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Genomic and molecular characterization of miltefosine resistance in Leishmania infantum strains with either natural or acquired resistance through experimental selection of intracellular amastigotes

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dc.contributor.author Mondelaers, A.
dc.contributor.author Sanchez-Cañete, M. P.
dc.contributor.author Hendrickx, S.
dc.contributor.author Eberhardt, E.
dc.contributor.author Garcia-Hernandez, R.
dc.contributor.author Lachaud, L.
dc.contributor.author Cotton, J.
dc.contributor.author Sanders, M.
dc.contributor.author Cuypers, B.
dc.contributor.author Imamura, H.
dc.contributor.author Dujardin, J. C.
dc.contributor.author Delputte, P.
dc.contributor.author Cos, P.
dc.contributor.author Caljon, G.
dc.contributor.author Gamarro, F.
dc.contributor.author Castanys, S.
dc.contributor.author Maes, L.
dc.date.accessioned 2016-06-02T13:56:49Z
dc.date.available 2016-06-02T13:56:49Z
dc.date.issued 2016
dc.identifier.issn 1932-6203
dc.identifier.doi http://dx.doi.org/10.1371/journal.pone.0154101
dc.identifier.other ITG-B9B
dc.identifier.other ITG-B10B
dc.identifier.other ITG-B11A
dc.identifier.other DBM
dc.identifier.other U-MOLPAR
dc.identifier.other JIF
dc.identifier.other DOI
dc.identifier.other FTA
dc.identifier.other OAJ
dc.identifier.other Abstract
dc.identifier.other UPD62
dc.identifier.uri http://hdl.handle.net/10390/8905
dc.description.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 DeltaLiRos3 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. en_US
dc.language English en_US
dc.subject Protozoal diseases en_US
dc.subject Kala azar en_US
dc.subject Visceral en_US
dc.subject Leishmaniasis en_US
dc.subject Leishmania infantum en_US
dc.subject Vectors en_US
dc.subject Sandflies en_US
dc.subject Phlebotomus argentipes en_US
dc.subject Drug resistance en_US
dc.subject Miltefosine en_US
dc.subject Molecular en_US
dc.subject Genomics en_US
dc.subject Characterization en_US
dc.subject Strains en_US
dc.subject Natural resistance en_US
dc.subject Acquired immunity en_US
dc.subject Intracellular en_US
dc.subject Amastigotes en_US
dc.title Genomic and molecular characterization of miltefosine resistance in Leishmania infantum strains with either natural or acquired resistance through experimental selection of intracellular amastigotes en_US
dc.type Article-E en_US
dc.citation.issue 4 en_US
dc.citation.jtitle PLoS ONE en_US
dc.citation.volume 11 en_US
dc.citation.pages e0154101 en_US
dc.identifier.pmid http://www.ncbi.nlm.nih.gov/pubmed/27123924
dc.citation.jabbreviation PLoS ONE en_US


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