Understanding the role of epistasis in Leishmania drug resistance

• Applicant: Tom Beneke, Ph.D.
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 532631727

Leishmania parasites are transmitted into their vertebrate host by the bite of a sand fly and can cause a range of clinical manifestations, including local (cutaneous leishmaniasis, CL) and visceral infections (visceral leishmaniasis, VL). Since there is no human vaccine available, anti-leishmanial drugs remain the first line of defence for years to come and it is important that we understand how Leishmania species become resistant against them. One drug resistance mechanism employed by Leishmania is genome instability, leading to the hypothesis that Leishmania rapidly adapts to changing environments by alternating gene dosages. However, I propose to extend this hypothesis to include genetic interactions (or epistasis), meaning that drug resistance is conferred by the combined effect of a simultaneous perturbation in two or multiple genes. In this project, we want to test this hypothesis by employing a newly developed CRISPR/Cas9 cytosine base editor (CBE) toolbox (“LeishBASEedit”) that enables for the first time reverse genetic screens via delivery of plasmid libraries in Leishmania. I propose to use this new method to perform a genome-wide loss-of-function drug resistance screen in a representative CL (L. mexicana) and VL (L. donovani) strain. In the first funding period, a PhD student and research assistant will pursue under my supervision this genome-wide CBE screen with four highly relevant anti-leishmanial drugs (liposomal amphotericin B, miltefosine, DNDI-6148 and DNDI-0690). In a second independent step, we will adapt the CBE targeting approach to target two or multiple genes at once and use this to perform a combinatorial loss-of-function and drug resistance screen in a consecutive second funding period. Drug resistance mechanisms of newly identified biomarkers will then be characterised by producing and studying individual mutants. To enable other researchers to pursue similar screens, we will organise a CRISPR screening workshop and share generated genome-wide single and combinatorial targeting libraries with the community. Our project will for the first time deliver data on Leishmania drug resistance for nearly all protein coding genes and their genetic partners, highlighting large gene regulatory networks relevant for a range of eukaryotic and parasitic systems. It will also yield genome-wide viability data for two Leishmania species, allowing to identify species-specific differences. We envision that here identified drug biomarkers will allow us to detect resistant parasites in the field, use selected treatments for these and in the future help to develop new drugs that can bypass resistance mechanisms.

• DFG Programme: Research Grants
• International Connection: Switzerland, United Kingdom
• Cooperation Partners: Professorin Dr. Eva Gluenz; Professor Dr. David Horn; Dr. Susan Wyllie