Sterol 14α-demethylase mutation leads to 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 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

LC-MS metabolomics analysis of WT and AmB resistant <i>L</i>. <i>mexicana</i> promastigotes.

<p>A. PCA plot of the LC-MS metabolomic analysis. Each circle represents a single replicate and shaded areas indicate respective 95% confidential intervals; light blue, WT cells; green, AmB resistant cells; red, fresh medium; magenta, WT spent medium; dark blue, AmB resistant cells spent medium. B. Representative sterols as detected by LC-MS metabolomic analysis significantly changing between WT and AmB resistant cells. Mass of these metabolites is listed since specific identification is not possible by LC-MS, but formulae of 410.3547 = C₂₉H₄₆O; 426.3497 = C₂₉H₄₆O₂; 394.3235 = C₂₈H₄₂0. Mean values of three replicates are plotted, error bars represent standard deviations.</p

Selection of AmB resistance in <i>L</i>. <i>mexicana</i> promastigotes.

<p>AmB resistance was selected by step-wise increase in AmB concentrations in the growth medium. The open circles and left <i>y</i> axis indicate the AmB concentration in the growth medium during the selection over 182 days as shown on <i>x</i> axis. The grey bars and the right <i>y</i> axis indicate the specific EC₅₀ values (representative single values of three are plotted) for AmB attained during the selection process at different times as indicated by the <i>x</i> axis.</p

Drug sensitivity of AmB resistance cell lines in this and previous studies.

<p>While <i>L</i>. <i>mexicana</i> was used here, the quoted values are for <i>L</i>. <i>donovani</i> from Mbongo et al. (1998), and Garcia-Hernandez et al. (2012). Fold change is calculated as the ratio of EC₅₀ value of the resistant cell line to WT for a given drug. EC₅₀ values are expressed as means of three replicates ± SEM.</p

Reversal in AmB sensitivity and sterol composition after WT CYP51 re-expression in the AmB resistant cell line.

<p>Red bars represent EC₅₀ values in WT, AmB resistant cells, and AmB resistant cells re-expressing WT CYP51. Mean values of three replicates are shown, error bars represent standard deviations, p < 0.0001. Blue bars represent intensity detected for a sterol C₂₈H₄₈O, consistent with ergosta-5,7,22E-trien-3β-ol.</p

Identification of the sterol peaks indicated in Fig 3.

<p>Identification is based on matches with the NIST library after fragmentation and the scores are indicated. Abundance relative to the total sterol content is indicated in each cell line, and the fold change of that.</p

Effect of anti-leishmanial drugs on AmB resistant cells expressing the WT CYP51.

<p>Mean values of three replicates are shown with SEM values.</p