Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae

Abstract

Ecotin, first described inEscherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in variousCampylobacterspecies, includingCampylobacter rectusandCampylobacter showaeresiding in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotinsin vitro, the orthologs fromC.rectusandC.showaewere recombinantly expressed and purified fromE.coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins fromC.rectusandC.showaeinhibit NE, factor Xa and trypsin, but not theCampylobacter jejuniserine protease HtrA or its ortholog inE.coli, DegP. To further evaluate ecotin functionin vivo, anE.coliecotin-deficient mutant was complemented with theC.rectusandC.showaehomologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of theE.coliecotin-deficient mutant can be rescued upon expression of ecotins fromC.rectusandC.showae. In addition, theC.rectusandC.showaeecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen,Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.