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

<jats:title>Abstract</jats:title><jats:p>The arsenal of drugs used to treat leishmaniasis, caused by <jats:italic>Leishmania</jats:italic> 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 revolutionised our understanding of drug mode-of-action in the related kinetoplastid parasite, <jats:italic>Trypanosoma brucei</jats:italic>. Therefore, we screened our genome-scale <jats:italic>T. brucei</jats:italic> RNAi library in the current anti-leishmanial drugs, sodium stibogluconate (antimonial), paromomycin, miltefosine and amphotericin-B. Identification of <jats:italic>T. brucei</jats:italic> orthologues of the known <jats:italic>Leishmania</jats:italic> 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 <jats:italic>T. brucei</jats:italic> orthologue of the <jats:italic>Leishmania</jats:italic> miltefosine transporter, a putative β-subunit/CDC50 co-factor, 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 anti-leishmanial drug action and potential resistance mechanisms, and should facilitate the development of improved therapies, as well as surveillance for drug-resistant parasites.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>Leishmaniasis is a devastating disease caused by the <jats:italic>Leishmania</jats:italic> parasites and is endemic to a wide swathe of the tropics and sub-tropics. While there are drugs available for the treatment of leishmaniasis, these suffer from various challenges, including the spread of drug resistance. Our understanding of anti-leishmanial drug action and the modes of drug resistance in <jats:italic>Leishmania</jats:italic> is limited. The development of genetic screening tools in the related parasite, <jats:italic>Trypanosoma brucei</jats:italic>, has revolutionised our understanding of these processes in this parasite. Therefore, we applied these tools to the anti-leishmanial drugs, identifying <jats:italic>T. brucei</jats:italic> orthologues of known <jats:italic>Leishmania</jats:italic> proteins that drive drug uptake, as well as a panel of novel proteins not previously associated with anti-leishmanial drug action. Our findings substantially advance our understanding of anti-leishmanial mode-of-action and provide a valuable starting point for further research.</jats:p></jats:sec>