The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes

Abstract

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

Fig. 1.

[³H]Pentamidine uptake in procyclic s427 trypanosomes at a permeant concentration of 30 nM (A) and 1 μM (B). Cells were incubated with [³H]pentamidine for 60 s in the presence or absence of various concentrations of unlabeled pentamidine as indicated. The insets depict the conversion of the inhibition plots to Michaelis-Menten saturation plots. Both graphs are representative of at least six independent experiments, each performed in triplicate. The data shown represent the average of these triplicate determinations ± S.E.M. When not shown, error bars fall within the symbols.

Fig. 2.

High-affinity pentamidine transport in bloodstream (A) and procyclic (B) forms of T. brucei brucei in the presence of various concentrations of unlabeled inhibitors:▴, pentamidine; ○, DB820; ■, CPD0801. [³H]Pentamidine concentration was 25 nM, and incubation time was 60 s. The experiments shown are representative of at least three independent experiments, each performed in triplicate. Error bars are standard errors.

Fig. 3.

Transport of 1 μM [³H]diminazene in procyclic trypanosomes. A, procyclic s427 were incubated for the indicated times with radiolabel in the presence (□) or absence (■) of 1 mM unlabeled diminazene aceturate. The correlation coefficient for the 1 μM line was 0.97 and it was highly significantly different from 0 (P < 0.00001; F test), whereas the 1 mM line was not significantly different from 0 (P = 0.50; F test). B, inhibition of [³H]diminazene transport by various concentrations of pentamidine (●), unlabeled diminazene (○), adenine (▴), and inosine (□). C, Michaelis-Menten saturation curve for [³H]diminazene in procyclic cells, representative of four independent experiments. All experiments were performed in triplicate, and error bars indicate S.E.M.

Fig. 4.

Transport of [³H]pentamidine in wild-type and ABR cell line lines. Transport of 30 nM (A and B) or 1 μM [³H]pentamidine (C and D) was assessed in bloodstream forms of the s427WT (A and C) and ABR (B and D) cell lines over 60 s in the presence or absence of various concentrations of propamidine (■) or unlabeled pentamidine (○). Pentamidine uptake was expressed as picomoles per 10⁷ cells per second. The experiments were performed in triplicate; error bars represent S.E. of internal replicates. Data shown are representative of at least three identical and independent experiments.

Fig. 5.

Transport of 1 μM [³H]pentamidine (■) or 1 μM [³H]diminazene (□) in four different cell lines. Transport rates were derived by linear regression from time courses with points (in triplicate) at 0, 30, 60, 120, 300, 450, and 600 s. Zero uptake levels and saturability were verified in the presence of 1 mM unlabeled permeant. Bars show the average transport of three to four experiments (each performed in triplicate) and S.E. Representative experiments are shown in Supplemental Fig. S4.

Fig. 6.

Saturation plot of [³H]diminazene transport in bloodstream forms of the tbat1(−/−) cell line. Transport of 1 μM label was determined in triplicate at 5 min of incubation; error bars represent S.E. The experiment shown is representative of three identical experiments.