Mechanism of action of Pseudomonas aeruginosa exotoxin Aiadenosine diphosphate-ribosylation of mammalian elongation factor 2 in vitro and in vivo

Abstract

Previous studies showed that P. aeruginosa exotoxin A (PA toxin) catalyzes NAD-dependent inhibition of protein synthesis in a rabbit reticulocyte lysate and transfer of radioactivity from [14C]adenine-labeled NAD to a protein having the same molecular weight as elongation factor 2 (EF-2). Such an inhibited protein-synthesizing lysate was restored to activity by addition of a protein from normal mouse liver which co-purifies with EF-2. EF-2 activity was almost totally absent in livers of mice injected 24 h earlier with PA toxin. EF-2 concentrations were only partially reduced in other organs and were normal in brains of intoxicated mice. Studies using NAD labeled in various positions show that PA toxin, like fragment A of diphtheria toxin, catalyzes transfer of the ADP-ribosyl moiety of NAD. Reversal occurred when the modified protein was incubated with excess concentrations of PA toxin and nicotinamide, and NAD was identified as a product of the reverse reaction. The protein modification catalyzed by PA toxin or by fragment A of diphtheria toxin could be reversed by incubation with the other toxin. These 2 toxins apparently ADP-ribosylate the same amino acid of EF-2 in a stereochemically identical fashion. PA toxin inactivates EF-2 in intoxicated mice to an extent which would ultimately result in death.