Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism, lipid transport and oxidative stress response

Abstract

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each line, the predominant ergostane-type sterol of wild-type cells was replaced by other sterol species. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). These differences appeared to relate to the presence of lipids in the former. Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several lines showed reduced virulence, at least one AmB resistant line displayed heightened virulence in mice whilst retaining its resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Raman Metabolomics of Candida auris Clades: Profiling and Barcode Identification

“…In either case, however, a key-role could be foreseen for the ergostane moiety. Modifications of the sterol composition of the plasma membrane could be interpreted as a strategy to stabilize the structure of the plasma membrane in order to obstruct the action of pore formation exerted by Amphotericin B [ 82 ]. On the other hand, the presence of ergostane molecules as glycosylated sterols in the outer layer of the cell walls could be construed as a ploy to dodge the pro-inflammatory mediators (i.e., nitric oxide, reactive oxygen species, and interleukin-1 beta) released by activated macrophages.…”

Genome deletions to overcome the directed loss of gene function in Leishmania

“…Although, interestingly, a similar deletion spanning both genes (ortholgoues of LmjF.13.1530 and LmjF.13.1540) was described in clinical isolates of L. donovani resistant to antimonials which were selected for miltefosine resistance in vitro (Shaw et al, 2016). In addition, several amphotericin B resistant laboratory strains of L. mexicana showed an identical deletion spanning both genes (~9 kb) in three lines, while in one line this deletion was more extensive (19 kb) resulting in the deletion of four contiguous genes (S4 Figure) (Fernandez-Prada et al, 2016;Pountain et al, 2019;Alpizar-Sosa et al, 2021).…”

New Glycosylated Polyene Macrolides: Refining the Ore from Genome Mining

“…Leishmania parasites are less strictly dependent on ergosterol and can resist amphotericin B by synthesizing alternative sterols that do not bind polyenes. In parasites, this resistance mechanism is not associated with a loss of fitness or virulence [7][8][9]. Despite its status as one of the leading antifungal antibiotics, amphotericin B has serious negative aspects.…”