Investigating the role of Ros3 in differential susceptibility of Leishmania to miltefosine using a CRISPR/Cas9 approach

Abstract

The first choice drug for cutaneous leishmaniasis treatment in Brazil, where Leishmania (Viannia) braziliensis is the most prevalent etiological agent, is meglumine antimoniate, administered intravenously for 20 days. Success rates for antimonial therapy can be as low as 50% in some areas. As part of an effort to evaluate whether miltefosine would be applicable to leishmaiasis chemotherapy in Brazil, we found that susceptibility to this drug varied among Brazilian isolates of L. (V.) braziliensis. This was an intriguing finding because these isolates were never exposed to this drug, not yet approved for use in Brazil. Aiming to understand what are the molecular bases of this differential susceptibility we studied in these Brazilian isolates some of the previously described pathways involved in miltefosine resistance in Leishmania parasites. We demonstrated the existence of differences in drug uptake between these isolates, with a reduced uptake in tolerant parasites compared to sensitive and reference strains. Polymorphisms in the miltefosine transport machinery and differences in efflux/metabolism rates, fitness and drug compartmentalization were not found to be associated to drug susceptibility in these isolates. Transcriptome analysis raised the hypothesis that differences in miltefosine susceptibility and uptake in these isolates might be caused by differential expression of Ros3 (a component of the miltefosine transport machinery). Thus, the aim of this project is to investigate the role of the protein Ros3 in the susceptibility of Leishmania parasites to miltefosine. This will be done by using CRISPR/Cas9 mediated genome edition to produce mutants with a tagged Ros3 protein or knocked out for Ros3 expression. The initial approach is to establish these mutants in a Leishmania major background, available at the laboratory in Oxford. The expertise acquired during this project will be of great importance for investigating Ros3 role in MF susceptibility in these isolates and to functionally validating the other target genes found differentially expressed among sensitive and tolerant L. (V.) braziliensis clinical isolates. (AU)