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Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

ABSTRACT: Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the Bacillus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutant elastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors (proelastases) were produced, no appreciable processing was observed with these mutants. The possibility that autocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, by immunizing mice with vaccines made from these mutant elastase, we were able to obtain good protection against an intraperitoneal P. aeruginosa challenge.

SUBMITTER: Kawamoto S

PROVIDER: S-EPMC281377 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

Kawamoto S S Shibano Y Y Fukushima J J Ishii N N Morihara K K Okuda K K

Infection and immunity 19930401 4

Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the Bacillus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the ...[more]

PMID: 8454342

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Project description:BackgroundPseudomonas aeruginosa (PA) is a common cause of respiratory infection and morbidity. Pseudomonas elastase is an important virulence factor regulated by the lasR gene. Whether PA elastase activity is associated with worse clinical outcomes in ICU patients is unknown.Research questionIs there an association between PA elastase activity and worse host outcomes in a cohort of ICU patients?MethodsPA respiratory isolates from 238 unique ICU patients from two tertiary-care centers within the University of Pittsburgh Medical Center health system were prospectively collected and screened for total protease and elastase activity, biofilm production, antimicrobial resistance, and polymicrobial status. The association between pathogen characteristics and 30-day and 90-day mortality was calculated using logistic regression. For subgroup analysis, two patterns of early (≤72 h) and late sample (>72 h) collection from the index ICU admission were distinguished using a finite mixture model. Lung inflammation and injury was evaluated in a mouse model using a PA high elastase vs low elastase producer.ResultsPA elastase activity was common in ICU respiratory isolates representing 75% of samples and was associated with increased 30-day mortality (adjusted OR [95% CI]: 1.39 [1.05-1.83]). Subgroup analysis demonstrated that elastase activity was a risk factor for 30- and 90-day mortality in the early sample group, whereas antimicrobial resistance was a risk factor for 90-day mortality in the late sample group. Whole genome sequencing of high and low elastase producers showed that predicted loss-of-function lasR genotypes were less common among high elastase producers. Mice infected with a high elastase producer showed increased lung bacterial burden and inflammatory profile compared with mice infected with a low elastase producer.InterpretationElastase activity is associated with 30-day ICU mortality. A high elastase producing clinical isolate confers increased lung tissue inflammation compared with a low elastase producer in vivo.

Project description:Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.

Dataset Information

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

Publications

Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection.

Similar Datasets

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