Alteration to one of three adenosine transporters is associated with resistance to Cymelarsan in Trypanosoma evansi

Abstract

Continuous exposure of Trypanosoma evansi bloodstream forms to Cymelarsan in vitro resulted in the induction of resistance to the drug over a period of 4.5 months. Induction of resistance to Cymelarsan was accompanied by increased resistance to both Arsobal and Berenil, but Cymelarsan-resistant trypanosomes remained sensitive to Suramin and Antrycide. Since resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake, the adenosine metabolism in drug-sensitive and drug-resistant clones was measured. The initial rate of [3H]-adenosine uptake was much higher in sensitive trypanosomes than in drug-resistant parasites, but the amount of radiolabel accumulated by each population was similar. Both adenine and inosine inhibited incorporation of [3H]-adenosine in each population, but in quite different ways. Adenine inhibited more than 80% of adenosine incorporation in drug-sensitive trypanosomes but suppressed less than half of this process in the resistant population. In contrast, inosine inhibited only 10-15% of adenosine incorporation in the sensitive parasites but was inhibitory to a much greater extent in resistant trypanosomes. Lysis of drug-sensitive trypanosomes by 1 microM Cymelarsan was inhibited by adenosine, adenine and Berenil but not by inosine. Furthermore, in drug-sensitive trypanosomes, adenosine uptake could be reduced in three stages by the sequential addition of inosine, Berenil and adenine, whereas in drug-resistant parasites a reduction in adenosine uptake was caused only by the addition of inosine and adenine. These observations provide evidence that the acquisition of resistance to Cymelarsan is accompanied by the loss of one of three adenosine transporters in T. evansi. In drug-sensitive trypanosomes, this transporter mediates the entry of adenine and also of Cymelarsan and Berenil into the parasites.

Mechanisms of Arsenical and Diamidine Uptake and Resistance in Trypanosoma brucei

“…Since Carter and Fairlamb showed that adenosine and adenine protect T. b. brucei from melarsen-induced lysis in vitro (6), several studies have pointed to a role for the P2 transport activity in the resistance of African trypanosomes to both melaminophenyl arsenicals and diamidines (2,5,27,29,30). A T. b. brucei gene, TbAT1, was found to encode an adenine-sensitive adenosine transporter when expressed in yeast (19), but formal proof that TbAT1 encodes the P2 activity in trypanosomes has awaited the gene deletion study presented here.…”

Purine and pyrimidine transport in pathogenic protozoa: From biology to therapy

“…Nucleoside transport has been extensively studied in the trypanosomatidae, particularly in T. b. brucei and L. donovani , but to a much lesser extent also in Trypanosoma evansi [54–56], Trypanosoma equiperdum [57], Trypanosoma congolense [58], Trypanosoma vivax [59] and Trypanosoma cruzi [10].…”

Multiple Genetic Mechanisms Lead to Loss of Functional TbAT1 Expression in Drug-Resistant Trypanosomes

“…However, all drugs currently registered for use carry significant problems related to administration, toxicity, increasing incidence of treatment failure, and in the case of animal trypanosomiasis, drug resistance (1). Previous work has shown that defects in the P2 aminopurine transporter, encoded by the TbAT1 gene (the same gene in Trypanosoma equiperdum is referred to as TeAT1 [Trypanosoma equiperdum AT1] in this article), are linked to drug resistance in Trypanosoma brucei brucei (11,21), Trypanosoma brucei gambiense (22), and Trypanosoma brucei rhodesiense (28), as well as in Trypanosoma evansi (24,32) and T. equiperdum (3), very close phylogenetic relatives of T. brucei (27). The P2 transporter has been shown to be capable of carrying both melaminophenyl arsenical (11) and diamidine (10,14,15,18) classes of drug into African trypanosomes in the T. brucei group.…”