Dataset Information

Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major.

ABSTRACT: Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.

SUBMITTER: Turner KG

PROVIDER: S-EPMC4521777 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major.

Turner Kimbra G KG Vacchina Paola P Robles-Murguia Maricela M Wadsworth Mariha M McDowell Mary Ann MA Morales Miguel A MA

PLoS neglected tropical diseases 20150731 7

Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less ...[more]

PMID: 26230675

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Project description:SbIII clonal mutants and an isogenic WT clonal line. Genomic DNA from clonal WT or mutants were digested and hybridized to whole genome DNA microarrays. Antimonials are still the mainstay of treatment against Leishmaniasis but in the past decade resistance has been a severe threat. We carried out short read next generation sequencing (NGS) and comparative genomic hybridization (CGH) of three independent Leishmania major antimony resistant mutants. Copy number variations were consistently detected in both NGS and CGH where several chromosomal aneuploidies were correlated to antimony resistance. A major attribute of antimony resistance was a novel terminal deletion of variable length (67kb-204kb) of the polyploid chromosome 31 in the three mutants and was experimentally validated. Terminal deletion in two mutants occurred at the level of inverted repeated sequences in chromosome 31. AQP1 (LmjF.31.0020), a gene encoding for an aquaglyceroporin, which facilitates uptake of trivalent metalloids, was a part of the deleted region. Transfection of AQP1 into resistant mutants rendered them hypersensitive to SbIII. CGH, NGS and Southern blot analysis also highlighted a novel stable, intrachromosomal amplification of a subtelomeric locus on chromosome 34 in one mutant. This region encoded redox enzymes like ascorbate dependent peroxidase (APX) and glucose-6-phosphate dehydrogenase (G6PDH) and overexpression of the genes coding for these enzymes in revertant backgrounds demonstrated resistance to SbIII and protection from reactive oxygen species (ROS) accumulation. Generation of G6PDH null mutant in one revertant exhibited SbIII sensitivity and protection from ROS which were rescued in the add back. Our genomic analyses and parallel functional validation highlighted novel genomic rearrangements, functionally important resistant loci and the implication of new genes in antimony resistance in Leishmania. This submission represents the microarray component of the study

Dataset Information

Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major.

Publications

Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major.

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