The role of calcium-dependent protein kinases in Toxoplasma gondii cell signalling

© 2015 Dr. James McCoyToxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, and can cause severe disease during pregnancy or in immunocompromised individuals. It also serves as a useful model organism for more experimentally intractable apicomplexans, such as the malaria-causing Plasmodium species. During lytic infection of humans apicomplexan parasites proceed through successive rounds of active host cell invasion, parasite replication, egress, and movement through tissue. Ca2+ signalling cascades are known to be essential to the regulation of these processes, and although the molecular composition of these signalling pathways is still poorly understood, the Ca2+- Dependent Protein Kinases (CDPKs) have arisen as key mediators. This work describes the role of several CDPKs in regulating Ca2+-driven processes in T. gondii. In Chapter 1 I thoroughly review the current literature, as it stands in this area and with regards to what will be pertinent to the coming chapters. In Chapter 3 I describe a role for TgCDPK3 in regulating Ca2+-stimulated host cell egress. Genetic deletion of TgCDPK3 left parasites unable to rapidly respond to egress stimuli, not activating parasite motility or secreting the microneme organelles in order to escape host cells. Requirement for TgCDPK3 activity in egress was found to rely on the local ionic concentration. Ca2+-stimulated egress is thought to be downstream of activation of Protein kinase G (PKG), and in Chapter 4 I extend our understanding of the regulation of Ca2+ signalling to up-regulate egress in response to stimuli by showing that both TgCDPK3 and another CDPK, TgCDPK4, are required for response to PKG activation. By generation of a double knockout I show that TgCDPK3 and TgCDPK4 work synergistically to control egress upon the agonism of this signal, demonstrating these kinases cooperate in regulating egress. TgCDPK3 and TgCDPK4 localise to the plasma membrane, and this localisation is key to activity of TgCDPK3. The contribution of subcellular localisation to the specificity of CDPK activity was then pursued. Relocalisation of TgCDPK3 to the cytoplasm allowed complete rescue of chemical inhibition of the cytosolically localised TgCDPK1. Meanwhile, addition of the N-terminal acylation motif of TgCDPK3 to TgCDPK1 conferred plasma membrane localisation, and allowed partial rescue of the ΔCDPK3 egress phenotype. This could be enhanced by mutation of key residues in the catalytic cleft site of TgCDPK1, to make it more closely resemble that of TgCDPK3, demonstrating that subcellular localisation and the catalytic cleft are key to regulating the specific function of apicomplexan CDPKs. In Chapter 5 I utilise a forward genetic strategy to further the understanding of the signalling pathway that TgCDPK3 regulates in Ca2+ stimulated egress. I identify a previously uncharacterised component of T. gondii egress signalling, Suppressor of Egress 1 (TgSE1). Knockout of TgSE1 in ΔCDPK3 parasites restored near-wild type egress levels. Making use of these various knockouts, a phosphoproteomics strategy was used to reveal further members of Ca2+-stimulated egress pathways in T. gondii. Lastly in Chapter 6 I show that CDPKs have diverse roles in T. gondii Ca2+ signalling, as revealed for TgCDPK2, which plays an essential a role in glucose storage. TgCDPK2 co-localises with several metabolic enzymes with putative roles in turnover of the storage polysaccharide amylopectin. ΔCDPK2 parasites showed unchecked accumulation of amylopectin, due to a hyper accumulation and a defect in an ability to degrade it in normal physiological settings. This had little effect on growth of lytic stage parasites, but aberrant amylopectin accumulation caused the death of differentiated cysts showing the importance of regulation of this storage compound in this therapeutically refractory stage. Together, this work demonstrates the diverse roles that CDPKs and Ca2+ signalling have in regulating T. gondii infection, and offers novel insights into the molecular mechanisms underlying these pathways