The dynamic nature and functions of actin in Toxoplasma gondii

Whitelaw, Jamie Adam (2017) The dynamic nature and functions of actin in Toxoplasma gondii. PhD thesis, University of Glasgow.

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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 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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Results chapter 3 and 4 has substantial work published in: Whitelaw et al (2016) Surface attachment, promoted by the Acto-myosin system of Toxoplasma gondii is important for efficient gliding motility and invasion. BMC Biology, 15(1). doi:10.1186/s12915-016-0343-5 Results Chapter 5 was has substantial work published in Periz et al (2016) Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation. eLife, 10.7554/eLife.24119
Keywords: Toxoplasma gondii, actin, gliding motility, migration, invasion, cell division, F-actin, molecular motors, retrograde flow, myoA motor complex, glideosome, Apicomplexa, parasitology, parasites, cytochalasin, latrunculin, jasplakinolide, phalloidin, Halo-tag, chromobody, nanobodies, genetics, knockout, DiCre.
Subjects: Q Science > Q Science (General)
Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Parasitology
Funder's Name: UNSPECIFIED
Supervisor's Name: Meissner, Prof. Markus
Date of Award: 2017
Depositing User: Dr Jamie A Whitelaw
Unique ID: glathesis:2017-8072
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 04 May 2017 13:30
Last Modified: 04 May 2017 13:30

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