Study of Natural Miltefosine-Resistance Mechanism in Leishmania (L.) chagasi Isolates from Visceral Leishmaniasis Patients with Different Treatment Outcomes

Visceral leishmaniasis (VL) treatment relies on a few chemotherapeutic drugs including Sb(V),amphotericin B and miltefosine. Miltefosine has been highly active against VL in India. However,susceptibility differences to miltefosine have been observed in clinically relevant Leishmaniaspecies. Miltefosine resistance mechanisms are being elucidated in laboratory Leishmania spp.strains but are less clear in clinical isolates. In this study, we used comparative proteomics (2DDIGE/MS)and genomics (Illumina) approaches to highlight molecular differences between L.(L.) chagasi (= L. (L.) infantum) isolates from VL patients in Brazil with different miltefosinetreatment outcomes. The high-resolution proteomics showed 46 proteins that exhibitedsignificantly different abundances (p<0.05) between the isolates from one cured and onerelapsed patient. Most of the proteins up-regulated in the proteome of the isolate from therelapsed patient were associated with redox homeostasis, stress response, and drugtranslocation. Whole genome sequencing was carried out with isolates from cured (n=14) andrelapsed (n=12) patients. 93 orthologs groups exhibited a significant difference (p<0.01) in genedosage between the two groups, including a deletion of a locus on chromosome 31 (containingfour genes; miltefosine sensitivity locus, MSL) which was associated with isolates from relapsedpatients. It was inferred that this deletion process occurs by homologous recombination usingrepetitive sequence flanking the MSL, and apparently is not induced by miltefosine pressure.Re-expression of individual MSL genes in a miltefosine resistant promastigote line did notrestore the in vitro miltefosine susceptibility phenotype. These data suggest that miltefosineresistancemechanisms in Leishmania spp. are complex and multifactorial.