Project description:<p><strong>BACKGROUND:</strong> Protozoan Leishmania parasites are responsible for a range of clinical infections that represent a substantial challenge for global health. Amphotericin B (AmB) is increasingly used to treat Leishmania infection, so understanding the potential for resistance to this drug is an important priority. Previously we described four independently-derived AmB-resistant L. mexicana lines that exhibited resistance-associated genetic lesions resulting in altered sterol content. However, substantial phenotypic variation between these lines, including differences in virulence attributes, were not fully explained by these changes.</p><p><strong>METHODS:</strong> To identify alterations in cellular metabolism potentially related to phenotypic differences between wild-type and AmB-resistant lines, we extracted metabolites and performed untargeted metabolomics by liquid chromatography-mass spectrometry.</p><p><strong>RESULTS:</strong> We observed substantial differences in metabolite abundance between lines, arising in an apparently stochastic manner. Concerted remodeling of central carbon metabolism was not observed; however, in three lines, decreased abundance of several oligohexoses was observed. Given that the oligomannose mannogen is an important virulence factor in Leishmania, this could relate to loss of virulence in these lines. Increased abundance of the reduced forms of the oxidative stress-protective thiols trypanothione and glutathione was also observed in multiple lines.</p><p><strong>CONCLUSIONS:</strong> This dataset will provide a useful resource for understanding the molecular basis of drug resistance in Leishmania, and suggests a role for metabolic changes separate from the primary mechanism of drug resistance in determining the phenotypic profile of parasite lines subjected to experimental selection of resistance.</p>
Project description:<p>Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of <em>Leishmania mexicana</em> and one <em>L. infantum</em> 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.</p>
Project description:To determine the modulation of gene expression of Leishmania mexicana(M379)-inoculated BALB/c ears in the presence of promastigote secretory gel (PSG) A genome-wide transcriptional analysis was performed by comparing the gene expression profiles of Leishmania mexicana- inoculated BALB/c ears and Leishmania mexicana plus PSG BALB/c ears. Leishmania mexicana amastigotes were purified from mouse cutaneous lesions and transformed in vitro in metacycic promastigotes (MT). After 6, 24 and 48 hours, ears were collected and processed for RNA extraction. Three Biological replicates per condition were run.
Project description:We examined the gene expression changes resulting from the evolution of resistance in experimental populations of the yeast Saccharomyces cerevisiae subjected to two antifungal drugs, fluconazole (FLC) and amphotericin B (AmB). Fluconazole resistance may involve increased efflux or changes in sterol metabolism, while AmB resistance generally involves changes in sterol metabolism; for all of these types of resistance, the gene expression changes are extensive. The goal of these experiments was to test whether failure of gene expression changes all downstream of the original mutation for drug resistance would affect the ability of a mutant cell to evolve and/or to support a drug-resistant phenotype.
Project description:To determine the modulation of gene expression of Leishmania mexicana(M379)-inoculated BALB/c ears in the presence of promastigote secretory gel (PSG)
