Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei

Abstract

217; importance, 135 23 Main text (intro, results, discussion), 4953. 24 Abstract 25The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited 26 and beset by toxicity and emergent resistance. Furthermore, our understanding of drug 27 mode-of-action and potential routes to resistance is limited. Forward genetic approaches 28 have revolutionised our understanding of drug mode-of-action in the related kinetoplastid 29 parasite, Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNAi 30 library in the current anti-leishmanial drugs, sodium stibogluconate (antimonial), 31 paromomycin, miltefosine and amphotericin-B. Identification of T. brucei orthologues of the 32 known Leishmania antimonial and miltefosine plasma membrane transporters effectively 33 validated our approach, while a cohort of 42 novel drug efficacy determinants provides 34 new insights and serves as a resource. Follow-up analyses revealed the antimonial 35 selectivity of the aquaglyceroporin, TbAQP3. A lysosomal major facilitator superfamily 36 transporter contributes to paromomycin/aminoglycoside efficacy. The vesicle-associated 37 membrane protein, TbVAMP7B, and a flippase contribute to amphotericin-B and 38 miltefosine action, and are potential cross-resistance determinants. Finally, multiple 39 phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania 40 miltefosine transporter, a putative β-subunit/CDC50 co-factor, and additional membrane-41 associated hits, affect amphotericin-B efficacy, providing new insights into mechanisms of 42 drug uptake and action. The findings from this orthology-based chemogenomic profiling 43 approach substantially advance our understanding of anti-leishmanial drug action and 44 potential resistance mechanisms, and should facilitate the development of improved 45 therapies, as well as surveillance for drug-resistant parasites.46 Importance 47 Leishmaniasis is a devastating disease caused by the Leishmania parasites and is 48 endemic to a wide swathe of the tropics and sub-tropics. While there are drugs available 49 for the treatment of leishmaniasis, these suffer from various challenges, including the 50 spread of drug resistance. Our understanding of anti-leishmanial drug action and the 51 modes of drug resistance in Leishmania is limited. The development of genetic screening 52 tools in the related parasite, Trypanosoma brucei, has revolutionised our understanding of 53 these processes in this parasite. Therefore, we applied these tools to the anti-leishmanial 54 drugs, identifying T. brucei orthologues of known Leishmania proteins that drive drug 55 uptake, as well as a panel of novel proteins not previously associated with anti-leishmanial 56 drug action. Our findings substantially advance our understanding of anti-leishmanial 57 mode-of-action and provide a valuable starting point for further research.58 101 efficacy (reviewed in (14)). In addition, the generation of drug resistant Leishmania in the 102 laboratory and various 'omi...

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

“…The copyright holder for this this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.08.471712 doi: bioRxiv preprint P a g e | 27 flippase, involved in the architecture of the plasma membrane lipids (93,(144)(145)(146). The combined roles of redox changes and sterol changes could influence the ability to select and sustain resistance in vivo.…”

“…flippase, involved in the architecture of the plasma membrane lipids (93,(144)(145)(146). The combined roles of redox changes and sterol changes could influence the ability to select and sustain resistance in vivo.…”

Establishment of quantitative RNAi-based forward genetics inEntamoeba histolyticaand identification of genes required for growth

“…The RNAi library has genome-wide coverage 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 DNAfragments inferred from read mapping results (Fig. S2), rather than on more traditional read mapping coverage or CDS overlap counting strategies.…”

“…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 [30,31], DNA repair [32], virulence [33], transmission [34,35], and drug therapies [36][37][38][39]. 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 [30,31].…”

“…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 [30,31]. More recent studies used next generation sequencing on non-clonal mutant populations in order to identify mutants and quantify their relative abundance [32][33][34][35][36][37][38][39]. 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.…”