Leveraging Inducible ORFeome Screens to Reveal New Mechanisms of Antileishmanial Drug Resistance

About This Grant

Project Summary Leishmaniasis, a disease caused by Leishmania parasites, poses a significant public health threat worldwide, and its reach is expanding to include parts of the United States. Treatment options remain limited, and the increasing emergence of drug-resistant parasites threatens the long-term effectiveness of existing therapies. Miltefosine, the only oral drug approved for leishmaniasis, is critical for treatment but increasingly compromised by resistance. The molecular mechanisms underlying miltefosine action and resistance remain poorly defined, limiting progress in combating treatment failures and developing more effective interventions. This project aims to address key gaps in our understanding of miltefosine resistance by leveraging advanced functional genomic tools developed in our laboratory. As proof of concept, we performed a pilot screen for miltefosine resistance in Leishmania using an inducible expression library of ~4000 open reading frames (ORFs) from the related parasite Trypanosoma brucei (the TbORFeome). We identified ZC3H18, an RNA-binding protein (RBP), as a novel resistance factor, making it the first RBP experimentally validated to mediate drug resistance in kinetoplastid parasites. This discovery highlights post-transcriptional regulation as a new, underexplored mechanism of antileishmanial drug resistance. In Aim 1, we will define the pathways regulated by Leishmania ZC3H18 to understand how it mediates miltefosine resistance. Building on our success with the TbORFeome library, we have developed a genome-scale, barcoded inducible overexpression library covering ~8,000 L. donovani genes (the LdORFeome). This library represents a major advance in Leishmania genetics, featuring near-complete coverage of protein coding genes, robust expression control through a novel split-Cre recombinase system, and an integrated barcoding platform for high- throughput analysis. In Aim 2, we will screen the LdORFeome library to uncover additional miltefosine resistance genes, which could provide a deeper understanding of the drug's mode of action. This work will uncover novel genetic determinants and regulatory pathways involved in miltefosine resistance, provide new insight into drug mechanism, and establish a versatile functional genomics platform for Leishmania. The findings have the potential to inform clinical strategies and support the development of next- generation therapies for leishmaniasis.