Whitelaw, Jamie Adam (2017) The dynamic nature and functions of actin in Toxoplasma gondii. PhD thesis. http://theses.gla.ac.uk/8072/ 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 http://theses.gla.ac.uk/ [email protected] The dynamic nature and functions of actin in Toxoplasma gondii By Jamie Adam Whitelaw B.Sc. (Hons), M.Res. Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy School of Life Sciences College of Medical, Veterinary & Life Sciences Institute of Infection, Immunity & Inflammation University of Glasgow ii Abstract Toxoplasma gondii is an obligate intracellular pathogen. Due to its experimental tractability it has acted as an excellent model system to understand the fundamental principles of pathogenic mechanisms within the group Apicomplexa, including Plasmodium spp. the causative agent of malaria. Work on T. gondii has provided the foundation to understanding how apicomplexan parasites power motility and invasion, which centres around the parasites gliding machinery. This movement depends on the parasite's acto-myosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system. Deletions of core components of the gliding machinery, such as parasite actin or subunits of the glideosome indicate that the parasites remain motile and invasive, albeit at significantly reduced efficiencies. These findings could be explained by different possibilities, such as functional redundancies or compensatory mechanisms for multiple components of the glideosome. Toxoplasma only encodes a single copy of ACT1, therefore redundancies for ACT1 are unlikely. Much of the research in to the role(s) of TgACT1 focuses on motility and invasion. Interestingly, while the conditional act1 KO shows a deficiency in gliding and invasion, severe defects affecting parasite survival were observed during intracellular replication and egress. The amount of actin remaining in the act1 KO parasites was disputed which led to alternate conclusions about actins role in the parasites. Therefore, this study provides a much more detailed characterisation of the conditional act1 KO and when the phenotypes are observed in relation to actin levels within the parasite. Furthermore, the study provides evidence of an alternative model for motility that is independent of the parasites acto-myosin system. Several studies assert that the polymerisation kinetics of TgACT1 is unusual, allowing the formation of only short, unstable actin filaments. However, to date, it has not been possible to study actin in vivo, therefore its physiological role has remained unclear. In order to investigate this, parasites expressing a chromobody that specifically binds to F-actin were generated and characterised. Importantly, TgACT1 forms a vast network during the intracellular life-stages that is important for parasite replication and egress. Moreover, these filaments iii allow vesicle exchange and produce F-actin connections between parasites in neighbouring vacuoles. This study also demonstrates that the formation of F- actin depends on a critical concentration of G-actin, implying a polymerisation mechanism akin to all other actins. This work is important for understanding the mechanisms used by Toxoplasma to move and invade with regards to the functions of the acto-myosin system. Moreover, it highlights a novel role of actin that is required to control the organisation of the parasitophorous vacuole during division. The role of actin during the lifecycle may have wider implications to other apicomplexan species, such as Plasmodium spp. and also much further in the field of parasitology where F-actin information is scarce. iv Table of contents Abstract ...................................................................................... ii List of figures ................................................................................ x List of tables ............................................................................... xii Acknowledgements ....................................................................... xiii Publications arising from this work ..................................................... xv Conference proceedings .................................................................. xv Author’s declaration ..................................................................... xvi Definitions/abbreviations ............................................................... xvii Chapter 1 Introduction ................................................................. 19 1.1 The phylum Apicomplexa .................................................... 19 1.2 History of Toxoplasma gondii ............................................... 20 1.3 Pathogenesis of Toxoplasma gondii ........................................ 21 1.4 Lifecycle of Toxoplasma gondii ............................................. 22 1.4.1 Lifecycle in the definitive host ............................................. 23 1.4.2 Lifecycle in the intermediate host ......................................... 24 1.4.2.1 The lytic lifecycle ............................................................ 24 1.4.2.1.1 Gliding motility ............................................................. 25 1.4.2.1.2 Invasion ...................................................................... 26 1.4.2.1.3 Replication .................................................................. 28 1.4.2.1.4 Egress ......................................................................... 29 1.4.2.2 Bradyzoite cysts ............................................................... 30 1.5 Morphology of Toxoplasma gondii .......................................... 30 1.5.1 The ultrastructure of the tachyzoite ...................................... 30 1.5.2 The apical complex and secretory organelles ............................ 32 1.5.2.1 The conoid and cytoskeleton ............................................... 32 1.5.2.2 Micronemes .................................................................... 32 1.5.2.3 Rhoptries ....................................................................... 33 1.5.2.4 Dense granules ................................................................ 34 1.5.3 Inner membrane complex ................................................... 35 1.5.4 The apicoplast................................................................. 35 1.6 Actin ............................................................................ 36 1.6.1 Overview and structure of actin in eukaryotes ........................... 36 1.6.2 Actin in apicomplexan parasites ............................................ 40 1.7 Actin binding proteins (ABPs) in Apicomplexa ............................ 43 1.7.1 Nucleation ..................................................................... 44 1.7.2 Monomer sequestration ...................................................... 45 1.7.3 Severing and filament depolymerisation .................................. 47 v 1.7.4 Crosslinking and bundling of F-actin ....................................... 47 1.7.5 Myosin motors ................................................................. 48 1.8 Small molecules that bind to actin ......................................... 49 1.8.1 Actin polymerisation drugs .................................................. 50 1.8.1.1 Phalloidins ..................................................................... 50 1.8.1.2 Jasplakinolide ................................................................. 50 1.8.2 Actin depolymerisation drugs ............................................... 52 1.8.2.1 Latrunculins ................................................................... 52 1.8.2.2 Cytochalasins .................................................................. 52 1.9 Actin-related and actin-like proteins in Apicomplexa ................... 53 1.9.1.1 Actin-related proteins ....................................................... 53 1.9.1.2 Actin-like proteins ............................................................ 54 1.10 The role of actin during cell motility ...................................... 55 1.10.1 The mechanisms behind cell motility ...................................... 55 1.10.2 Crawling motility ............................................................. 55 1.10.2.1 Swimming ...................................................................... 59 1.10.2.2 Osmotic engine model ....................................................... 60 1.10.3 Apicomplexan gliding motility .............................................. 61 1.10.3.1 Molecular basis of the MyoA-motor complex ............................. 62 1.10.3.2 The linear motor model...................................................... 63 1.11 Actin during apicomplexan invasion ........................................ 66 1.12 Toxoplasma gondii as a model organism .................................. 68 1.13 Reverse genetics in Apicomplexa ..........................................