Veterinary Science - Theses

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Author: Ansell, Brendan Robert Edward × End date: 2017 × Start date: 2013 × Type: PhD thesis ×

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    Mechanisms of drug response and resistance in Giardia duodenalis
    Ansell, Brendan Robert Edward ( 2017)
    Giardia duodenalis is a parasitic protist and the most common intestinal parasite of humans, causing 200-300 million cases of diarrhoeal disease annually. Metronidazole is a widely administered anti-giardial drug, but treatment failure is relatively common, attributed in part to metronidazole resistance in the infecting parasites. This drug enters parasitic cells as a pro- drug, which is activated to cytotoxic intermediate forms via oxidoreductase enzymes in the parasite’s antioxidant system. Metronidazole-resistant lines — defined here as those that exhibit significantly greater tolerance to the drug in vitro compared to wild-type cells, show perturbed transcription and activity of various oxidoreductase enzymes. However inconsistencies in these features suggest that yet-undiscovered genes and biochemical mechanisms may also contribute to resistance. Progress in understanding the biology of resistance, and of Giardia in general, is further hampered by a lack of functional information for around half of the proteins encoded in the genome. Therefore in this work, high-throughput protein structure prediction was used to attribute putative functions to hypothetical proteins in Giardia, and over-expression of as yet uncharacterized oxidoreductase enzymes was found to increase parasite tolerance to metronidazole. RNA sequencing was used to profile transcriptional changes in parasites over time under standard culture conditions, and in response to sub-lethal concentrations of metronidazole, hydrogen peroxide and thermal stress. Genetically controlled investigation of transcriptional changes in three drug-resistant parasite lines relative to their susceptible parent lines, revealed down-regulation of drug-activating enzymes and of genes that function proximal to pyruvate:ferredoxin oxidoreductase – a nexus of glycolysis and electron transport systems. Changes specific to each resistant line suggested that active drug detoxification and efflux systems may also contribute to metronidazole resistance. In all, this work provides substantial insight into mechanisms of response and resistance to a vital treatment for a major global pathogen.