Visceral leishmaniasis (VL) is estimated to cause over 300,000 deaths annually, with over 90% of these cases attributed to infection by Leishmania donovani. It is therefore a significant global health problem and a major problem in endemic areas such as Nepal and northern India. Drug resistance in the parasite population is reducing the efficacy of clinical drugs, especially antimonials, and new methods to use existing drugs more carefully or develop new drugs are required.In this study, promastigotes of three Nepalese clinical isolates with different inherent susceptibilities to antimony (Sb) based drugs (i.e. sensitive, Sb-S; intermediate, Sb-I; or resistant, Sb-R) were grown in increasing concentrations of drugs until they expressed tolerance to 74 μM MIL (termed MIL-R) or 97 μM PMM (termed PMM-R). The drug resistant parents were then cloned to assess the impact of resistance to miltefosine (MIL) or paromomycin (PMM) on the ‘fitness’ and metabolomics/lipidomic profile, thereby elucidating the mechanism(s) responsible for drug resistance and potentially the development of screening assays for MIL or PMM resistance. MIL selected resistance gave rise to significant changes in the lipidome (p <0.05), particularly in lipids involved in the Kennedy pathway, responsible for the biosynthesis of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs). Importantly there were no mutual alterations in the MIL-R lipidomes. MIL resistance in Sb-S parasites was associated with a significant upregulation of PCs (p <0.05) whereas Sb-R MIL-R clones appeared to have significant depletions in their PC content (P <0.05) likely as a consequence of maintaining their PE content. MIL uptake in Sb-S and Sb-R MIL-R clones was significantly reduced compared to their corresponding wild-type (WT, p <0.05) and MIL resistance was associated with disruptions in the gene encoding the Leishmania donovani miltefosine transporter (LdMT) protein in both MIL-R clones.PMM resistance had significantly higher amounts of proline, glutamine, methionine and serine, which have all been associated with increased resistance to oxidative stress. MIL-R clones were significantly more susceptible to killing by superoxide (p <0.05) and NO induced killing compared to their corresponding WT strain and were significantly less infective (p <0.05) to macrophages. In contrast, PMM-R was associated with an increase in parasite ‘fitness’ as PMM-R promastigotes and intracellular amastigotes were more resistant to NO (p <0.05) and killing by interferon gamma/liposaccharide stimulated macrophage killing compared to their WT parents (p <0.05). However the effects of MIL resistance or PMM resistance were strain dependant indicating that there was no simple assay to identify drug resistant strains. Additionally, a library of novel minor groove binding (MGB) molecules, developed at the University of Strathclyde, were screened for their potential as new antileishmanial drugs. Twelve out of twenty-nine compounds tested had inhibitory effects on L. donovani in macrophage screening studies, causing > 75% suppression in parasite burdens compared to controls. One compound, MGB-3-NO, was tested in a murine model of VL. Treatment with MGB-3-NO (50 mg/kg) solution had no antileishmanial activity but treatment with MGB-3-NO encapsulated into non-ionic surfactant vesicles to increase its targeting to macrophages, caused a significant reduction in liver parasite burdens compared to controls (p <0.05).The results of this study identified similar types of metabolic and lipidomic changes in MIL-R and PMM-R parasites compared to WTs but the changes were clone-specific indicating that there was no clear and predictable mechanism to either MIL or PMM resistance selected in promastigotes.
|Date of Award||30 Nov 2016|
- University Of Strathclyde
|Sponsors||University of Strathclyde|
|Supervisor||Katharine Carter (Supervisor) & Martin Wiese (Supervisor)|