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

Abstract

The P2 aminopurine transporter, encoded by Tb AT1 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 Tb AT1 to be in the subtelomeric region of chromosome 5 and determined the status of the Tb AT1 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, Tb AT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, Tb AT1 is present, but expression at the RNA level is no longer detectable. Further characterization of Tb AT1 in T. equiperdum revealed that a loss of heterozygosity at the Tb AT1 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 ΔTb at1 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.