title: The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase.
abstract: Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143-92280, USA.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Eggers
authors.forename: Iain A
authors.initials: IA
authors.lastname: Murray
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Delmar
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Day
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008969:Molecular Sequence Data; D020636:Pantoea / Q000235:genetics / Q000378:metabolism; D033903:Periplasmic Proteins / Q000235:genetics / Q000502:physiology*; D010802:Phylogeny; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000502:physiology*; D013045:Species Specificity; D015010:Yersinia pestis / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D015842:Serine Proteinase Inhibitors; C487722:ecotin protein, Pseudomonas aeruginosa; C487721:ecotin protein, Yersinia pestis; D019272:Leukocyte Elastase