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

• Published in: PloS one 2016-04, Vol.11 (4), p.e0154101-e0154101
• Main Authors: Mondelaers, Annelies, Sanchez-Cañete, Maria P, Hendrickx, Sarah, Eberhardt, Eline, Garcia-Hernandez, Raquel, Lachaud, Laurence, Cotton, James, Sanders, Mandy, Cuypers, Bart, Imamura, Hideo, Dujardin, Jean-Claude, Delputte, Peter, Cos, Paul, Caljon, Guy, Gamarro, Francisco, Castanys, Santiago, Maes, Louis
• Format: Article
• Language: English
• Subjects: Aberration; Acquired immune deficiency syndrome; AIDS; Amastigotes; Antiprotozoal Agents - pharmacology; Biological Transport; Biology and Life Sciences; Carrier Proteins - genetics; Carrier Proteins - metabolism; Clinical isolates; Cloning; Corrosion resistance; Coupling (molecular); Deactivation; Deoxyribonucleic acid; DNA; Dosage and administration; Drug resistance; Drug Resistance - genetics; Drug therapy; Gene deletion; Gene expression; Gene Expression Regulation; Gene sequencing; Genetic aspects; Genetic Complementation Test; Genome, Protozoan; Genomes; Genomic analysis; Genotype; High-Throughput Nucleotide Sequencing; HIV; Human immunodeficiency virus; Inactivation; Intracellular; Laboratory animals; Leishmania; Leishmania donovani; Leishmania infantum; Leishmania infantum - drug effects; Leishmania infantum - genetics; Leishmania infantum - growth & development; Leishmania infantum - metabolism; Life Cycle Stages - drug effects; Life Cycle Stages - genetics; Life Sciences; Microbial drug resistance; Microbiology and Parasitology; Miltefosine; Mutation; Parasites; Parasitic diseases; Parasitic Sensitivity Tests; Parasitology; Phenotype; Phosphorylcholine - analogs & derivatives; Phosphorylcholine - pharmacology; Physiological aspects; Promastigotes; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Research and Analysis Methods; Selection, Genetic; Stop codon; Transfection; Vector-borne diseases; Visceral leishmaniasis
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0154101

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 LdR