Giardia intestinalis is a protozoan parasite that causes diarrhea, and due to its 200 million human cases each year it has become important to try and reduce the infected. Giardia intestinalis expresses so called variant surface proteins (VSPs) on its surface. VSPs are surface antigens that the immune system can detect and react to. In order to hide from the immune system and protect itself Giardia is able to perform antigenic variation, where it switches the expressed VSP to another one of the 200 VSPs in its genomic repertoire. Understanding the regulation and expression of the VSPs is an important steppingstone towards being able to create a vaccine or a better treatment to try to reduce the negative health effects created by this parasite. The main components that regulate VSPs that are being expressed on the surface are Dicer and Argonaute. They are both involved in the RNA interference (RNAi) machinery that has been suggested to silence the VSPs that are not being expressed post-transcriptionally. The aim of this study is to create mutants of Giardia that will no longer express Argonaute or Dicer and thereby be able to analyze the VSP expression and to see if a difference in expression can be detected. This can lead to an understanding of the regulatory mechanism of antigenic variation. We were able to create mutants that most likely did not express Argonaute or Dicer, however since PCR verification of successful knockout did not work, except for mutant Dicer nr. 12, genome sequencing is needed for verification of all the knockout mutants. When analyzing the VSP expression using immunostaining the only surviving Argonaute mutant and the two Dicer mutants that had gone through an initial limiting dilution showed that there was a significant difference in VSP expression for the Argonaute mutants and one of the Dicer mutants. There was a higher fluorescence detected in the nr. 8 Argonaute mutant and the 12#2 Dicer mutant, which indicate a higher VSP expression for those two strains compared to the Cas9 expressing wild type. Furthermore, fluorescence dots could be detected around the parasites especially for Dicer 12#2 which could indicate that this mutant has a higher turnover of VSPs and is releasing more of them into the surrounding area. Due to time limitations many follow up experiments were not able to be performed, however the results obtained give an important insight into the regulation of VSPs in Giardia intestinalis.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-530732 |
| Date | January 2024 |
| Creators | Lundström, Andrea |
| 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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