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The cytotoxin of Pseudomonas aeruginosa: cytotoxicity requires proteolytic activation
G Orlik-Eisel1, F Lutz, A Henschen
1Institut für Pharmakologie and Toxikologie, Universität Giessen, Federal Republic of Germany.
Abstract:
The primary structure of a cytotoxin from Pseudomonas aeruginosa was determined by sequencing of the structural gene. The cytotoxin (31,700 Mr) lacks an N-terminal signal sequence for bacterial secretion but contains a pentapeptide consensus sequence commonly found in prokaryotic proteins which function in a TonB-dependent manner. The cytotoxin gene has a [G + C]-content of 53.8% which is considerably lower than generally observed for genes from Pseudomonas aeruginosa. The cytotoxin gene was exclusively detected in strain 158 but not in three other clinical isolates, as determined by Southern and Northern hybridization. The latter technique revealed that the toxin is translated from monocistronic mRNA. The promoter of the cytotoxin is inactive in Escherichia coli. Upon site-directed modification of the 5'-noncoding region by the polymerase chain reaction the gene was expressed under control of the trc-promoter. The gene product obtained in Escherichia coli was nontoxic. Toxicity was induced by subsequent treatment with trypsin. [35S]methionine-labeled cytotoxin with high specific radioactivity was obtained by in vitro transcription/translation. Like [125I] labeled material from Pseudomonas aeruginosa this polypeptide bound to membrane preparations from Ehrlich ascites cells, as evidenced by sedimentation through a sucrose gradient at neutral pH.
Insights
Researchers sequenced a Pseudomonas aeruginosa cytotoxin gene, finding it requires trypsin activation for toxicity. This toxin binds to host cell membranes, offering insights into bacterial pathogenesis and potential therapeutic targets.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Chemistry
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen that produces various toxins.
- Understanding cytotoxin structure and function is crucial for developing targeted therapies.
- The specific cytotoxin studied here lacks a typical secretion signal but shares features with TonB-dependent proteins.
Purpose of the Study:
- To determine the primary structure of a cytotoxin from Pseudomonas aeruginosa.
- To investigate the gene's characteristics, expression, and the toxin's functional properties.
- To elucidate the mechanism of cytotoxin activation and cellular interaction.
Main Methods:
- Gene sequencing and Southern/Northern hybridization to analyze the cytotoxin gene.
- Site-directed mutagenesis and polymerase chain reaction (PCR) for gene expression studies.
- In vitro transcription/translation and cell binding assays using radiolabeled cytotoxin.
Main Results:
- The cytotoxin (31,700 Mr) gene has a lower G+C content than typical for P. aeruginosa.
- The gene was detected only in strain 158 and is transcribed as monocistronic mRNA.
- The gene product expressed in E. coli was nontoxic until activated by trypsin; it binds to Ehrlich ascites cell membranes.
Conclusions:
- The cytotoxin's primary structure and gene organization were elucidated.
- Toxicity is dependent on post-translational modification (trypsin activation).
- The cytotoxin interacts with host cell membranes, suggesting a mechanism for cellular entry or activity.