Abstract
Pseudomonas aeruginosa secretes several endopeptidases, including elastase, alkaline proteinase (Apr), a lysine-specific endopeptidase (LysC), and an aminopeptidase (PaAP), all of which are important virulence factors. Activation of the endopeptidases requires removal of an inhibitory N-terminal propeptide. Activation of pro-PaAP, in contrast, requires C-terminal processing. The activating proteases of pro-PaAP and their cleavage site(s) have not yet been defined. Studying pro-PaAP processing in a wild type P. aeruginosa strain and strains lacking either elastase or both elastase and Apr, we detected three processing variants, each ~56 kDa in size (AP56). Activity assays and N- and C-terminal sequence analyses of these variants pointed at LysC as the principal activating protease, cleaving a Lys512-Ala513 peptide bond at the C-terminal end of pro-PaAP. Elastase and/or Apr are required for activation of LysC, suggesting both are indirectly involved in activation of PaAP. To shed light on the function(s) of the N-terminal domain of AP56, we purified recombinant AP56 and generated from it the 28 kDa catalytic domain (AP28). The kinetic constants (Km and Kcat) for hydrolysis of Leu-, Lys-, Arg- and Met-p-nitroanilide (pNA) derivatives by AP56 and AP28 were then determined. The catalytic coefficients (Kcat/Km) for hydrolysis of all four substrates by AP28 and AP56 were comparable, indicating that the non-catalytic domain is not involved in hydrolysis of small substrates. It may, however, regulate hydrolysis of natural peptides/proteins. Lys-pNA was hydrolyzed 2 to 3-fold more rapidly than Leu-pNA and ~8-fold faster than Arg- or Met-pNA, indicating that Lys-pNA was the preferred substrate.
Highlights
Pseudomonas aeruginosa is an opportunistic pathogen that causes acute infections in immunocompromised and burn patients and in corneal infections of traumatized eyes [1,2]
AP58 was not detected in the medium whether or not it was pre-treated with proteases, and the only PaAP related protein observed in all samples was ~56 kDa (Fig 1B, bottom left)
This suggested that, in addition to Ela [30], activation of PaAP can be mediated by other proteases, such as alkaline proteinase (Apr), lysine-specific endopeptidase (LysC) or both
Summary
Pseudomonas aeruginosa is an opportunistic pathogen that causes acute infections in immunocompromised and burn patients and in corneal infections of traumatized eyes [1,2]. Ela is the most abundant and potent protease of P. aeruginosa [11] It has a broad cleavage specificity it favors hydrophobic or aromatic amino acid residues, with preference to Phe and Leu at the P10 position, i.e., cleavage occurs on the amino side of these residues [12]. The proteolytic activity of Apr is limited and its cleavage specificity remains unclear it has been reported to cleave peptide bonds on the carboxyl side of arginine residues [13]. LasA protease cleaves exclusively peptide bonds after Gly-Gly pairs that are abundant in the cell wall peptidoglycan of Staphylococci and present in elastin. This unique specificity accounts for its ability to lyse Staphylococci and nick elastin [14,15]. LysC is a lysine specific serine protease that cleaves peptide bonds on the carboxyl side of lysine residues exclusively and is highly sensitive to the lysine-specific serine protease inhibitor Tosyl-lysyl-chloromethylketone (TLCK) [16,17]
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