Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.

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HePC]), an antitumoral drug, is the only successful oral treatment for VL. In the current study, we describe the phenotypic traits of L. donovani clonal lines that have acquired resistance to HePC. We performed whole-genome and RNA sequencing of these resistant lines to provide an inclusive overview of the multifactorial acquisition of experimental HePC resistance, circumventing the challenge of identifying changes in membrane-bound proteins faced by proteomics. This analysis was complemented by assessment of the in vitro infectivity of HePC-resistant parasites. Our work underscores the importance of complementary "omics" to acquire the most comprehensive insight for multifaceted processes, such as HePC resistance.