Ros3 (Lem3p/CDC50) Gene Dosage Is Implicated in Miltefosine Susceptibility in Leishmania (Viannia) braziliensis Clinical Isolates and in Leishmania (Leishmania) major

The Ros3 protein is a component of the MT-Ros3 transporter complex, considered as the main route of miltefosine entry in Leishmania. L. braziliensis clinical isolates presenting differences in miltefosine susceptibility and uptake were previously shown to differentially express ros3. In this work, we showed that the ros3 gene copy number was increased in the isolate presenting the highest rates of miltefosine uptake and, thus, the highest susceptibility to this drug. The role of the ros3 gene dosage in miltefosine susceptibility was then investigated through a modulation of the gene copy number using two distinct approaches: through an overexpression of ros3 in a tolerant L. braziliensis clinical isolate and in L. major and by generating mono- and diallelic knockouts of this gene in L. major using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 (Cas = CRISPR-associated). Although the levels of ros3 mRNA were increased at least 40-fold in overexpressing clones, no significant reduction in the half-maximal effective concentration (EC50) for miltefosine was observed in these parasites. The partial or complete deletion of ros3 in L. major, in turn, resulted in a significant increase of 3 and 20 times, respectively, in the EC50 to miltefosine. We unequivocally showed that the ros3 copy number is one of the factors involved in the differential susceptibility and uptake of miltefosine.

Automated summary

The ros3 gene copy number was found to be associated with increased miltefosine uptake and susceptibility in Leishmania. Modulating the gene copy number showed significant impact on miltefosine susceptibility, with overexpression leading to increased EC50 values and deletion resulting in higher EC50 values. This study confirms that ros3 copy number is a factor affecting differential susceptibility and uptake of miltefosine.