Reduced temperature alters Pseudomonas exotoxin A entry into the mouse LM cell

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.