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Characterization of DNA Methylation in Giardia intestinalis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. (Svärd Group)
2021 (English)Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
Abstract [en]

Abstract

G. intestinalis is an intestinal protozoan parasite that causes 180 million symptomatic diarrheacases (giardiasis) and more than 0.5 billion asymptomatic infections per year. Symptomaticinfections are usually treated with metronidazole (Flagyl). However, resistance is emerging, andan alternative treatment is required. The mechanism to which the parasite causes the disease is notunderstood and, neither is the regulation of encystation (a process where trophozoites differentiateto cyst) However, preliminary data suggest that an epigenetic mechanism is involved. DNAmethylation is an important epigenetic regulator in many organisms, but it is not known if theDNA is methylated in Giardia. The main goal of this project was to characterize DNA methylationin G. intestinalis and, if it exists, study if it is linked to cell differentiation and use it as a target fordrug treatment. We found out using the dot blot technique complemented withimmunofluorescence assays, that 6mA and 5mC DNA methylation exists on the genomic DNA ofthe assemblage A G. intestinalis isolate WB. 6mA methylation was also found on RNA. However,no major differences were detected between trophozoites and cysts. Assemblage B Giardia isolates(GS and H3) also have methylated genomic DNA, but we detected lower levels of methylation. Abioinformatic search was performed in the G. intestinalis WB genome in an attempt to identifyDNA methylases. Expression levels through-out the life cycle, sequence similarities and structuralmodelling using iTASSER identified six putative DNA methylases in the WB genome. The sixDNA methylases were over-expressed in Giardia, three were lethal and three localized to thenucleus. 5-azacytidine and 5-aza-2’-deoxycytidine nucleoside analog drugs prevent methylationand are incorporated into RNA and DNA, respectively. We tested these two drugs on Giardiatrophozoites, and both have effects on the trophozoite stage (IC50 1.46±0.46 μM for 5-aza-2’-deoxycytidine and 111±24 μM for 5-Azacytidine). The 5-aza-2’-deoxycytidine drug is actuallymore effective than metronidazole, showing that nucleoside analogs affecting DNA methylationcould be alternative drugs for treatment of giardiasis.

Place, publisher, year, edition, pages
2021. , p. 31
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-444560OAI: oai:DiVA.org:uu-444560DiVA, id: diva2:1561973
Educational program
Master Programme in Biology
Presentation
2021-06-02, Zoom, Uppsala, 13:00 (English)
Supervisors
Examiners
Available from: 2021-06-09 Created: 2021-06-07 Last updated: 2021-06-10Bibliographically approved

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