Abstract
At high bacterial cell density the gene expression program of Pseudomonas aeruginosa is regulated by quorum sensing. Among the gene products highly up-regulated by this system is an exoprotease, leucine aminopeptidase (PA-LAP), which is coexpressed with several known virulence factors and secreted as a proenzyme. We undertook a study of its activation by expressing the full-length proform of PA-LAP recombinantly in Escherichia coli (here termed, rLAP55) and characterizing individual steps in its conversion to an active enzyme. Activation is initiated with the proteolytic removal of a C-terminal prosequence. Removal of approximately 20 amino acids is accomplished by Pseudomonas elastase, which is also positively regulated by quorum sensing. Activation is also mediated by other proteases that cleave rLAP55 near its C terminus. The importance of the C terminus was confirmed by showing that C-terminal deletions of 1-24 amino acids produce a fully active enzyme. The removal of C-terminal prosequences either by proteolysis or deletion leads to an unusual autoprocessing event at the N terminus. Autoprocessing is apparently an intramolecular event, requires the active site of LAP, and results in the removal of 12 N-terminal amino acids. Furthermore, a detailed analysis of the C-terminal prosequence suggests that the proenzyme state is dependent on the presence of a basic side chain contributed by the last amino acid, lysine 536. Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus.
Highlights
Pseudomonas aeruginosa is an opportunistic pathogen of humans causing acute life-threatening infections in immunocompromised and burned patients and chronic infections in individuals affected by cystic fibrosis (1)
Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus
We propose that PA-LAP is secreted to the growth media as a proenzyme
Summary
AP⌬sig seq forward (FOR), 5Ј-CATATGCCTTCGGAAGCGCAACAGT-3Ј; AP39 forward, 5Ј-CATATGAAACCCAACCCGTCGAT-3Ј; AP537 reverse (REV), 5Ј-GAATTCTTACTTGATGAAGT-3Ј; AP512K reverse, 5Ј-ATGAATTCAAGCTTTTATTTCTGGCCGGCGGCGGCGATCTC-3Ј; AP529R reverse, 5Ј-ATGAATTCAAGCTTTTAGCGTTCGATCTGGCTGGCGCTC-3Ј; AP⌬536 reverse, 5Ј-ATGAATTCAAGCTTTTAGATGAAGTCGTGACCCCAGC-3Ј; D308A forward, 5Ј-GCGCGCACCTCGACTCGGTGTTCGAAGGCCCCGGTATCAACGCCAACGGTTCGGGCAGC-3Ј; SPH wild type (WT) reverse, 5Ј-AATAAGTGATAATAAGCGGACCTGCA-3Ј; D370A-1 forward, 5Ј-AAGAAGAAGATCAAGGCCTACCTGAACTTCGCCATGATCGGCTCG-3Ј; D370-2 WT reverse, 5ЈAAGTTCAGGTAGGCCTTGATCTTCTTC-3Ј
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