title: Inhibition of outer membrane proteases of the omptin family by aprotinin.
abstract: Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: John R
authors.initials: JR
authors.lastname: Brannon
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
authors.forename: David L
authors.initials: DL
authors.lastname: Burk
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: Jean-Mathieu
authors.initials: JM
authors.lastname: Leclerc
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: Jenny-Lee
authors.initials: JL
authors.lastname: Thomassin
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
authors.forename: Andrea
authors.initials: A
authors.lastname: Portt
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
authors.forename: Albert M
authors.initials: AM
authors.lastname: Berghuis
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada Microbiome and Disease Tolerance Centre, McGill University, Montreal, Quebec, Canada.
authors.forename: Samantha
authors.initials: S
authors.lastname: Gruenheid
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada Microbiome and Disease Tolerance Centre, McGill University, Montreal, Quebec, Canada Faculty of Dentistry, McGill University, Montreal, Quebec, Canada herve.le-moual@mcgill.ca.
authors.forename: Hervé
authors.initials: H
authors.lastname: Le Moual
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides; D007611:Aprotinin / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D054804:Cathelicidins / Q000378:metabolism; D044082:Citrobacter rodentium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D057057:Serine Proteases / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013045:Species Specificity
chemical_list: D023181:Antimicrobial Cationic Peptides; D001425:Bacterial Outer Membrane Proteins; D054804:Cathelicidins; D029968:Escherichia coli Proteins; D015842:Serine Proteinase Inhibitors; C499588:ompT protein, E coli; D007611:Aprotinin; D010447:Peptide Hydrolases; D057057:Serine Proteases
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