A conditional CRISPR/Cas9 system gives novel insights into actin dynamics in Toxoplasma gondii

Stortz, Johannes Felix (2020) A conditional CRISPR/Cas9 system gives novel insights into actin dynamics in Toxoplasma gondii. PhD thesis, University of Glasgow.

Abstract

Actin is a highly abundant structural protein in eukaryotes that is critical for several cellular processes. In the apicomplexan parasite Toxoplasma gondii, actin is critical for the completion of the lytic cycle and, thus, parasite survival. Only recently, actin structures were visualised in Toxoplasma by exploiting actin-chromobodies, revealing an extensive actin network within the parasitophorous vacuole (PV) (Periz et al. 2017). This network consists of intravacuolar filamentous structures that connect individual parasites within the PV. In addition, parasites possess a cytosolic actin centre (cAC) anterior to the nucleus.

The study presented here aimed at exploiting actin visualisation to investigate actin dynamics in unprecedented detail in vivo. For this purpose, I established a conditional CRISPR/Cas9 that allows for rapid and efficient gene disruption in Toxoplasma. Combining this system with the actin-chromobody technology granted detailed insights into the actin dynamics in intracellular parasites. I identified the actin depolymerisation factor (TgADF) as an important factor in the disassembly of the intravacuolar F-actin filaments prior to parasite egress from the host cell. Furthermore, this study revealed TgFormin2 to be critical for maintaining the cAC. Since cAC loss severely impaired actin distribution and peripheral actin flow in intracellular parasites, I concluded that TgFormin2 represents a major key player in mediating proper actin dynamics. TgFormin2 also appeared to be important for apicoplast inheritance and positioning.

In summary, data presented in this thesis significantly contribute to the understanding of actin dynamics in Toxoplasma. Further insights into apicomplexan actin dynamics will be gained by exploiting the conditional CRISPR/Cas9 technology for phenotypic screening approaches.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Molecular biology, microbiology, parasitology, apicomplexans, toxoplasma, actin, actin-binding-proteins, formins.
• Subjects: Q Science > Q Science (General); Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Parasitology
• Supervisor's Name: Meissner, Prof. Dr. Markus and Waters, Prof. Andy
• Date of Award: 2020
• Depositing User: Mr Johannes Felix Stortz
• Unique ID: glathesis:2020-77880
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Jan 2020 16:15
• Last Modified: 05 Oct 2022 15:32
• Thesis DOI: 10.5525/gla.thesis.77880
• URI: https://theses.gla.ac.uk/id/eprint/77880

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