In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization

Abstract

SummaryIn this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb‐S; and antimony resistant Sb‐R). MIL‐R was easily induced in both strains using the promastigote‐stage, but a significant increase in MIL‐R in the intracellular amastigote compared to the corresponding wild‐type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain‐specific genetic changes were discovered in MIL‐adapted parasites, including deletions at the LdMT transporter gene, single‐base mutations and changes in somy. The most obvious lipid changes in MIL‐R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL‐R parasites, with more genetic changes occurring in Sb‐R compared with Sb‐S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb‐R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

Miltefosine Susceptibility and Resistance in Leishmania: From the Laboratory to the Field

“…Additionally, single mutations in Ros3 gene alleles were also observed in a MF resistant line leading to a high resistance level as observed in selected resistant lines containing mutations in MT alleles [33]. These data indicate that both proteins are selected during the drug pressure, although a higher recurrence of mutations in MT gene has been observed in resistant parasites [35,[51][52][53]. Besides, once the MT gene is inactivated, the resistance phenotype persists in amastigotes in vitro and in vivo in animal models of VL and CL [35,54], indicating that this machinery is functional throughout the life cycle of the parasite.…”

“…Parasites resistant to MF can be obtained as promastigotes in vitro by increasing drug concentration (stepwise selection) [32,35,41,49] or by chemical mutagenesis followed by selection of MF [32,50]. In general, the mechanism of resistance is related to a defect in drug internalization due to mutations in the MT gene [32,35,49,51,52]. This defect in drug accumulation can be restored after functional expression of the MT gene in the resistant line [32,49].…”

“…Two clinically derived L. (L.) donovani strains with different inherent antimonial sensitivities were also selected in vitro as promastigotes and changes in the number of copies of chromosomes, single-base mutations and deletions of MT gene were observed in these resistant lines [52]. Changes in the content of phosphatidylcholines and lysophosphatidylcholines were also associated with MF resistance in these two clinical lines [52].…”

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection

“…Both were selected for their background susceptibility to antimony (Sb), with BPK275/0/-cl18 having a Sb-resistant background and BPK282/0-cl4 having a Sb-susceptible background. Although a Sbresistance background may alter the interaction with several drugs [30], it does not impact on the experimental selection for MIL resistance [31], hence allowing the use of both strains without challenging our stated research aims. The intrinsically low susceptibility of BPK282/0-cl4 towards paromomycin, a feature that has been associated with increased infectivity, could possibly explain its relatively more easy adaptation to the hamster model [22].…”

Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei

“…Miltefosine uptake in Leishmania is dependent on a phospholipid-transporting flippase (the miltefosine transporter, MT) and its β-subunit, Ros3 (17, 18); both in vitro selected lines and miltefosine resistant L. donovani clinical isolates harbour mutations in the MT (7, 64, 65). Consistent with this, T. brucei RNAi library selection in miltefosine led to enrichment for RNAi fragments mapping to the syntenic sequence in T. brucei ( Tb927.11.3350 ).…”