Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei

Abstract

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative β-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance inTrypanosoma brucei

“…All T. brucei AQP paralogs are predicted as topologically similar, but nevertheless possess distinct properties and subcellular localisations [13,18–23,73]. TbAQP2 is essential for pentamidine and melarsoprol uptake [13], while TbAQP3 is associated with susceptibility to antimonial compounds including sodium stibogluconate [71], indicating transport specificity.…”

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

“…Illumina sequencing was used to analyze the pilot and final libraries, and the fragmented gDNA that was used to construct each library. Notably, our approach for analysis of the sequencing data is novel and differs from previous approaches for analysis of RNAi screens [33][34][35][36][37][38][39][40] since all results presented here are based on full-length DNA-fragments inferred from read mapping results (S2 Fig), rather than on more traditional read mapping coverage or CDS overlap counting strategies. Our approach was designed to better represent the actual DNA fragments present in the plasmid library, instead of just the short ends of these fragments that were sequenced on the Illumina platform.…”

“…In other parasites, RNAi knockdown has been successfully adapted for forward genetic approaches, and this has been very effective in uncovering genes relevant to parasite metabolism [31,32], DNA repair [33], virulence [34], transmission [35,36], and drug therapies [37][38][39][40]. Initially, these approaches relied on the generation of clonal lines after selection, and Sanger sequencing analysis of individual clones to identify the knocked down gene [31,32].…”

“…Initially, these approaches relied on the generation of clonal lines after selection, and Sanger sequencing analysis of individual clones to identify the knocked down gene [31,32]. More recent studies used next generation sequencing on non-clonal mutant populations in order to identify mutants and quantify their relative abundance [33][34][35][36][37][38][39][40]. The latter approaches are useful because they can quantitatively identify mutants that are enriched by selection, and mutants that are less abundant or are lost after selection.…”

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei

“…All T. brucei AQP paralogs are predicted as topologically similar, but nevertheless possess distinct properties and subcellular localisations [12,[17][18][19][20][21][22]81]. TbAQP2 is essential for pentamidine and melarsoprol uptake [12], while TbAQP3 is associated with susceptibility to antimonial compounds including sodium stibogluconate [71], indicating transport specificity.…”