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

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. Author summary Bloodstream infections are major cause of morbidity and mortality in many countries around the globe. As the number of multi-drug resistant pathogenic strains is growing, it is urgent to identify their virulence factors and unveil the corresponding mechanisms of action that enable the pathogen to avoid potent immune response. 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. However, the interaction of ecotin with the complement system, which provides a first line defense against pathogens, remained unexplored. We found that ecotin blocks activation of the complement lectin pathway by inhibiting its key activator enzymes, MASP-1 and MASP-2. Furthermore, by inhibiting MASP-3, ecotin also disrupts a fundamental link between the lectin- and the alternative pathways. We provide evidence that E. coli cells devoid of ecotin are extremely vulnerable to complement-mediated lysis and they are also potently killed by some complement-independent antimicrobial factors of human serum. These findings could explain the observations of other research groups reporting that ecotin is crucial for the survival of pathogenic microbes in the host. Our results therefore also highlight ecotin as a potential target of future antimicrobial therapies.