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Reduced temperature alters Pseudomonas exotoxin A entry into the mouse LM cell
Abstract:
The movement of Pseudomonas exotoxin A (PE) into the cytoplasm of mouse LM fibroblasts was followed by using inhibition of protein synthesis as a biochemical index of toxin activity; biotinyl-PE and avidin-gold colloids were used for electron microscopy. At 37 degrees C both specific antitoxin and pronase-trypsin protected cells against PE toxicity when added within seconds of warming cells, whereas methylamine was protective when added during the first 7 min of endocytosis. Lowering the temperature to 19 degrees C afforded protection when the temperature transition was accomplished within 15 min of the original endocytic event. These data suggest that PE enters an acidic compartment before reaching a step blocked by shifting cells from 37 to 19 degrees C. PE expressed toxicity for LM cells at 19 degrees C, but at a concentration 1 order of magnitude higher than that required at 37 degrees C. At 19 degrees C, antitoxin or trypsin-pronase protection was rapidly ablated. In contrast cells were fully protected by methylamine for 90 min. Using electron microscopy we demonstrated that toxin moved normally (30 s) to coated areas at 19 degrees C, but remained at this site for up to 20 min before being internalized. The majority of the toxin internalized at 19 degrees C remained in endosomes or in Golgi-associated vesicles and was not delivered to lysosomes. The results suggest that, under physiological conditions (37 degrees C), PE rapidly enters cells through coated areas, moves to an acidic compartment (i.e., the endosome), and then probably to the Golgi region en route to lysosomes. The evidence suggests that movement of toxin from endosomes or Golgi vesicles to lysosomes is blocked at 19 degrees C. We hypothesize that the active form of PE enters the cytosol, where it expresses its toxicity during fusion of Golgi-derived, toxin-laden vesicles with lysosomes.
Insights
Pseudomonas exotoxin A (PE) enters cells via endocytosis. Lowering temperature blocks PE transport from endosomes to lysosomes, preventing toxicity. This reveals a critical step in toxin entry.
Area of Science:
- Cell Biology
- Molecular Toxicology
- Protein Trafficking
Background:
- Pseudomonas exotoxin A (PE) is a potent protein toxin that inhibits protein synthesis.
- Understanding the cellular entry pathway of PE is crucial for developing targeted therapeutics.
- Endocytosis is a primary mechanism for cellular uptake of toxins and other molecules.
Purpose of the Study:
- To investigate the intracellular trafficking of Pseudomonas exotoxin A (PE) in mouse LM fibroblasts.
- To identify temperature-sensitive steps in the PE intoxication pathway.
- To elucidate the role of endosomal acidification and transport in PE toxicity.
Main Methods:
- Inhibition of protein synthesis was used as a biochemical marker for PE toxicity.
- Biotinylated PE and avidin-gold conjugates were employed for electron microscopy.
- Temperature shifts (37°C to 19°C) and chemical treatments (methylamine, trypsin-pronase) were used to probe toxin transport.
Main Results:
- PE entry into the cytoplasm was blocked at 19°C, requiring a higher toxin concentration for toxicity.
- Methylamine protected cells from PE toxicity at 19°C, suggesting involvement of an acidic compartment.
- Electron microscopy showed PE accumulating in endosomes and Golgi-associated vesicles at 19°C, with impaired lysosomal delivery.
Conclusions:
- PE rapidly enters cells via coated pits at 37°C, proceeds through an acidic endosomal compartment, and moves towards lysosomes.
- A temperature-sensitive step, likely involving transport from endosomes/Golgi vesicles to lysosomes, is essential for PE toxicity.
- The active form of PE may enter the cytosol during the fusion of Golgi-derived vesicles with lysosomes.