title: Inhibition of Pseudomonas aeruginosa virulence: characterization of the AprA-AprI interface and species selectivity.
abstract: Pseudomonas aeruginosa secretes the virulence factor alkaline protease (AprA) to enhance its survival. AprA cleaves one of the key microbial recognition molecules, monomeric flagellin, and thereby diminishes Toll-like receptor 5 activation. In addition, AprA degrades host proteins such as complement proteins and cytokines. P. aeruginosa encodes a highly potent inhibitor of alkaline protease (AprI) that is solely located in the periplasm where it is presumed to protect periplasmic proteins against secreted AprA. We set out to study the enzyme-inhibitor interactions in more detail in order to provide a basis for future drug development. Structural and mutational studies reveal that the conserved N-terminal residues of AprI occupy the protease active site and are essential for inhibitory activity. We constructed peptides mimicking the N-terminus of AprI; however, these were incapable of inhibiting AprA-mediated flagellin cleavage. Furthermore, we expressed and purified AprI of P. aeruginosa and the homologous (37% sequence identity) AprI of Pseudomonas syringae, which remarkably show species specificity for their cognate protease. Exchange of the first five N-terminal residues between AprI of P. syringae and P. aeruginosa did not affect the observed specificity, whereas exchange of only six residues located at the AprI surface that contacts the protease did abolish specificity. These findings are elementary steps toward the design of molecules derived from the natural inhibitor of the virulence factor AprA and their use in therapeutic applications in Pseudomonas and other Gram-negative infections.
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, HP G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. b.w.bardoel@umcutrecht.nl
authors.forename: Bart W
authors.initials: BW
authors.lastname: Bardoel
authors.forename: Kok P M
authors.initials: KP
authors.lastname: van Kessel
authors.forename: Jos A G
authors.initials: JA
authors.lastname: van Strijp
authors.forename: Fin J
authors.initials: FJ
authors.lastname: Milder
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005408:Flagellin / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D044224:Pseudomonas syringae / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D037521:Virulence Factors; D005408:Flagellin; D010450:Endopeptidases; C087822:alkaline protease