title: Polyomic characterisation of polyene drug resistance in Leishmania spp.
Creators: name: Alpizar Sosa, Edubiel Arturo
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: Leishmaniasis, drug discovery, drug resistance, polyene antifungals, amphotericin B, nystatin, sterols, ergosterol, omics, metabolomics, genomics, WGS, NGS, molecular parasitology, neglected tropical diseases, NTDs.

description: Amphotericin B is the compound of choice for the treatment of leishmaniasis, however a definitive mode of action and full knowledge of causes of resistance to this polyene are still poor. The aim of this project is to use a polyomic approach to characterise laboratory generated mutant lines of Leishmania spp., selected for resistance against the polyene antifungals, amphotericin B and nystatin. While previous work has characterised multiple lines of L. mexicana resistant to amphotericin B, this is the first report of resistant lines selected against nystatin in Leishmania spp. Ergosta-7,22-dien-3-ol and cholesta-5,7,22-trienol, were the two main sterol intermediates replacing ergosterol in all eight polyeneresistant lines of Leishmania spp. The former sterol intermediate was associated with five novel mutations in sterol C5-desaturase, in two and six AmBR- and NysR-mutants and the latter resulted from changes in C24-sterol methyl transferase, along with deletion of the miltefosine transporter and its neighbouring gene downstream in two AmBR lines. Interestingly, switching from ergosterol to these two sterol intermediates was associated with an increased and an attenuated inflammatory response in vivo, respectively. In all cases, viable parasites were recovered post-infection and the retention of resistance in vivo was confirmed. In addition, response to treatment with amphotericin B was observed only in wild type parasites. Untargeted metabolomics provided hints towards modes of action in addition to the binding to ergosterol. Upregulation of the pentose phosphate pathway plays a central role as a key provider of NADPH suggesting an immediate pulse of oxidative stress associated with addition of the drug. Amphotericin B treatment rapidly altered lipid metabolism, decreasing the abundance of Acetyl-CoA, NADPH, leucine and mevalonate. In all mutants, the total or partial loss of the key membrane sterol ergosterol lead to amphotericin B resistance. All polyene resistant mutants were more susceptible to pentamidine and paromomycin. Conversely, miltefosine resistance was found in all mutants, with this increase being more pronounced in two lines showing a deletion of the miltefosine transporter. The grounds of cross-resistance to a new library of sterol inhibitors, 1,2,3-triazolylsterols, was also assessed. The most active hits showed a micromolar potency, albeit a mode of action independent of the inhibition of sterols is suggested. Considering the increase of resistance against the antileishmanials and the limited therapies available, this thesis provides valuable information on the MoA and resistance of polyenes in Leishmania, should the resistance against AmB, the drug of choice for leishmaniasis, increases in clinical settings, and to improve the discovery of potential new drug targets.