Abstract
Ecotin, first described in Escherichia 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 various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins 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.
Highlights
Bacteria have developed various strategies to survive in their ecological niche
To gain insight into the conservation of this ecotin homolog among Campylobacter species, homology searches against the protein sequence database (BlastP, [57]) using either the amino acid sequence of the C. rectus or the E. coli ecotin as a query, revealed that potential ecotin proteins were present in C. showae, C. curvus, C. concisus, C. hominis, C. gracilis and C. ureolyticus
C. rectus is a recognized oral pathogen implicated in causing periodontitis [20], while C. showae has been linked to causing gingivitis and periodontitis, and more recently with inflammatory bowel disease [68]
Summary
Proteases are vital players of the immune system for protecting against and clearing pathogens [1]. One of these particular areas is the oral cavity where polymorphonuclear. Campylobacter ecotins neutrophils (PMNs) of the innate immune system are the first line of defense against invading microorganisms. Neutrophils can engulf invading bacteria via phagocytosis to form a phagolysosome where microbial killing takes place by the action of these proteases, reactive oxygen species and additional antimicrobial mechanisms. Aside from assisting in pathogen destruction, NETs and NSPs are involved in human inflammatory conditions including chronic lung diseases like cystic fibrosis [4,5,6,7,8]. Overstimulation e.g. by Pseudomonas aeruginosa or delayed apoptosis of neutrophils results in excessive accumulation of NETs and NSPs and in subsequent lung tissue degradation [8,9]
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