Cloning and transcriptional regulation of the elastase lasA gene in mucoid and nonmucoid Pseudomonas aeruginosa

Goldberg, Joanna B.; Ohman, Dennis E.

doi:10.1128/jb.169.3.1349-1351.1987

Cited by 26 publications

(23 citation statements)

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“…Animal model infection studies comparing the lasA1 mutant and wild-type strains suggest that LasA contributes substantially to virulence in acute (2) and chronic (41) lung infections. Transcriptional activation of lasA occurs near stationary phase, indicating that expression of lasA is cell density dependent (9). The expression of both lasA and lasB is under the control of lasR (38) and rhlR (3), members of two autoinducerresponsive regulatory systems in P. aeruginosa that respond to cell density.…”

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confidence: 99%

“…To resolve this and to define the preproenzyme domain structure of the LasA precursor, we began this study by determining the sequence of a lasA-encoding 3.74-kb fragment that was previously cloned from P. aeruginosa FRD1 (9). Expression studies were performed, and the signal peptidase cleavage site was identified, allowing the precise size determination of individual domains within pre-proLasA.…”

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

1996

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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...

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

1996

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“…In contrast, it has been reported that mucoid strains of P . aeruginosa secrete less elastase than their non-mucoid counterparts (Ohman & Chakrabarty, 1982;Goldberg & Ohman, 1987). However, this probably reflects transcriptional control in the latter example rather than the release of latent enzyme from the surface of the outer membrane (Goldberg & Ohman, 1987;Mohr et al, 1990).…”

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confidence: 99%

Heterologous expression of an alginate lyase gene in mucoid and non-mucoid strains of Pseudomonas aeruginosa

Gacesa

Goldberg²

1992

Journal of General Microbiology

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~ ~~~ A 1.95 kb DNA fragment containing the d y gene from KZebsieZZupneumoniue, which encodes an alginate lyase, has been ligated into the broad-host-range vector pLAFR3. Transfer of the resultant recombinant plasmid, pALY8, into mucoid and non-mucoid strains of Pseudomonas ueruginosu resulted in expression of the alginate lyase. The heterologously expressed alginate lyase, which had the same isoelectric point and substrate specificity as the native enzyme, altered the morphology of mucoid strains. Analysis of the extracellular material from mucoid strains revealed that lyase expression reduced the M, and overall yield of alginate produced. The mature form of the recombinant enzyme was the same as that produced extracellularly by KZebsieZZupneumoniae; however, most of the alginate lyase was retained intracellularly by P. ueruginosu.

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“…which also belong to the two-component-system superfamiiy. In this respect it is interesting that algB (Goldberg and Ohman, 1987), a site of mutations reducing alginate synthesis, also turned out to belong to the signai-transduction regulatory systems (Wozniak and Ohman, 1991).…”

Section: Mutations In the Muc Loci And Upregulation Of The Alginate Smentioning

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1991

Molecular Microbiology

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SummaryThe profuse production of the exopolysaccharide alginate results in mucoidy, a critical virulence factor expressed by Pseudomonas aeruginosa during chronic respiratory tract infections in cystic fibrosis. Studies of the regulation of this pathogenic determinant have unravelled at least two levels of control, including bacterial signal transduction systems and histone-like eiements. Although only in its initial phase, an understanding of the dual control of mucoidy may help to illuminate adaptive processes that depend on the combination of these regulatory factors. Integration of specific signals transduced by the two-component systems with inputs generated by the general state of bacterial nucleoids may govern the expression of certain virulence determinants and provide a framework facilitating selection of phenotypes successful under particular environmental conditions and selective pressures.

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Cloning and transcriptional regulation of the elastase lasA gene in mucoid and nonmucoid Pseudomonas aeruginosa

Cited by 26 publications

References 24 publications

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

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

Heterologous expression of an alginate lyase gene in mucoid and non-mucoid strains of Pseudomonas aeruginosa

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