Giardia intestinalis is a unicellular protozoan parasite, causing giardiasis – a gastro-intestinal disease of variable outcome and severity, in a broad range of mammalian species. The parasite has a comparatively simple life cycle with two stages, proliferative trophozoites and infectious cysts, as well as a reduced set of eukaryotic-specific organelles. Its metabolic pathways are simplified if compared to non-parasitic organisms. Giardia compensates for this apparent simplicity with unique inventions and complex regulation of metabolic processes. Transition from a fragile trophozoite to a protected, compact cyst is called encystation. This process starts upon changes in the growing conditions: cholesterol deprivation and elevated pH, and leads to changes in membrane lipids, elevated cAMP, and induction of encystation-specific gene expression, starting with activation of Myb-like protein expression. Within hours postencystation induction, cyst wall components (GalNAc and CWPs) are produced and transported to the cell membrane, flagella and adhesive disc are disassembled and stored in cytoplasm, followed by DNA replication and diplomixis. One of the encystation-specific upregulated genes in Giardia (assemblage A, isolate WB) is GL50803_1470 (termed ORF1470). Its predicted protein product has an Alba_2 domain, binding nucleic acids, presumably DNA. To study its function in G. intestinalis, we created knockout mutants, using CRISPR/Cas9 technique, Cas9- HA cell line and pGdelp-BbsI-B00826, with integrated pac- and gRNA cassettes. Transfected Giardia Cas9-HA cells were PCR verified for the complete knockout of the gene, encysted, and effect of ORF1470 was studied using cyst counting, morphometric analysis, cell imagining and Western blot for detection of CWPs. We have found minor phenotypical differences between the parental strain Cas9-HA and wild-type WB and ORF1470 deficient cells. Future plans are further experiments with obtained ΔORF1470 strains including further KO verification, visualization of ORF1470 product during encystation and determination of its binding site in the genome using ChIP-seq technology.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-530658 |
| Date | January 2024 |
| Creators | Wochinger, Yevgeniya |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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