Resistance to dl-α-difluoromethylornithine by clinical isolates of Trypanosoma brucei rhodesiense: Role of S-adenosylmethionine

Abstract

The ornithine decarboxylase (ODC) inhibitor dl-α-difluoromethylornithine (DFMO) has emerged as a new treatment for West African sleeping sickness but is less effective against East African sleeping sickness. We examined uncloned clinical isolates of Trypanosoma brucei rhodesiense, agent of the disease in East Africa, which were refractory to DFMO in laboratory infections, for characteristics that would explain their resistance. None of the isolates were from patients treated with DFMO. Two isolates took up [ 3H]DFMO at 50–70% lower rates than drug-sensitive strains but ODC activities, K i values for DFMO, spermidine and spermine uptake rates, polyamine content and inhibition of polymamine metabolism by DFMO were statistically (P < 0.05) similar between sensitive and refractory isolates. One cloned strain, continuously passaged in vivo under DFMO pressure and included for comparison, had >85% lower ODC activity and up to 14-fold higher putrescine uptake rates than sensitive controls. A statistically important trend was the metabolism of S-adenosylmethionine (AdoMet): activities of AdoMet synthetase and AdoMet decarboxylase were 2- to 5-fold and 3- to 40-fold lower in resistant strains, respectively, while intracellular AdoMet pools (AdoMet + decarboxylated AdoMet) that were >60-fold elevated in sensitive strains during DFMO treatment, increased only 9-fold in refractory isolates. The extreme elevation of the AdoMet pool in sensitive isolates from 0.7 to 44 nmol/mg protein and an intracellular pool concentration of >5 mM may lead to an imbalance in methylation of proteins or other cell constituents as a consequence of DFMO action. These studies indicate that the metabolism of AdoMet is altered significantly in DFMO refractory isolates and suggest that differences in AdoMet metabolism may be responsible for increased tolerance to DFMO.