A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion

Abstract

The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism. LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity. We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli. The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da. Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E. coli. The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity. However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P. aeruginosa lasA⌬ mutant. Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease. Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis. Under the control of an inducible trc promoter, lasA was overexpressed in P. aeruginosa and the processing intermediates were examined. Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease. Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed. To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity. This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus. Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele. The product of lasA5 expressed from the chromosome of P. aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity. This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P. aeruginosa can proceed via mechanisms which do not involve autoproteolysis.Pseudomonas aeruginosa is a potent opportunistic pathogen, and its virulence is largely associated with the organism's ability to secrete numerous toxic and degradative enzymes into its environment. Among these enzymes are a variety of proteases that can contribute to pathogenicity by breaking down host defenses and nonspecific physical barriers to the bacteria. The exact number of proteases produced by P. aeruginosa is not yet known. However, two proteases, elastase and LasA protease, have received much attention because they can degrade elastin, a major component of connective tissue, blood vessels, and lung tissue. Elastin is relatively resistant to hydrolysis by most...

Inhibitors and Specificity of Pseudomonas aeruginosa LasA

“…This supports LasA as a zinc metalloendopeptidase and is consistent with the recent demonstration that efficient production of LasA by P. aeruginosa requires zinc ions (29). LasA as well as the Lysobacter and Achromobacter ␤-lytic endopeptidases (15,26,27) does not contain the HEXXH zinc-binding motif typical of most zinc proteinases. An HXH motif noted in A. lyticus ␤-lytic endopeptidase (27) and shared with both the Lysobacter enzyme and LasA (positions 120 -122) was proposed as a potential zinc ligand (27).…”

“…The assay of staphylolytic activity that we used for this purpose is sensitive and specific. Although the rate of staphylolysis by LasA may be increased by up to 2.5-fold in the presence of P. aeruginosa elastase or alkaline proteinase (observed by us (15) and others (17)), neither elastase nor alkaline proteinase alone show staphylolytic activity. In addition, for significant enhancement of LasA action on S. aureus cells, relatively large amounts of elastase or alkaline proteinase are required.…”

“…An HXH motif noted in A. lyticus ␤-lytic endopeptidase (27) and shared with both the Lysobacter enzyme and LasA (positions 120 -122) was proposed as a potential zinc ligand (27). Substitution in LasA of His-120 blocks LasA activity (15), suggesting that His-120 may be one of the zinc ligands in LasA.…”

“…Subsequently, elastase-negative mutants of P. aeruginosa were found to retain some elastolytic activity (12,13), and the purified LasA fragment was shown to possess basal elastolytic activity (14). The 40-kDa product of lasA was identified as a precursor of the extracellular LasA protein (15), and because of its action on elastin, the secreted 22-kDa form of LasA has been referred to as a second elastase (12)(13)(14).…”

Peptidoglycan Amidase MepA Is a LAS Metallopeptidase

“…The LAS hypothesis further implied that the conserved histidines and the aspartate in the HX(3,6)D and HXH motifs of MepA should be catalytically important (9,26). To test this assumption for MepA, each of the four conserved residues was separately mutated to alanine.…”