Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion

The opportunistic pathogen Toxoplasma gondii is an obligate intracellular parasite part of the phylum Apicomplexa, able to infect all warm-blooded animals including humans. Invasion by apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii to host cells requires the establishment and crossing through of a small ring-like junctional structure serving as an interface and stabiliser between the parasite and host cell plasma membrane. During the invasion process, the host cell possibly resist invasion to some degree, exerting force on the parasite’s entry point as de novo actin polymerisation has been characterised in this location (Gonzalez et al., 2009). Additionally, the parasite is required to generate force via an actomyosin motor to achieve host cell membrane penetration successfully, leading to mechanical deformation when the parasite is squeezing through the junctional ring. This actomyosin motor depends on a protein complex termed the glideosome, that pulls actin to achieve forward motility. Actin plays a key role in the parasite’s biology with important functions not only during invasion but also during replication, apicoplast maintenance and egress. Until recently, the lack of reliable F-actin sensors hampered the characterisation of actin dynamics during these processes. With the use of nanobodies with the potential to recognise actin (Periz et al., 2017), a complex actin behaviour was uncovered allowing the assessment of in vivo dynamics through the parasite’s lytic cycle. The uncovered flow of F-actin presented new opportunities to address debate over stablished hypothesis on parasite’s actin and to extend the initial roles attributed to actin including the establishment of cytoplasmic actin pool through the parasite’s life. Additionally, these F-actin dynamics were shown to be affected by traditional actin modulating drugs, as well as interference with actin binding factors resulting in abrogation of these dynamics and phenotypes associated with motility. Additionally in this thesis, it is suggested that F-actin’s role in invasion goes beyond powering the glideosome via force traction, but to facilitate nucleus passage and deformation. Real time and super resolution microscopy highlighted that during invasion events, the junction ring can oppose nucleus passage as parasites deficient of core components of the acto-myosin system have been shown to be incapable of withstand pressure exerted at the junction ring, leading to blebbing and collapse of the invading parasite (Bichet et al., 2016). Although some of these parasites are able to complete invasion, the dynamics are visibly affected suggesting more systems are at play during invasion. The literature shows that other eukaryotic systems deploy nucleus protection and displacement mechanisms to facilitate migration through tight spaces by the concerted action of actomyosin complexes and cytoskeletal structures (Petrie et al., 2012; Petrie and Yamada, 2015; McGregor, Hsia and Lammerding, 2016). This thesis proposes that the F-actin machinery facilitates nucleus passage through the junctional ring, offering a model fort the dual contribution of F-actin forces by constricting and pushing/pulling the nucleus during host cell invasion by these apicomplexan parasites, sharing similar mechanism with those of larger eukaryotes.