Abstract
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.
Highlights
Ecotin is a canonical serine protease (SP) inhibitor first isolated from Escherichia coli [1]
A microbial inhibitor of serine proteases, ecotin was previously implicated in protecting various pathogenic bacteria and eukaryotic Leishmania species against the host immune system by inhibiting leukocyte elastase
We found that ecotin blocks activation of the complement lectin pathway by inhibiting its key activator enzymes, MASP-1 and MASP-2
Summary
Ecotin is a canonical serine protease (SP) inhibitor first isolated from Escherichia coli [1]. Ecotin inhibits all three major pancreatic SPs, trypsin, chymotrypsin and elastase, and its function was first assumed to protect E. coli in its natural habitat, the colon [1]. Ecotin orthologs were found in several microbes including human pathogens, such as Yersinia pestis Pseudomonas aeruginosa and Burkholderia pseudomallei [8,9] and even in eukaryotic organisms such as the pathogenic protozoa Trypanosomatida, including Leishmania major [10]. Ecotin orthologs from E. coli, Y. pestis, and P. aeruginosa were shown to inhibit neutrophil elastase (NE) secreted by neutrophils and macrophages during inflammation, and this activity was interpreted as a potential defense mechanism [11]
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