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

Abstract

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.

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

“…In Candida lusitaniae , the Amp B-resistant clinical isolates showed reduced ERG3 gene expression, suggesting that the C5-C6 double bond contributes to the binding of Amp B to membrane sterol (22). In another report, mutations in sterol C5-desaturase (LSO) were found to be associated with Amp B resistance in Leishmania mexicana (15). Here we measured the sensitivity of lso – promastigotes to Amp B in liquid culture by growing cells in various concentrations of Amp B for 48 hours (Fig.…”

“…It is interesting that LSO activity appears to contribute to the binding affinity between Amp B and ergostane-based sterols. The C5-C6 double bond is conserved among all major sterols including cholesterol (mammals), ergosterol (fungi and trypanosomatids) and stigmasterol (plants) (15, 23-26). Without this double bond, the A ring could twist/rotate more freely from the B ring, potentially making the sterol core less flat and reducing its binding capacity to Amp B (Fig.…”

“…In addition to C14DM and SMT, mutations in the gene encoding lathosterol oxidase (LSO) are also implicated in Amp B resistance (15, 21, 22). LSO (aka sterol C5-desaturase) catalyzes the formation of C5-C6 double bond in the B ring of sterol intermediates, a late step in sterol synthesis (Fig.…”

“…S1)(23). Orthologs of LSO have been identified in mammals, yeast, protozoans, plants and algae (15, 23-26). In Saccharomyces cerevisiae , the activity of LSO (encoded by ERG3 ) is sensitive to cyanide and requires iron, NAD(P)H as well as molecular oxygen (27).…”

Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei

“…Red denotes known Leishmania drivers of anti-leishmanial efficacy whose loss-of-function reduces drug efficacy (see text for details). The strain prefix for the truncated gene IDs is at the top of each panel, with the exception of the sterol biosynthetic enzymes recently shown to contribute to amphotericin-B efficacy against L. mexicana (70). Grey-filled circles (endosomes) and ellipses (lysosome) represent the endocytic system.…”