Modulation of mitochondrial electrical potential: a candidate mechanism for drug resistance in African trypanosomes

Abstract

Bloodstream forms of four populations of the livestock pathogen Trypanosoma congolense, isolated from different natural infections, have been shown to exhibit a wide range of sensitivities to the trypanocide isometamidium chloride (Samorin®). In mice the 50% curative doses (CD50) for Samorin range from 0.007 to 20 mg/kg body weight. Uptake of isometamidium chloride demonstrated Michaelis–Menten-type kinetics in all the populations, with Km values in the range 0.35–0.87 μM, and Vmax varied from 17 to 216 pmol/min per 108 cells. The magnitude of Vmax was correlated with sensitivity to the drug. In contrast, no correlation was observed between Km values and drug sensitivity. Pulse–chase experiments indicated two compartments for accumulation of drug. The first consists of freely diffusible drug that is invariant between populations; the other consists of retained isometamidium, which is of variable magnitude between the populations and is correlated with drug sensitivity. Autoradiography and fluorescence microscopy demonstrated initial, rapid accumulation of the drug within the mitochondrion, specifically the kinetoplast. In a drug-sensitive population of T. congolense, agents affecting mitochondrial function were shown to produce dose-dependent inhibition of mitochondrial membrane potential (ΔΨmito), as measured by the accumulation of the lipophilic cations [3H]methyltriphenylphosphonium iodide or rhodamine 123. The agents also produced parallel inhibition of isometamidium uptake, suggesting an involvement of ΔΨmito in the accumulation of the drug. When characterized in each of the four populations, the spontaneous ΔΨmito was shown to be characteristic of each population and was correlated with Vmax for drug uptake and sensitivity to the drug in vitro and in vivo. We therefore conclude that in T. congolenseΔΨmito is an important determinant of the rate and accumulation of the trypanocide isometamidium chloride. Populations of this trypanosome species vary with respect to ΔΨmito, which is correlated with sensitivity to isometamidium. We suggest that when exposed to drug, the selection of such populations represents a novel mechanism of drug resistance in protozoan parasites.