Genomic instability at the locus of sterol C24-methyltransferase promotes amphotericin B resistance in Leishmania parasites

Amphotericin B is an increasingly important tool in efforts to reduce the global disease burden posed by Leishmania parasites. With few other chemotherapeutic options available for the treatment of leishmaniasis, the potential for emergent resistance to this drug is a considerable threat. Here we characterised four novel amphotericin B-resistant Leishmania mexicana lines. All lines exhibited altered sterol biosynthesis, and hypersensitivity to pentamidine. Whole genome sequencing demonstrated resistance-associated mutation of the sterol biosynthesis gene sterol C5-desaturase in one line. However, in three out of four lines, RNA-seq revealed loss of expression of sterol C24-methyltransferase (SMT) responsible for drug resistance and altered sterol biosynthesis. Additional loss of the miltefosine transporter was associated with one of those lines. SMT is encoded by two tandem gene copies, which we found to have very different expression levels. In all cases, reduced overall expression was associated with loss of the 3' untranslated region of the dominant gene copy, resulting from structural variations at this locus. Local regions of sequence homology, between the gene copies themselves, and also due to the presence of SIDER1 retrotransposon elements that promote multi-gene amplification, correlate to these structural variations. Moreover, in at least one case loss of SMT expression was not associated with loss of virulence in primary macrophages or in vivo. Whilst such repeat sequence-mediated instability is known in Leishmania genomes, its presence associated with resistance to a major antileishmanial drug, with no evidence of associated fitness costs, is a significant concern. Author summary Leishmania parasites are a significant threat, causing some 0.9-1.6 million new infections and 20,000 deaths annually. Amphotericin B is gaining importance in control of leishmaniasis, as other treatment options are increasingly compromised by emergent drug resistance. We sought to identify mechanisms by which resistance to amphotericin B could emerge through experimental selection and characterisation of four novel resistant L. mexicana lines. Whilst in all cases, sterol metabolism was dramatically affected, in three lines specific sterol changes resulted from decreased expression of the sterol biosynthesis enzyme sterol C24-methyltransferase (SMT). We found that this resulted from structural variation events at the genome level, resulting in specific loss of the more highly expressed SMT gene copy. These events appear to arise due to repetitive sequence elements in the genomic region, suggesting that SMT could be particularly prone to loss of expression in this manner. Moreover, in at least one line, we found that disruption of SMT function was not associated with any loss of fitness. These findings are significant in demonstrating that an apparently common mechanism of resistance-associated mutation does not compromise parasite virulence, thus representing a possible route to emergent resistance in the clinical setting.