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

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...

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Main Authors: Annelies Mondelaers, Maria P Sanchez-Cañete, Sarah Hendrickx, Eline Eberhardt, Raquel Garcia-Hernandez, Laurence Lachaud, James Cotton, Mandy Sanders, Bart Cuypers, Hideo Imamura, Jean-Claude Dujardin, Peter Delputte, Paul Cos, Guy Caljon, Francisco Gamarro, Santiago Castanys, Louis Maes
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2016-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC4849676?pdf=render
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author Annelies Mondelaers
Maria P Sanchez-Cañete
Sarah Hendrickx
Eline Eberhardt
Raquel Garcia-Hernandez
Laurence Lachaud
James Cotton
Mandy Sanders
Bart Cuypers
Hideo Imamura
Jean-Claude Dujardin
Peter Delputte
Paul Cos
Guy Caljon
Francisco Gamarro
Santiago Castanys
Louis Maes
author_facet Annelies Mondelaers
Maria P Sanchez-Cañete
Sarah Hendrickx
Eline Eberhardt
Raquel Garcia-Hernandez
Laurence Lachaud
James Cotton
Mandy Sanders
Bart Cuypers
Hideo Imamura
Jean-Claude Dujardin
Peter Delputte
Paul Cos
Guy Caljon
Francisco Gamarro
Santiago Castanys
Louis Maes
author_sort Annelies Mondelaers
collection DOAJ
description 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.
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spelling doaj.art-fa841d66e44940beb8de83b083d6da952022-12-21T18:18:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01114e015410110.1371/journal.pone.0154101Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.Annelies MondelaersMaria P Sanchez-CañeteSarah HendrickxEline EberhardtRaquel Garcia-HernandezLaurence LachaudJames CottonMandy SandersBart CuypersHideo ImamuraJean-Claude DujardinPeter DelputtePaul CosGuy CaljonFrancisco GamarroSantiago CastanysLouis MaesDuring 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.http://europepmc.org/articles/PMC4849676?pdf=render
spellingShingle Annelies Mondelaers
Maria P Sanchez-Cañete
Sarah Hendrickx
Eline Eberhardt
Raquel Garcia-Hernandez
Laurence Lachaud
James Cotton
Mandy Sanders
Bart Cuypers
Hideo Imamura
Jean-Claude Dujardin
Peter Delputte
Paul Cos
Guy Caljon
Francisco Gamarro
Santiago Castanys
Louis Maes
Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.
PLoS ONE
title Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.
title_full Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.
title_fullStr Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.
title_full_unstemmed Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.
title_short Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes.
title_sort genomic and molecular characterization of miltefosine resistance in leishmania infantum strains with either natural or acquired resistance through experimental selection of intracellular amastigotes
url http://europepmc.org/articles/PMC4849676?pdf=render
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