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

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 pathwayrelated 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.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 o...

Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

“…In contrast, ErpB and ErpQ bind to both C1r and C1s but selectively recognize activated forms of the proteases (Figs 2, S5). Further, we showed that ErpQ in incapable of blocking C1r activity (Fig S7 Finally, ErpQ did not prevent cleavage of small peptide substrates, and is unusual among microbial-derived serine protease inhibitors, such as ecotin or BBK32 (49), which typically target the active site (79,80).…”

Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study

“…The regulator of the mucoid phenotype rmp genes were detected in K. pneumoniae ( n = 14/218; 6.4%) and K. quasipneumoniae ( n = 1/83; 1.2%). Ecotin has been described as being able to modulate the host immune response [ 56 ] and was detected in isolates belonging to all species ( n = 445/548; 81.2%).…”

Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae

“…Bacterial strategies to avoid neutrophil-mediated killing include launching a general survival response, avoiding contact, preventing phagocytosis, surviving inside the neutrophil, and inducing cell death [ 10 , 11 ]. Specific countermeasures against the action of proteases are the production of protease inhibitors, including serine protease inhibitors such as ecotin [ 12 ], or masking proteolytic cleavage sites through glycosylation of protease target proteins [ 13 ]. Protein glycosylation is the most common post-translational modification in nature and exists in all three domains of life.…”

“…coli as a periplasmic protease inhibitor that exhibits a broad specificity toward exogenous serine proteases including trypsin, chymotrypsin, factor Xa, NE, kallikrein, urokinase and factor XII, but not against metallo-, aspartyl and sulfhydryl proteases, or its own proteases [ 28 – 32 ]. Ecotin homologs have since been found in more than 300 organisms, predominantly in Gram-negative bacterial pathogens such as Pseudomonas aeruginosa , Shigella flexneri , Yersinia pestis , Burkholderia pseudomallei and Klebsiella pneumoniae [ 12 , 31 – 33 ], but also in two eukaryotic protozoan parasites within the Trypanosomatidae genus ( Leishmania major and Trypanosoma cruzi [ 12 , 34 , 35 ]), and in some plant pathogens e. g. Pantoea citrea [ 31 ]. In the latter case, ecotin was found to be less potent in binding to NE when compared to the E .…”