ena-STUDY-WTCMP-16-09-2015-12:39:40:923-209
A non-active site mutation in lanosterol 14a demethylase leads to loss of ergosterol biosynthesis and underlies Amphotericin B resistance in Leishmania mexicana
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 show that promastigote Leishmania mexicana can be selected for resistance to amphotericin B 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 4,4 dimethylcholesta 8,14,24 trien-3ß-ol becomes prevalent. This indicates that the resistance phenotype is related to loss of drug-binding. Comparing sequences of the parasites' genomes revealed 192 single nucleotide polymorphisms that distinguish wild type and resistant cells, but only one of these was found associated with a gene encoding an enzyme in the sterol biosynthetic pathway, lanosterol 14a 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 lanosterol 14a 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 other leishmanicidal drugs including paromomycin and pentamidine and also agents that induce oxidative stress. This work reveals the power of combining a Polyomics approach to discover the mechanism of resistance selection to drugs as well as offering novel insights into the selection of resistance to amphotericin B itself.
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 show that promastigote Leishmania mexicana can be selected for resistance to amphotericin B 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 4,4 dimethylcholesta 8,14,24 trien-3ß-ol becomes prevalent. This indicates that the resistance phenotype is related to loss of drug-binding. Comparing sequences of the parasites' genomes revealed 192 single nucleotide polymorphisms that distinguish wild type and resistant cells, but only one of these was found associated with a gene encoding an enzyme in the sterol biosynthetic pathway, lanosterol 14a 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 lanosterol 14a 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 other leishmanicidal drugs including paromomycin and pentamidine and also agents that induce oxidative stress. This work reveals the power of combining a Polyomics approach to discover the mechanism of resistance selection to drugs as well as offering novel insights into the selection of resistance to amphotericin B itself.