Generation and Implementation of a whole-genome Gain-of-Function Library in Trypanosoma brucei: Insights into Melarsoprol Resistance
Open AccessTrypanosoma brucei and kinetoplastida relatives are early branching eukaryotic protozoa with highly divergent genomes from established model eukaryotes. Forward genetics approaches are the best tools for uncovering novel aspects Trypanosomatid biology, pathogenesis, and therapeutic approaches against this family of parasites that effects the health of 1 billion people on Earth. Here we have generated a T. brucei cloned ORFeome consisting of over 90% of the targeted genome and used it to make an inducible Gain-of-Function library for broadly applicable forward genetic screening. Treating parasitic infections requires the identification of medicines that target aspects of their biology that are distinct from the host. One such pathway that has been widely investigated for drug development and is shared among Trypanosomatids is the biosynthesis of trypanothione, a form of glutathione critical in Trypanosomatid redox metabolism. Melarsoprol is a classic drug of last resort against African trypanosomiasis inhibits trypanothione, yet its mode of cell killing has remained unclear. It is widely speculated that melarsoprol killing arises from its effects on more than one cellular target. Thus, we conducted an ORFeome-based Gain-of-Function genetic screen to identify genes whose induced expression can promote survival in the presence of melarsoprol. Gain-of-Function library induction resulted in the isolation of robust survivor populations in all three biological replicates. 57 genes were overrepresented in survivor populations compared to input library controls. Among the most highly overrepresented genes was γ-glutamylcysteine synthetase, a biosynthetic precursor of trypanothione whose overexpression promotes melarsoprol resistance in vitro. Additionally, we found that GSH1 overexpression alleviates melarsoprol’s cell cycle stall phenotype and restores the actively replicating cell population. This result provides critical proof of Gain-of-Function library functionality and further supports the importance of trypanothione as a key target of melarsoprol. In addition, we found significant clusters of genes that function in gene expression regulation and largely unstudied genes that localized to either the mitochondria or flagellum. Single gene overexpression constructs for the top mitochondrial, flagellum, and gene expression hits promoted melarsoprol resistance in vitro. It is unclear at this time if mitochondrial and flagellar hits arising from the gain-of-function screen protect the cell from melarsoprol by way of the trypanothione pathway, alternative redox pathways, or complex indirect effects (such as transport). Thus, this study has produced two powerful new tools for kinetoplastida research, a T. brucei cloned ORFeome and an inducible Gain-of-Function library. Collectively, the tools and discoveries described herein are expected to promote major advances in kinetoplastida biology and therapeutic development in the years to come.
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