title: The impact of medium on drug action and resistance in Leishmania mexicana
Creators: name: Ithnin, Nur Raihana
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: Leishmania mexicana, defined medium, drug action, drug resistance.

description: The Leishmaniases are parasitic diseases caused by intracellular infectious agents – the kinetoplastid Leishmania spp. parasites. Few effective treatments are available to combat the leishmaniases, thus the development of new drugs is urgently required. Studying the metabolism of Leishmania spp. offers a route to assist the identification of new drug candidates that target critical leishmanial metabolic pathways. The research of this thesis focused on the development of defined medium (DM), which was used to culture Leishmania promastigotes and characterise various aspects of these cells. DM consists exclusively of known chemical compounds, meaning that serum and protein supplements have been omitted. The development of DM offers fully-characterised culture conditions for Leishmania promastigote growth, removing the effects of unidentified growth factors/compounds which are usually present in serum. DM use also prevents variability that commonly arises from serum batch variations. The cultivation of Leishmania promastigotes in DM was successful, with growth rates being comparable with those of cells grown in HOMEM (rich) medium. Phenotypic analysis indicated that DM-cultured cells are significantly reduced in size relative to HOMEM-cultured cells. The DM-cultured cells were more sensitive to several oxidative stress inducing agents. Metabolomics analysis revealed that DM-cultured cells display aspects of a ‘stringent metabolic response,’ consuming fewer nutrients and secreting lower levels of the metabolic end-products associated with glucose, arginine and tryptophan metabolism. Interestingly, DM-cultured cells are hypersensitive to miltefosine, but less susceptible to amphotericin B. The difference in miltefosine sensitivity between DM- and HOMEM-cultured cells is likely due to miltefosine having a high protein-binding capability – high levels of serum-derived protein are present in HOMEM medium only. Metabolic changes were not marked when comparing cells grown in HOMEM and DM and expression levels of the miltefosine transporter did not differ between HOMEM- and DM-cultured cells. The application of RNA-seq to determine any changes in the expression of other genes expression that may be associated with miltefosine sensitivity was also conducted. ATG8 and cytochrome C are highly expressed in DM-cultured cells. Both of these genes are related to nutrient starvation and apoptotic events. The DM-cultured cells were also used to study mechanisms of amphotericin B resistance. Resistance took a longer timescale to select in DM relative to HOMEM-cultured cells. Moreover, on short term exposure to AmB, metabolomics experiments showed that some changes, including the increased flux through the pentose phosphate pathway in HOMEM, were not seen in DM grown cells. This indicated differences in oxidative stress related events. This may relate to the difference in resistance levels in cells selected in HOMEM (~5-fold) versus DM(~2-fold) since genetic analysis revealed that in both cases mutations to sterol methyltransferase (SMT) genes related to resistance. The sterol profile also changed between the AmBr lines cultured in different media. High accumulation of ergosta-5,7,24 trienol is found in AmBr resistant DM-cultured cells, which decreased in AmBr resistant HOMEM-cultured cells. In contrast, the accumulation of cholesta-5,7,22-dienol is increased in AmBr resistant HOMEM-cultured cells, but is decreased in AmBr DM-cultured cells.