Cyclic nucleotide signalling in malaria parasite differentiation

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Strategic Research Priority: World Class BiosciencesAbstract Cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) are important signalling molecules which regulate critical events across all stages of the malaria parasite lifecycle. To further our understanding of how this signalling pathway governs important events involved in egress and invasion, this study investigates two different members of this signalling cascade in Plasmodium falciparum parasites.Project Guanylyl Cyclase a (GCa) is a large predicted bifunctional enzyme which is a key component of the signalling pathway. It consists of an ATPase domain, which is thought to flip phospholipids; and a GC domain, which is responsible for generating cGMP. Using the dimerisable Cre recombinase system (DiCre), an inducible GCa knockout line was generated, confirming that GCa is essential and required for egress. GCa-deficient parasites can be rescued by chemically complementing with a cGMP analogue. Although the exact role of the ATPase remains unknown, mutagenesis of this domain indicate that it performs an essential function.Activation of this signalling cascade culminates in the phosphorylation of several effector proteins, many of which form part of the glideosome. This includes the actomyosin motor protein, Myosin A (MyoA), which is phosphorylated at a single site, Ser19. 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. A DiCre MyoA knockout line was generated, revealing that MyoA is essential for red blood cell invasion. This conditional KO line was then used to investigate whether phosphorylation of Ser19 is important for motor function by complementing with wild type or mutant versions of the protein that either mimic or ablate phosphorylation of Ser19. In vitro motility assays using material derived from parasites expressing either wildtype or mutant Myosin A were also performed in order to understand the effects of this phosphorylation event on motor activity.