Investigation of the role of GCα in activating cGMP signalling in Plasmodium falciparum and how cyclic nucleotide signalling modulates molecular motor function.

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.