Abstract
Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance.
Highlights
Animal African Trypanosomiasis (AAT), called nagana, is an infection of livestock and wild animal species with African trypanosomes, principally Trypanosoma congolense, Trypanosoma brucei sspp, Trypanosoma vivax and Trypanosoma suis, which is spread by the bite of infected tsetse flies
We have studied the phenomenon of diminazene resistance in T. congolense
This veterinary parasite does not have an equivalent of the T. brucei diminazene transporter TbAT1 (Munday et al, 2013) and it is clear that the T. brucei model for diminazene resistance, based on loss of TbAT1 function (De Koning et al, 2004; Graf et al, 2013), is not applicable to T. congolense
Summary
Animal African Trypanosomiasis (AAT), called nagana, is an infection of livestock and wild animal species with African trypanosomes, principally Trypanosoma congolense, Trypanosoma brucei sspp, Trypanosoma vivax and Trypanosoma suis, which is spread by the bite of infected tsetse flies. Accumulation of 0.1 μM [3H]-DA was monitored over 30 min in IL3000 and two of the derived DA-resistant clones, 4C2 and 6C3 This showed that even over half an hour there was no clear difference in the rate of accumulation of [3H]-DA in any of the three cell lines and that resistance is very unlikely to be the result of induced changes in drug uptake rate (Figure 6a). The most significantly upregulated (Helicase associated domain (HA2) putative: TcIL3000.A.H_000255300.1) and downregulated (Histone H4 putative: TcIL3000.A.H_000205600.1) genes are shown as circled asterisks to illustrate reproducibility across clones some cases, there was significant differential expression in only two of the DA-resistant clones, for example, a predicted long-chain fatty acid-CoA ligase (TcIL3000.A.H_00065500.1; mean q-value: .0356; mean Log fold change: −0.297)
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