Diminazene resistance inTrypanosoma congolenseis linked to reduced mitochondrial membrane potential and not to reduced transport capacity

Abstract

Trypanosoma congolense is one of the principal agents causing livestock trypanosomiasis in sub-Saharan Africa. This wasting disease is costing these developing economies billions of dollars and undermining food security. Only two old drugs, the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induce diminazene resistance in T. congolense laboratory strain IL3000 in vitro. Resistance was stable and not deleterious to in vitro growth. There was no cross-resistance with the phenanthridine drugs, with melaminophenyl arsenicals, with two promising new oxaborole trypanocides, nor with other diamidine trypanocides such as pentamidine, except the close structural analogues DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3H]-diminazene was also partly inhibited by folate, as well as by competing diamidine drugs, albeit at quite high concentrations, and uptake of tritiated diminazene and pentamidine was slow and low affinity. Uptake of [3H]-folate was in turn partly inhibited by diminazene, and inhibition of diminazene uptake by folate and pentamidine appeared to be additive, indicating multiple low affinity transport mechanisms for the drug. Expression of the T. congolense folate transporters TcoFT1-3 in diminazene-resistant T. b. brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3H]-diminazene uptake was not different in T. congolense IL3000 and its diminazene resistant clones and RNAseq and whole-genome sequencing of multiple resistant clones did not reveal major changes in folate transporter sequence or expression. Instead, flow cytometry revealed a strong and stable reduction in the mitochondrial membrane potential Ψm in all resistant clones. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters and that resistance is the result of a reduction in Ψm that limits mitochondrial accumulation of the drug.

Antiparasitic and Cytotoxic Activity of Bokkosin, A Novel Diterpene-Substituted Chromanyl Benzoquinone From Calliandra portoricensis

“…It is difficult to speculate at this point as to the reasons for this. However, it is known that the diamidine transporters of both trypanosome species are completely different, and thus those drug transporters are highly unlikely to be involved in the uptake of bokkosin; however, in both species these cationic diamidines target the mitochondrion aided by the mitochondrial membrane potential, which is reduced in T. congolense 6C3 (Carruthers et al, 2020). A possible interpretation of these observations would be that the target for bokkosin is not mitochondrial.…”