Requirement of Thiols in the Adenosine-diphosphate Ribosylation of Elongation Factor-2 by Pseudomonas Aeruginosa Exotoxin A

The extent of the transfer of the adenosine 5'-diphosphate ribose (ADPR) moiety of nicotinamide adenine dinucleotide onto elongation factor 2 (EF-2) catalyzed by Pseudomonas aeruginosa exotoxin A (PA-toxin) was dependent upon the presence of a reducing agent, dithiotheritol (DTT). The reaction requires DTT in low concentration (1 to 10 mM) and in the absence of DTT less product, ADPR-EF 2, was formed. PA-toxin was fully activated by treatment with a denaturing agent, sodium dodecyl sulphate (SDS), in conjunction with DTT. In the presence of activated toxin, the maximum transfer of ADPR onto EF-2 was observed when EF-2 had been previously reduced with DTT. Denaturation of EF-2 prior to reduction did not produce a further increase in its ability to act as a substrate for PA-toxin. OHIO J. SCI. 81(2): 74, 1981 Most strains of Pseudomonas aeruginosa produce a lethal exotoxin, PA-toxin, that may represent a major virulence factor of this organism (Bjorn et al 1977; Pavlovskis et al 1977). This toxin is a heat labile enzyme that can inhibit eucaryotic protein synthesis by catalyzing the transfer of the adenosine 5' diphosphate ribose (ADPR) moiety of nicotinamide adenine dinucleotide (NAD) onto elongation factor-2 (EF-2) by a reaction identical to that catalyzed by diphtheria toxin (Iglewski et al 1976, Iglewski et al 1977, Iglewski and Kabat 1975). In its native state, the toxin is an inactive proenzyme that can be activated by treatment with urea and dithiothreitol (DTT) (Leppla et al 1978) or by freezing at -20 °C and thawing (Vasil et al 1977). Activation by the latter method results in the production of an active fragment presumably due to the presence of a contaminating protease. Vasil's study reported that following activation, DTT was not required in the reaction. Our study presents evidence that a requirement exists for the presence of thiols in the ADP ribosylation of EF-2 by PA^Tanuscript received 3 October 1979 and in revised form 14 February 1980 (#79-51). toxin and that this requirement manifests itself as an increase in the extent of the reaction and is due to the reduction of EF-2. MATERIALS AND METHODS Production and Purification of PA-Toxin Pa-toxin was produced and purified from P. aeruginosa strain PA-103 as described previously (Callahan 1974, Liu et al 1973) with some modifications. The organisms were grown in the dialysate of Trypticase Soy Broth (BBL), supplemented with 1% glycerol and 0.05 M monosodium glutamate, for 18-22 hr at 32 °C with shaking. The remaining steps were carried out at 5 °C. Bacteria were removed by centrifugation at 10,000 g for 40 min and the supernatant fluid of the culture was concentrated to 0.1 its original volume in an Amicon Model DC-2 hollow fiber dialyzer/concentrator fitted with a PM-10 Diaflo cartridge (Amicon). The concentrate was dialyzed 2 to 3 hr in the same unit against 0.01 M tris (hydroxymethyl) aminomethanehydrochloride (Tris-HCl) (pH 8.0 at 25 °C), and another ten-fold concentration was achieved by precipitation with 80% saturated (NH4) 2SO4 and dissolution in 0.01 M Tris-HCl (pH 8.0 at 25 °C). After dialysis, this material was applied to a diethylaminoethyl (DEAE) cellulose column (24 x 1.5 cm) which had been equilibrated with 0.01 M Tris-HCl (pH 8.0 at 25 °C). Elution was achieved by a stepwise increase in ionic strength. Elution concentrations were 0.10 M, .23 M, and 1.0 M NaCl in 0.01 M Tris-HCl (pH 8.0 at 25 °C).