Selection and characterization of cells resistant to diphtheria toxin and pseudomonas exotoxin A: presumptive...

Area of Science:

• Cell Biology
• Molecular Biology
• Toxicology

Background:

• Diphtheria toxin and Pseudomonas exotoxin are potent protein synthesis inhibitors.
• These toxins function by ADP-ribosylating elongation factor 2 (EF-2).
• Understanding resistance mechanisms is crucial for developing therapeutic strategies.

Purpose of the Study:

• To investigate the mechanisms of resistance to diphtheria toxin in Chinese hamster ovary (CHO-K1) cells.
• To characterize novel classes of toxin-resistant variants.
• To elucidate the role of EF-2 in toxin resistance.

Main Methods:

• Selection of diphtheria toxin-resistant variants from CHO-K1 cell lines.
• Analysis of toxin uptake and intracellular processing.
• Biochemical assays to assess EF-2 ADP-ribosylation.
• Cross-resistance studies with Pseudomonas exotoxin.

Main Results:

• Two distinct classes of resistant variants were identified: permeability and cytoplasmic.
• Cytoplasmic variants exhibited cross-resistance to Pseudomonas exotoxin.
• EF-2 isolated from cytoplasmic variants was resistant to ADP-ribosylation by both toxins.
• Evidence suggests these are translational variants with altered EF-2.

Conclusions:

• A novel class of cytoplasmic variants confers resistance to both diphtheria toxin and Pseudomonas exotoxin.
• The resistance is attributed to a mutationally altered EF-2 product, impacting its susceptibility to ADP-ribosylation.
• These findings highlight the central role of EF-2 in the mechanism of action of these toxins.