-binding lectin associated with MASP2 in the common carp (Cyprinus carpio). J. Immunol. Baltim. Md 1950 177, 5471–5479 (2006).

26. Nonaka M. & Kimura A. Genomic view of the evolution of the complement system. Immunogenetics 58, 701–713 (2006). doi: 10.1007/s00251-006-0142-1 16896831

27. Kocsis A. et al. Selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (MASP)-1 and -2: significant contribution of MASP-1 to lectin pathway activation. J. Immunol. 185, 4169–4178 (2010). doi: 10.4049/jimmunol.1001819 20817870

28. Tada R., Nagi-Miura N., Adachi Y. & Ohno N. An unambiguous assignment and structural analysis using solution NMR experiments of O-antigen from Escherichia coli ATCC23505 (Serotype O9). Chem. Pharm. Bull. (Tokyo) 55, 992–995 (2007). doi: 10.1248/cpb.55.992 17603187

29. Zhao L. et al. LPS-induced platelet response and rapid shock in mice: contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system. Blood 100, 3233–3239 (2002). doi: 10.1182/blood-2002-01-0252 12384422

30. Lindberg B., Lindh F. & Lönngren J. Structural studies of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O 55. Carbohydr. Res. 97, 105–112 (1981). doi: 10.1016/s0008-6215(00)80528-5 7030487

31. Inagi R. et al. FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a. Immunol. Lett. 27, 49–52 (1991). doi: 10.1016/0165-2478(91)90243-4 2019419

32. Harboe M. & Mollnes T. E. The alternative complement pathway revisited. J. Cell. Mol. Med. 12, 1074–1084 (2008). doi: 10.1111/j.1582-4934.2008.00350.x 18419792

33. Berends E. T. M., Mohan S., Miellet W. R., Ruyken M. & Rooijakkers S. H. M. Contribution of the complement Membrane Attack Complex to the bactericidal activity of human serum. Mol. Immunol. 65, 328–335 (2015). doi: 10.1016/j.molimm.2015.01.020 25725315

34. Fujimori Y. et al. Effects of a highly selective plasma kallikrein inhibitor on collagen-induced arthritis in mice. Agents Actions 39, 42–48 (1993). doi: 10.1007/bf01975713 8285139

35. Hansson K. M., Nielsen S., Elg M. & Deinum J. The effect of corn trypsin inhibitor and inhibiting antibodies for FXIa and FXIIa on coagulation of plasma and whole blood. J. Thromb. Haemost. JTH 12, 1678–1686 (2014). doi: 10.1111/jth.12707 25142753

36. Lambris J. D., Ricklin D. & Geisbrecht B. V. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142 (2008). doi: 10.1038/nrmicro1824 18197169

37. Zipfel P. F., Hallström T. & Riesbeck K. Human complement control and complement evasion by pathogenic microbes—tipping the balance. Mol. Immunol. 56, 152–160 (2013). doi: 10.1016/j.molimm.2013.05.222 23810413

38. Abreu A. G. & Barbosa A. S. How Escherichia coli Circumvent Complement-Mediated Killing. Front. Immunol. 8, (2017).

39. Stubben C. J. et al. Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens. BMC Genomics 10, 501 (2009). doi: 10.1186/1471-2164-10-501 19874620

40. Verma S. et al. Leishmania donovani Inhibitor of Serine Peptidases 2 Mediated Inhibition of Lectin Pathway and Upregulation of C5aR Signaling Promote Parasite Survival inside Host. Front. Immunol. 9, 63 (2018). doi: 10.3389/fimmu.2018.00063 29434593

41. Tseng B. S. et al. A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack. mBio 9, (2018).

42. Beloin C. et al. Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Mol. Microbiol. 51, 659–674. doi: 10.1046/j.1365-2958.2003.03865.x 14731270

43. Fung C. et al. Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum. J. Med. Microbiol. 59, 1089–1100 (2010). doi: 10.1099/jmm.0.019984-0 20522626

44. Costerton J. W., Stewart P. S. & Greenberg E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999). doi: 10.1126/science.284.5418.1318 10334980

45. Paula C. Lima A. & Mottram JC. Trypanosomatid-Encoded Inhibitors of Peptidases: Unique Structural Features and Possible Roles as Virulence Factors. Open Parasitol. J. 4, (2010).

46. Alam M. N., Das P., De T. & Chakraborti T. Identification and characterization of a Leishmania donovani serine protease inhibitor: Possible role in regulation of host serine proteases. Life Sci. 144, 218–225 (2016). doi: 10.1016/j.lfs.2015.12.004 26656469

47. Svensjö E., Nogueira de Almeida L., Vellasco L., Juliano L. & Scharfstein J. Ecotin-like ISP of L. major promastigotes fine-tunes macrophage phagocytosis by limiting the pericellular release of bradykinin from surface-bound kininogens: a survival strategy based on the silencing of proinflammatory G-protein coupled kinin B2 and B1 receptors. Mediators Inflamm. 2014, 143450 (2014). doi: 10.1155/2014/143450 25294952

48. Verma S. et al. Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut. Parasit. Vectors 10, 303 (2017). doi: 10.1186/s13071-017-2239-9 28645315

49. Goundry A., Romano A., Lima A. P. C. A., Mottram J. C. & Myburgh E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. fj201700797R (2018) doi: 10.1096/fj.201700797R 29097502

50. Nakajima S., Baba A. S. & Tamura N. Complement system in human colostrum: presence of nine complement components and factors of alternative pathway in human colostrum. Int. Arch. Allergy Appl. Immunol. 54, 428–433 (1977). 885627

51. Yamamoto G. K. & Allansmith M. R. Complement in tears from normal humans. Am. J. Ophthalmol. 88, 758–763 (1979). doi: 10.1016/0002-9394(79)90679-2 116549

52. Schwartz L. B. et al. Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase. J. Immunol. Baltim. Md 1950 130, 1891–1895 (1983).

53. Datsenko K. A. & Wanner B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97, 6640–6645 (2000). doi: 10.1073/pnas.120163297 10829079

54. Ambrus G. et al. Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments. J. Immunol. 170, 1374–1382 (2003). doi: 10.4049/jimmunol.170.3.1374 12538697

55. Dobó J. et al. MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity. J. Immunol. 183, 1207–1214 (2009). doi: 10.4049/jimmunol.0901141 19564340

56. Szabó A. et al. High-affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4’ acidic residues. J. Biol. Chem. 286, 22535–22545 (2011). doi: 10.1074/jbc.M111.235754 21515688

57. Williams J. A., Luke J. & Hodgson C. Strain engineering by genome mass transfer: efficient chromosomal trait transfer method utilizing donor genomic DNA and recipient recombineering hosts. Mol. Biotechnol. 43, 41–51 (2009). doi: 10.1007/s12033-009-9177-5 19455439

58. Lacroix M. et al. Assembly and enzymatic properties of the catalytic domain of human complement protease C1r. J. Biol. Chem. 276, 36233–36240 (2001). doi: 10.1074/jbc.M105688200 11445589

59. Luo C. et al. Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex. Biochemistry 31, 4254–4262 (1992). doi: 10.1021/bi00132a015 1533159

60. Jameson G. W., Roberts D. V., Adams R. W., Kyle W. S. A. & Elmore D. T. Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration. Biochem. J. 131, 107–117 (1973). doi: 10.1042/bj1310107 4737291

61. Green N. M. & Work E. Pancreatic trypsin inhibitor. II. Reaction with trypsin. Biochem. J. 54, 347–352 (1953). doi: 10.1042/bj0540347 13058883

62. Empie M. W. & Laskowski M. Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases. Biochemistry 21, 2274–2284 (1982). doi: 10.1021/bi00539a002 7046785

63. Yi L. et al. A highly sensitive fluorescence probe for fast thiol-quantification assay of glutathione reductase. Angew. Chem. Int. Ed Engl. 48, 4034–4037 (2009). doi: 10.1002/anie.200805693 19388016

64. Greco W. R. & Hakala M. T. Evaluation of methods for estimating the dissociation constant of tight binding enzyme inhibitors. J. Biol. Chem. 254, 12104–12109 (1979). 500698

65. Kim S., Narayana S. V. & Volanakis J. E. Crystal structure of a complement factor D mutant expressing enhanced catalytic activity. J. Biol. Chem. 270, 24399–24405 (1995). doi: 10.1074/jbc.270.41.24399 7592653

66. Rossi V. et al. Substrate specificities of recombinant mannan-binding lectin-associated serine proteases-1 and -2. J. Biol. Chem. 276, 40880–40887 (2001). doi: 10.1074/jbc.M105934200 11527969

67. Muschel L. H., Chamberlin R. H. & Osawa E. Bactericidal activity of normal serum against bacterial cultures. I. Activity against Salmonella typhi strains. Proc. Soc. Exp. Biol. Med. Soc. Exp. Biol. Med. N. Y. N 97, 376–382 (1958). doi: 10.3181/00379727-97-23748 13518280

68. El-Gebali S. et al. The Pfam protein families database in 2019. Nucleic Acids Res. 47, D427–D432 (2019). doi: 10.1093/nar/gky995 30357350

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