Mechanism of action of Pseudomonas aeruginosa exotoxin A: adenosine diphosphate ribosylation of mammalian elongation factor 2 in vitro and in vivo

Previous studies showed that P. aeruginosa exotoxin A (PA toxin) catalyzes nicotinamide adenine dinucleotide (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. In addition, EF 2 activity was almost totally absent in livers of mice which had been injected 24 hr earlier with PA toxin. On the contrary, 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 adenosine 5' diphosphate ribosyl moiety of NAD. Furthermore, 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 either by PA toxin or by fragment A of diphtheria toxin could be reversed by incubation with the other toxin. These results support the proposal that these two toxins adenosine 5' diphosphate ribosylate the same amino acid of EF 2 in a stereochemically identical fashion. Furthermore, PA toxin inactivates EF 2 intoxicated mice to an extent which would ultimately result in death.