Cyclic nucleotide signalling in malaria parasite differentiation

环鸟苷单磷酸（cGMP）和环腺苷单磷酸（cAMP）是重要的信号分子，在疟原虫生命周期的所有阶段调节关键事件。为了进一步了解这一信号通路是如何控制入侵和输出的重要事件，本研究调查了恶性疟原虫中这一信号级联的两个不同成员。GCa是一种大型双功能酶，是信号通路的关键组成部分。它由一个atp酶结构域组成，被认为可以翻转磷脂；和一个GC结构域，负责生成cGMP。使用二聚体Cre重组酶系统（DiCre），产生了一个可诱导的GCa敲除系，证实了GCa是必需的和必需的。缺乏gca的寄生虫可以通过化学补充cGMP类似物来拯救。虽然atp酶的确切作用尚不清楚，但该结构域的突变表明它起着重要的作用。这个信号级联的激活在几个效应蛋白的磷酸化中达到高潮，其中许多形成了滑体的一部分。这包括肌动球蛋白运动蛋白Myosin A (MyoA)，它在一个位点Ser19磷酸化。由于滑翔体产生分裂子侵入宿主细胞所需的力，这些cgmp依赖性磷酸化事件可能参与调节运动活动。产生了一个DiCre MyoA敲除系，揭示了MyoA对红细胞入侵至关重要。然后使用这个条件KO细胞系来研究Ser19的磷酸化是否对运动功能很重要，通过与野生型或突变型的蛋白质互补来模拟或去除Ser19的磷酸化。为了了解这种磷酸化事件对运动活性的影响，我们还使用表达野生型或突变型Myosin A的寄生虫材料进行了体外运动测定。

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.