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Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana

Mwenechanya, Roy; Kovarova, Julie; Dickens, Nicholas J.; Mudaliar, Manikhandan; Herzyk, Pawel; Vicent, Isabel M.; Weidt, Stefan K.; Burgess, Karl E.; Burchmore, Richard J.S.; Pountain, Andrew W.; Smith, Terry K.; Creek, Darren J.; Kim, Dong-Hyun; Lepesheva, Galina I.; Barrett, Michael P.

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana Thumbnail


Authors

Roy Mwenechanya

Julie Kovarova

Nicholas J. Dickens

Manikhandan Mudaliar

Pawel Herzyk

Isabel M. Vicent

Stefan K. Weidt

Karl E. Burgess

Richard J.S. Burchmore

Andrew W. Pountain

Terry K. Smith

Darren J. Creek

Galina I. Lepesheva

Michael P. Barrett



Abstract

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself.

Citation

Mwenechanya, R., Kovarova, J., Dickens, N. J., Mudaliar, M., Herzyk, P., Vicent, I. M., …Barrett, M. P. (2017). Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana. PLoS Neglected Tropical Diseases, 11(6), Article e0005649. https://doi.org/10.1371/journal.pntd.0005649

Journal Article Type Article
Acceptance Date May 18, 2017
Publication Date Jun 16, 2017
Deposit Date Sep 8, 2017
Publicly Available Date Sep 8, 2017
Journal PLOS Neglected Tropical Diseases
Electronic ISSN 1935-2735
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 11
Issue 6
Article Number e0005649
DOI https://doi.org/10.1371/journal.pntd.0005649
Public URL https://nottingham-repository.worktribe.com/output/866649
Publisher URL http://journals.plos.org/plosntds/article/authors?id=10.1371/journal.pntd.0005649
Additional Information Mwenechanya R, Kovárová J, Dickens NJ, Mudaliar M, Herzyk P, Vincent IM, et al. (2017) Sterol 14a-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana. PLoS Negl Trop Dis 11(6): e0005649. https://doi.org/10.1371/journal.pntd.0005649
Contract Date Sep 8, 2017

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