The pellicle of the apicomplexan parasite Toxoplasma gondii is a shared structure among the supergroup Alveolata, which consists of a patchwork of flattened vesicles underneath the plasma membrane. The apical complex, from which the phylum obtains its name, is the defining feature of apicomplexans. The apical complex is a dedicated invasion-related structure that is integrated into the parasite pellicle architecture, and these two structures are both essential for the pathogenesis of T. gondii. The aim of this study is to discover and characterize new components of the apical complex and the pellicle in T. gondii by implementing a three-pronged strategy that involves the study of the peripheral apical annuli, the exocytic organelle microneme and the membrane occupation and recognition nexus (MORN) domain-containing proteins.

The peripheral apical annuli of T. gondii, which consist of 5-7 ring-like structures at the lower edge of the apical cap, are intriguing compartments but their function and significance remain unknown. We have identified a novel apical annulus protein AAP6, and I have then demonstrated that it is an integral plasma membrane protein, which makes AAP6 the only membrane component found exclusively at the annuli so far. Depletion of AAP6 revealed that this protein is essential for the parasite lytic cycle in vitro, as the loss of AAP6 caused defects in parasite gliding motility, invasion and intracellular replication. A possible explanation for these defects is that the annuli might participate in the regulation of secretion processes as constitutive microneme exocytosis was impaired after AAP6 knockdown. Furthermore, I found that AAP6 is required to maintain the peripheral distribution of a transmembrane transporter HP03 which is sorting from the post-Golgi endosome-like compartment (ECL) to the parasite plasma membrane. Next, to uncover novel components and protein interactions of the T. gondii apical annuli, proximity-dependent biotin identification (BioID) was applied using AAP6 as bait. Two novel annuli proteins, which may be related to vesicle trafficking, named TgSnare and TgSyntaxin were discovered from the BioID study. Collectively, these studies tell us for the first time that the annuli span the plasma membrane to the extracellular environment, that they are important structures for parasite growth and invasion, and that the annuli are likely to function as vesicle delivery structures which maintain homeostasis of PM proteins. Preliminary investigations have been made into two other components of the pellicle and apical complex by two further approaches. Micronemes are one of the secretory organelles of the apical complex, and their exocytosis promotes parasite motility and invasion. The MORN domain is a common motif that is considered to have a role in lipid- binding and membrane-targeting. I have sought to identify new microneme proteins based on the new proteomic method LOPIT and to identify if MORN domain-containing proteins will comprise further elements of the pellicle beyond those already known. The locations of six novel microneme proteins (MC1-MC6) and five MORN domain- containing proteins (MORN2-MORN6) were revealed by protein endogenous tagging and microscopy. Preliminary functional investigations of some of these proteins were performed via inducible protein depletion studies.