Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes

Abstract

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.

Iron superoxide dismutase contributes to miltefosine resistance in Leishmania donovani

“…Recently, Mondelaers et al . have also shown lower expression of LiMT in MIL‐resistant Leishmania infantum parasites but did not find any change in LiRos3 expression compared to that of sensitive parasites indicating that Milt resistant is independent of LiMT expression in these isolates. Although there were reports as above indicating different genes that trigger resistance, there was not a definite target studied to overcome the miltefoisne resistance.…”

“…The factors dwindling the expression levels of P-type ATPase, LdMT-LdRos3-dependent flippase activity in translocation machinery [18,19,29], and genes involved in neutralization of oxidative stress [28,[46][47][48][49] generate Milt-resistant parasites. Recently, Mondelaers et al [50] have also shown lower expression of LiMT in MIL-resistant Leishmania infantum parasites but did not find any change in LiRos3 expression compared to that of sensitive parasites indicating that Milt resistant is independent of LiMT expression in these isolates. Although there were reports as above indicating different genes that trigger resistance, there was not a definite target studied to overcome the miltefoisne resistance.…”

Different Mutations in a P-type ATPase Transporter in Leishmania Parasites are Associated with Cross-resistance to Two Leading Drugs by Distinct Mechanisms

“…An average of 50-fold genome coverage was obtained for both the wild-type and the mutant. Read depth coverage analysis did not identify specific gene amplification or deletion in the mutant (S1 Dataset), although aneuploidy was observed for 6 chromosomes (S1 Table), a phenomenon often observed in drug resistant mutants [18, 37–39]. A search for point mutations revealed 18 homozygous single nucleotide polymorphisms (SNPs) in the AmB1000.1 mutant (S2 Table), 3 of which occurred within coding sequences (CDS) and caused an amino acid change (S2 Dataset).…”

“…donovani AmB-resistant promastigotes analysed by gas chromatography coupled to mass spectrometry (GC-MS) revealed an enrichment in cholesta-5,7,24-trien-3 β -ol [11], which suggests a more fluid cellular surface. On the other hand, resistance to MF primarily implies a transport defect with inactivation of the P-type ATPase miltefosine transporter (MT), or of its regulatory subunit LdRos3, causing a decrease in the uptake of lyso -phospholipids [16–18]. A recent study has reported changes to the metabolism of lipids in L .…”

Flavonoids as efficient scaffolds: Recent trends for malaria, leishmaniasis, Chagas disease, and dengue

“…Resistance of Leishmania spp. to antileishmanial drugs is related to some extent to metabolic pathways, including glucose and thiol metabolism, the overexpression of certain enzymes, and increased membrane fluidity, resulting in altered interactions with antileishmanial drugs (Ghosh et al, ; Mondelaers et al, ; Vacchina, Norris‐Mullins, Carlson, & Morales, ). These biochemical and biophysical changes must be taken into consideration while investigating potential novel therapeutic strategies for the treatment of leishmaniasis.…”