Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F ₁F _o-ATPase

Abstract

Background

Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine.

Methodology/Principal Findings

Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F ₁ ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance.

Conclusions/Significance

Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F ₁ ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential.

Author Summary

Isometamidium is the only prophylactic treatment of Animal African Trypanosomiasis, a wasting disease of livestock and domestic animals in sub-Saharan Africa. Unfortunately resistance threatens the continued utility of this drug after decades of use. Not only does this disease have severe impacts on agriculture, but some subspecies of Trypanosoma brucei are human-infective as well (causing sleeping sickness) and there is concern that cross-resistance with trypanocides of the diamidine class could further undermine treatment of both veterinary and human infections. It is therefore essential to understand the mechanism of isometamidium resistance and the likelihood for cross-resistance with other first-line trypanocides. Here, we report that isometamidium resistance can be caused by a mutation in an important mitochondrial protein, the γ subunit of the F ₁ ATPase, and that this mutation alone is sufficient for high levels of resistance, cross-resistance to various drugs, and a strongly reduced mitochondrial membrane potential. This report will for the first time enable a structural assessment of isometamidium resistance genes in T. brucei spp.