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

The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [ 3 H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a Tbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells. Organisms of the genus Trypanosoma cause a range of infectious diseases, including human African trypanosomiasis (HAT), nagana in cattle, and dourine in equines (2), and are of major public health and economic importance in sub-Saharan Africa. Chemotherapy is used against many of the human- and animal-infective parasites. 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. Furthermore, a series of drug-resistant parasites from both the laboratory and the field have all been shown to be defective in P2-mediated transport using a novel fluorescence test (28), and a restriction fragment length polymorphism (RFLP)-based approach has been used to infer the presence of drug-resistant alleles in populations of human-infective parasites from Uganda (22). Previous work had indicated that two independent genetic mechanisms, namely, introduction of point mutations (21) and deletion of the TbAT1 gene (21, 18), are associated with the acquisition of drug resistance, which implies that several mechanisms can alter P2 transporter activity, causing drug resistance. A more complete understanding of these mechanisms will be essential to underpinning the development of better molecularly based diagnostic tools to monitor drug resistance.