Characterisation of molecular regulation and intracellular signalling of the Toxoplasma gondii lytic cycle

Much of the pathogenesis associated with the apicomplexan parasites Toxoplasma and Plasmodium, responsible for toxoplasmosis and malaria respectively, arise due to the destructive lytic cycle of Apicomplexa wherein parasites invade host cells, undergo intracellular replication before actively exiting (egress) the host cell destroying it in the process. In this thesis I attempt to understand some of the regulatory mechanisms controlling key stages of the Toxoplasma gondii lytic cycle specifically focusing on signal transduction pathways, including second messenger signalling, kinase activation and protein phosphorylation. In Chapter 3, I examine the biological function of alpha Soluble NSF Attachment Protein (αSNAP) phosphorylation. Here, I show Ser-6 phosphorylation is required for the correct function of αSNAP in controlling secretory traffic. I demonstrate that during intracellular development conditional expression of a non-phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, I show that the vestigial plastid (termed apicoplast) is also affected upon overexpression of αSNAP. This work highlights the importance of phosphorylation control of αSNAP function in Toxoplasma organelle biogenesis and exposes a hereto-unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites. In Chapter 4, I investigate the signal transduction pathways controlling Toxoplasma egress and motility. I attempt to understand the signal hierarchy and complexities of the intracellular signalling network by assessing the contribution of activators (Ca2+, cGMP, pH and K+) and protein kinases (PKG, CDPK1, and CDPK3) to egress and motility. As intracellular Ca2+ is required for egress and motility I first assess, in real time, intracellular Ca2+ dynamics by applying genetically encoded biosensor technology, GFP-Calmodulin-M13-6 (GCaMP6). I expand my analysis to include cGMP signal transduction using the recently developed cGMP agonist BIPPO (Appendix A). Importantly, I delineated a signal transduction hierarchy where cGMP signalling precedes Ca2+ release. I further incorporate Ca2+-Dependent Protein Kinases TgCDPK1 and TgCDPK3 into my analysis where I find redundancies between the functions of cGMP kinase Protein Kinase G (TgPKG) and TgCDPK3. This work develops a hierarchical understanding of signal transduction in Toxoplasma egress and motility, identifying key intersections between signalling modules. In my concluding discussion I propose an updated model for intracellular signalling incorporating work presented here with the body of published work. In Chapter 5, I identify a novel contribution of cAMP responsive Protein Kinase A (TgPKA) to invasion. Functional analysis of TgPKA3cat identifies an important role in creation of the intracellular replicative niche during invasion. Conditional knockdown of TgPKA3cat results in a severe invasion defect where after invasion, tachyzoites reactivate and host cell deformation prevents successful parasitism. I show that TgPKA3cat has potential roles in moving junction formation and/or reactivation of Ca2+ signalling and motility. This emphasises the importance of signal transduction through cAMP/PKA during invasion. Taken together this body of work demonstrates the diverse and complex requirement of signal transduction pathways in the Apicomplexan lytic cycle. The data presented here provides novel insights into the identity, hierarchy, and interplay of signal transduction modules and contributes to the understanding of apicomplexan intracellular signalling pathways