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Stortz, Johannes Felix (2020) a Conditional CRISPR/Cas9 System Gives Novel Insights Into Actin Dynamics in Toxoplasma Gondii

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Stortz, Johannes Felix (2020) 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. http://theses.gla.ac.uk/77880/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] A conditional CRISPR/Cas9 system gives novel insights into actin dynamics in Toxoplasma gondii By Johannes Felix Stortz B.Sc., M.Sc. Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy School of Life Sciences College of Medical, Veterinary & Life Science Institute of Infection, Immunity & Inflammation 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. Table of Contents Abstract ...................................................................................... 2 Table of Contents .......................................................................... 3 List of Tables ................................................................................ 7 List of Figures ............................................................................... 8 List of Supplement Material.............................................................. 11 Table of copyright permissions .......................................................... 12 Acknowledgements ........................................................................ 13 Author’s Declaration ...................................................................... 14 Publications arising from this work and collaborations .............................. 16 Conference Proceedings .................................................................. 16 Definitions/abbreviations ................................................................ 17 1 Introduction ........................................................................... 21 1.1 Taxonomie ........................................................................ 21 1.2 Health Impact of apicomplexan parasites ................................... 22 1.3 The life cycle of Toxoplasma .................................................. 22 1.3.1 Definitive host – sexual replication ...................................... 23 1.3.2 Environmental stage ....................................................... 24 1.3.3 Intermediate host – asexual lytic cycle .................................. 25 1.4 The asexual lytic cycle of Toxoplasma tachyzoites: a closer look ....... 25 1.4.1 Tachyzoite structure ....................................................... 25 1.4.1.1 The apicoplast ....................................................................................................... 29 1.4.2 Gliding motility ............................................................. 31 1.4.3 Invasion ...................................................................... 35 1.4.4 Asexual replication ......................................................... 38 1.4.5 Egress ......................................................................... 39 1.5 Molecular Tools to dissect Toxoplasma biology ............................. 42 1.5.1 The type II CRISPR/Cas9 system .......................................... 43 1.6 Actin in eukaryotes .............................................................. 47 1.6.1 Structure of actin monomers and filaments ............................ 47 1.6.2 Function of Actin binding proteins (ABPs) .............................. 50 1.6.2.1 Actin treadmilling .................................................................................................. 50 1.6.2.2 Actin nucleation .................................................................................................... 52 1.7 Actin in Toxoplasma ............................................................. 56 1.7.1 Actin binding proteins (ABPs) in Toxoplasma ........................... 60 1.8 Aims of this study ................................................................ 63 2 Materials and Methods ............................................................... 65 2.1 Equipment ........................................................................ 65 2.2 Computer Software .............................................................. 66 2.3 Consumables, biological and chemical reagents ............................ 66 2.4 Kits ................................................................................. 67 2.5 Buffers, solutions and media................................................... 68 2.6 Antibodies ........................................................................ 69 2.7 Oligonucleotides ................................................................. 70 2.8 Plasmids ........................................................................... 71 2.9 Cell strains ........................................................................ 72 2.9.1 Bacteria strains and mammalian cell lines .............................. 72 2.9.2 Toxoplasma strains ......................................................... 72 2.10 Microbiology Methods ......................................................... 74 2.10.1 Liquid cultures and cryopreservation stocks of E. coli ............. 74 2.10.2 Transformation of chemically competent E. coli .................... 74 2.11 Molecular Biology Methods ................................................... 74 2.11.1 Polymerase Chain Reaction (PCR) ..................................... 74 2.11.2 Agarose Gel Electrophoresis (AGE) .................................... 75 2.11.3 DNA restriction ........................................................... 75 2.11.4 DNA purification .......................................................... 76 2.11.5 DNA ligation ............................................................... 76 2.11.6 Isolation of plasmid DNA from E.coli .................................. 76 2.11.7 Alcohol precipitation of plasmid DNA for Toxoplasma transfections 77 2.11.8 Isolation of genomic DNA (gDNA) from Toxoplasma ................ 77 2.11.9 Sub-cloning of the split-Cas9 plasmids ................................ 78 2.11.10 Sub-cloning of gRNA plasmids .......................................... 79 2.11.11 DNA sequencing .......................................................... 79 2.12 Biochemistry Methods ........................................................ 80 2.12.1 Indirect Immunofluorescence Analysis (IFA) ......................... 80 2.13 Cell Culture .................................................................... 81 2.13.1 Culturing host cells (HFFs) and Toxoplasma ......................... 81 2.13.2 Cryopreservation of Toxoplasma ....................................... 81 2.13.3 Stable and transient transfections of Toxoplasma .................. 81 2.13.4 Generation of the parental split-Cas9 (sCas9) strain ............... 82 2.13.5 Generation of sCas9-sgRNA strains .................................... 83 2.13.6 Generation of RHsCas9-CbEmerald strain ............................ 83 2.13.7 Serial dilution of transfected Toxoplasma parasites ................ 83 2.13.8 Induction of the split-Cas9 or DiCre system in Toxoplasma ........ 84 2.13.9 Egress Assay ............................................................... 85 2.14 Microscopy ....................................................................
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