Genetic and metabolic mechanisms of amphotericin B resistance in Leishmania parasites

Pathogenic protozoa of the genus Leishmania pose a significant burden on global health. Control relies on a limited range of chemotherapeutic options, with amphotericin B of increasing importance. Understanding how resistance may emerge to this drug is therefore of some concern. As amphotericin B acts through its affinity for leishmanial sterols, altered sterol composition has been described. However, little is known about the genetic basis of these changes. In this thesis, selection and characterisation of four novel amphotericin B-resistant L. mexicana lines are described. Changes to parasite drug sensitivity and fitness were profiled, as well as alterations in metabolism. This revealed heterogeneity between lines, suggesting that many changes arise stochastically during selection of resistance. In one line, no fitness costs to infectivity and replication in primary macrophages were found. Hypersensitivity to the drug pentamidine, however, was a consistent phenotype. All parasites demonstrated altered sterol composition. Genetic and transcriptomic profiling revealed associated changes in sterol biosynthesis genes, with mutation in sterol C5-desaturase observed in one line and loss of sterol C24-methyltransferase expression apparent in the other three. Broader analysis revealed extensive and apparently stochastic transcriptome remodelling, associated with chromosomal copy number changes. Deletion of the miltefosine transporter was found in one line, associated with miltefosine cross-resistance. Through reintroduction of candidate genes into resistant lines, a role for these changes was demonstrated in both amphotericin B resistance and pentamidine hypersensitivity. Reintroduction of sterol biosynthesis genes, but not the miltefosine transporter, was associated with at least partial restoration of wild-type sterol composition. Finally, changes to the sterol C24-methyltransferase locus were investigated, revealing that structural variations are the basis of expression changes, possibly mediated by repetitive sequences at this locus. These data demonstrate multiple routes to drug resistance, and suggest that at least some of these routes allow retention of parasite infectivity. Evidence of structural instability at the locus of sterol C24-methyltransferase, associated with altered sterol composition and drug resistance, is of particular concern