Related Experiment Videos
Effects of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A
1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
Cysteines 265 and 287 of Pseudomonas aeruginosa exotoxin A (ETA) were substituted by serine, thereby eliminating a disulfide bridge within domain II, the putative membrane insertion-translocation domain. Purified mutant toxin was 80-fold less toxic for mouse L cells than was wild-type ETA while retaining the same specific activity in the ADP-ribosyltransferase reaction as did wild-type toxin. Binding of the nonionic detergent Triton X-114 by mutant ETA occurred at a slightly higher pH than did binding by wild-type ETA, suggesting that the mutant protein more readily undergoes a conformational change exposing hydrophobic regions. Data are presented supporting the notion that the mutant and wild-type toxins enter from the same intracellular compartment. The lower cytotoxicity of the mutant protein could be due to accelerated intracellular degradation or abortive, premature membrane insertion.
Insights
Mutating Pseudomonas aeruginosa exotoxin A (ETA) reduced its toxicity by 80-fold. This suggests the disulfide bridge in domain II is crucial for ETA
Area of Science:
- Microbiology
- Molecular Biology
- Toxicology
Background:
- Pseudomonas aeruginosa exotoxin A (ETA) is a potent toxin responsible for various infections.
- Domain II of ETA is implicated in membrane insertion and translocation, crucial steps for its cytotoxic activity.
Purpose of the Study:
- To investigate the role of the disulfide bridge within domain II of ETA in its toxicity.
- To elucidate the mechanism by which ETA exerts its cytotoxic effects.
Main Methods:
- Site-directed mutagenesis was used to substitute cysteines 265 and 287 with serine, eliminating the disulfide bridge in domain II.
- Purified wild-type and mutant ETA were tested for cytotoxicity on mouse L cells.
- ADP-ribosyltransferase activity and Triton X-114 binding were assessed to evaluate toxin function and conformational changes.
Main Results:
- The mutant ETA exhibited an 80-fold reduction in toxicity compared to wild-type ETA.
- The ADP-ribosyltransferase activity of the mutant toxin remained comparable to the wild-type.
- The mutant ETA showed altered nonionic detergent binding, indicating changes in hydrophobic region exposure.
Conclusions:
- The disulfide bridge in domain II of ETA is essential for its high cytotoxicity.
- The reduced toxicity may result from accelerated intracellular degradation or premature membrane insertion of the mutant toxin.
- These findings provide insights into the molecular mechanisms of ETA-mediated cell entry and toxicity.