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Transcriptional differentiation of Trypanosoma brucei during in vitro acquisition of resistance to acoziborole

ABSTRACT: Purpose: Acoziborole is a recently developed benzoxaborole class compound, currently in clinical trials, for stage 1 and stage 2 treatment of Human African Trypanosomiasis. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. By characterising in vitro drug-resistance, this study aimed to gain a better understanding of the mechanisms involved in acoziborole resistance in the clinicaly relevant Trypanosoma brucei Methods: Drug resistance was generated in vitro through incremental dosage of acoziborole. RNA was isolated from axenic cultures of drug-resistant and parental drug sensitive cells and submitted for RNA-seq Results: Transcriptomics analysis revealed widepread downregulation of transcripts associated with mammalian-infective bloodstream-form parasites. Conversely, transcripts associated with insect-stage procyclic form parasites were increased, indicating that the resistant cells had undergone an unspecified "differentiation event", albeit on a transcriptomic level Conclusions: Trypanosoma brucei resistance to acoziborole can be generated under in vitro axenic conditions, and "transcriptional differentiation" is a mechanism of resistance. However, it is unknown whether this phenomenon is relevant to an in vivo setting

ORGANISM(S): Trypanosoma brucei brucei

PROVIDER: GSE168394 | GEO | 2021/03/07

REPOSITORIES: GEO

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Project description: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 sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage 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. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for 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.

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Transcriptional differentiation of Trypanosoma brucei during in vitro acquisition of resistance to acoziborole

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