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Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner.

Apicomplexan parasites invade host cells and immediately initiate cell division. The extracellular parasite discharges transmembrane proteins onto its surface to mediate motility and invasion. These are shed by intramembrane cleavage a process associated with invasion but otherwise poorly understood. Functional analysis of Toxoplasma rhomboid 4 a surface intramembrane protease by conditional overexpression of a catalytically inactive form produced a profound block in replication. This was completely rescued by expression of the cleaved cytoplasmic tail of Toxoplasma or Plasmodium apical membrane antigen 1 (AMA1). These results reveal an unexpected function for AMA1 in parasite replication and suggest that invasion proteins help to promote parasite switch from an invasive to a replicative mode.

Cyclic guanosine monophosphate (cGMP) is an important signalling molecule which regulates critical events across all stages of the malaria parasite lifecycle. To further our understanding of how cGMP signalling governs important events involved in blood stage egress and invasion as well as gametocyte development and gametogenesis, this study investigates two different members of this signalling cascade in Plasmodium falciparum parasites. Guanylyl Cyclase a (GC-alpha) is a large, seemingly bifunctional enzyme, which consists of a type IV P-type ATPase domain, which is predicted to flip aminophospholipids between lipid bilayer leaflets; and a GC domain, which is thought to be responsible for generating cGMP. Using a combination of approaches including mutagenesis and conditional disruption of PfGC-alpha, work presented in this thesis confirms that GC-alpha is responsible for cGMP synthesis and that both ATPase and GC domains are required for asexual blood stage growth. Although the exact role of the ATPase domain remains unknown, chemical complementation of GC-alpha-deficient parasites, which lack both ATPase and GC domains, with a cGMP analogue has revealed that the ultimate role of the ATPase is in modulating cGMP synthesis. Activation of this cGMP signalling cascade culminates in the phosphorylation of numerous effector proteins, many of which form part of the glideosome. This includes the actomyosin motor protein, Myosin A (MyoA), which is phosphorylated at serine 19 (S19). Since the glideosome generates the force required for merozoites to invade host cells, these cGMP-dependent phosphorylation events may be involved in modulating motor activity. In the second part of this thesis, conditional disruption of MyoA has confirmed that the actomyosin motor is essential for red blood cell invasion, however it is dispensable during gametocyte development and gametogenesis. Complementation of the MyoA knockout line with mutant versions of the protein that either mimic or ablate phosphorylation of S19 revealed that phosphorylation of this residue is important but not essential for parasite viability. In vitro motility assays using material derived from parasites expressing either wild type or mutant MyoA demonstrate that non-phosphorylated MyoA displays slower motility, providing evidence that cGMP-dependent phosphorylation is involved in modulating the activity of the actomyosin motor.