Giardia intestinalis is a unicellular protozoan parasite responsible for 280 million gastrointestinal infections every year. When colonizing its host, Giardia interacts closely with the small intestine epithelium by attaching to enterocytes and releasing multiple proteins to the extracellular environment. Some of the released proteins have been shown to aid the parasite’s survival in the intestine by disrupting various host defense mechanisms. Here, we attempt to characterize the specific localization of five proteins after their secretion by Giardia. In parallel we aim to produce and identify parasite’s molecules potentially working as triggers of the immune response built during infection. To study the localization of specific secreted proteins during in vitro interactions with differentiated Caco-2 cells, we started by creating transgenic parasites expressing the ADI, EF1α and G3PD proteins with a downstream detectable tag. To identify candidate proteins from Giardia, thought by our lab to be involved in immune system activation, we established a mammalian expression system for the production of recombinant versions of the selected candidate giardial PAMPs. We achieved the expression of the VSP1267 protein, natively present on the parasite’s surface. However, we found that this protein was not secreted after expression, thus complicating its purification and later use in TLR-activation experiments. In the future, we aim to localize the tagged proteins, expressed by the produced transgenic trophozoites, and optimize the mammalian expression system in order to identify candidate immune triggers during giardiasis.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-449099 |
| Date | January 2021 |
| Creators | Marques, Rafael |
| Publisher | Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi |
| 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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