Abstract
ObjectivesTrypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.MethodsThe TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.ResultsAll MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical.ConclusionsTbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
Highlights
The protozoan parasite Trypanosoma brucei is the aetiological agent of human African trypanosomiasis (HAT or sleeping sickness)
Despite the recent introduction of nifurtimox/eflornithine combination therapy for the late, cerebral stage of HAT,[1] there is an urgent need for new drugs, driven in part by resistance to the diamidines, phenanthridines and melaminophenyl arsenicals (MPAs) that have been the central pillars of African trypanosomiasis treatment for decades.[2]
We report that loss of the wild-type TbAQP2 open reading frame (ORF) was observed in all MPXR strains (T. b. brucei, T. b. gambiense and T. b. rhodesiense), whether they were selected for resistance to MPAs or pentamidine, including strains selected in vivo and able to be transmitted by tsetse flies
Summary
The protozoan parasite Trypanosoma brucei is the aetiological agent of human African trypanosomiasis (HAT or sleeping sickness). Brucei is one of the pathogens that cause animal African trypanosomiasis, a wasting disease of livestock. Despite the recent introduction of nifurtimox/eflornithine combination therapy for the late, cerebral stage of HAT,[1] there is an urgent need for new drugs, driven in part by resistance to the diamidines, phenanthridines and melaminophenyl arsenicals (MPAs) that have been the central pillars of African trypanosomiasis treatment for decades.[2] An understanding of the mechanisms of resistance,.
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