A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment

Abstract

Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library’s usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research. IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library’s usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition

“…The GSH1 enzyme is essential and represents the rate-limiting step of trypanothione biosynthesis (Huynh et al ., 2003; Agnihotri et al ., 2016). Induced expression of GSH1 was identified in a gain-of-function screen for genes whose expression can promote survival during melarsoprol treatment and resulted in a 1.5-fold increase in the relative EC 50 of melarsoprol (Carter et al ., 2020). To determine if the cell cycle and DNA synthesis defects observed during melarsoprol treatment were associated with the trypanothione pathway, we overexpressed the gene encoding GSH1 ( Tb927.10.12370 ) and analysed the effects during melarsoprol treatment (Supplementary Fig.…”

“…The matter of cell viability is always a concern when conducting experiments under toxifying conditions. From previous studies, we know that complete cell death during melarsoprol treatment requires 3 days in 35 n m , 4 days in 26 n m and 5 days in 17 n m (Carter et al ., 2020). Thus, it was notable that we observed a 20% decrease in DNA synthesis in 17 n m melarsoprol within 24 h (Fig.…”

“…Trypanosoma brucei cell lines, culture methods and cell counting Cell lines were generated from Lister 427 bloodstream-form parasites derived from the single marker line (Wirtz et al, 1999) and maintained in HMI-9 medium (Hirumi and Hirumi, 1994) supplemented with serum plus (Sigma-Aldrich Serum Plus Medium Supplement 14008C). GSH1 overexpression cell line was generated by cloning Tb927.10.12370 into pLEW100v5-BSD, followed by transfection into SM by AMAXA Nucleofector (Burkard et al, 2007), and evaluated in a previous publication (Carter et al, 2020). Antitrypanosomal drugs were resuspended in DMSO (melarsoprol, pentamidine and fexinidazole) or water (eflornithine) prior to dilution in HMI-9 at the indicated concentrations and evaluated, generally 24 h posttreatment (unless otherwise indicated in supplemental materials).…”

Genome-scale RNAi screens in African trypanosomes

“…A number of other functional genetic screening platforms have been established in the trypanosomatids and these are briefly outlined below. Several genome-scale over-expression or gain-of-function libraries have been screened to identify drug targets or drug resistance determinants; these include bloodstream form T. brucei libraries [76,87,88] and Leishmania cosmid libraries [89][90][91][92][93][94][95][96]. In addition, a novel genome-scale screen in T. brucei involved expression of proteins fused to an RNA-binding domain in a strain with a reporter incorporating a tether for the RNA-binding domain in the 3'-untranslated region; this screen implicated approx.…”

Structure, function and druggability of the African trypanosome flagellum

“…Similarly, whole‐genome gain‐of‐function libraries (Begolo et al, 2014; Carter et al, 2020) have been deployed to identify drug resistance mechanisms that might occur in the field. Combined with powerful genetic tools for functional analysis, such as inducible RNAi, CRISPR knockouts, and inducible ectopic‐expression, gene‐function can be rapidly addressed, allowing the design of combination therapies to target different pathways, reducing the risk of resistant strains being selected.…”