Biology subjects

De Koning, H.

Publications and source records attributed to De Koning, H..

2 recordsLinked to original sources

bioRxiv2021

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential drug sensitivity

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon drug sensitivity. Like T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly reduced in T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Comparative transcriptomics analysis showed higher levels of activity associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation and the succinate shunt in T. congolense. However, based on omics analysis and chemical inhibition, there does not appear to be significant levels of oxidative phosphorylation. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with pharmacological inhibitors, confirming a lack of essential electron transport chain activity in T. congolense, but increased sensitivity to inhibition of mitochondrial pyruvate import. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold greater EC50 against the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

microbiology↗

bioRxiv2020

Diminazene resistance in Trypanosoma congolense is linked to reduced mitochondrial membrane potential and not to reduced transport capacity

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 {Psi}m 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 {Psi}m that limits mitochondrial accumulation of the drug.

microbiology↗