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.

Structure and evolution of the Ivy protein family, unexpected lysozyme inhibitors in Gram-negative bacteria

“…The Ivy protein could play a similar role against lysozyme, whereas the Ivy paralogue might inhibit another of the many microbicidal agents present in human nasal and salivatory secretions (18) such as PLUNC proteins, ubiquitous in vertebrate genomes, and known to be active against P. aeruginosa (19). Interestingly, the mechanism of Ivy inhibition is similar to the one observed for ecotin against serine proteases, in a substrate-like fashion, through a critical protruding loop (15).…”

“…Finally, both Ivy and ecotin are part of the 25 genes significantly up-regulated by higher concentrations of acetyl phosphate known to promote the free living state where bacteria are more sensitive to the vertebrate innate immune system (15,20). Indeed more work is needed to identify the physiological function of Ivy genes that might lead to a reappraisal of the physiological role of C-type lysozyme and related enzymes in the control of opportunistic bacterial infections.…”

“…In synergy, these protective agents (i) make pores in the membrane of the pathogen through proteases (e.g., perforins, defensins, bacterial permeability-increasing proteins), (ii) opsonize bacteria through serum proteins (e.g., lipopolysaccharide-binding protein, lysozyme), and (iii) chemoattract leukocytes. In turn, Gram-negative bacteria have evolved protective mechanisms, such as ecotins, a family of periplasmic proteins inhibiting serine proteases, including the permeabilizing neutrophil elastase (15). The Ivy protein could play a similar role against lysozyme, whereas the Ivy paralogue might inhibit another of the many microbicidal agents present in human nasal and salivatory secretions (18) such as PLUNC proteins, ubiquitous in vertebrate genomes, and known to be active against P. aeruginosa (19).…”

“…Similar circumstances might actually be encountered in the natural environment of Gram-negative bacteria. The innate vertebrate immune system, increasingly recognized as the first line of defense against bacterial infection, produces a number of proteins highly toxic to Gram-negative but not to Gram-positive bacteria (13)(14)(15)(16)(17). In synergy, these protective agents (i) make pores in the membrane of the pathogen through proteases (e.g., perforins, defensins, bacterial permeability-increasing proteins), (ii) opsonize bacteria through serum proteins (e.g., lipopolysaccharide-binding protein, lysozyme), and (iii) chemoattract leukocytes.…”

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

“…This hypothesis is strongly supported by the fact that miropin is predicted to be a secretory lipoprotein, which is likely associated with the outer membrane. A similar function has been described for the canonical, non-covalent protease inhibitor ecotin located in the periplasm of E. coli (47). Alternatively, the main function of miropin could be related to control of the activity of the bacteria's own proteases.…”