Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom.
mBio. 2021 Jan 26;12(1):e02694-20. doi: 10.1128/mBio.02694-20.
Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here, we investigate the role of guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood-stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesize cGMP or mobilize calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that P4-ATPase activity is upstream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress. The clinical manifestations of malaria arise due to successive rounds of replication of parasites within red blood cells. Once mature, daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that the activation of cyclic GMP (cGMP) signaling is critical for initiating egress. Here, we demonstrate that GCα, a unique bifunctional enzyme, is the sole enzyme responsible for cGMP production during the asexual blood stages of and is required for the cellular events leading up to merozoite egress. We further demonstrate that in addition to the GC domain, the appended ATPase-like domain of GCα is also involved in cGMP production. Our results highlight the critical role of GCα in cGMP signaling required for orchestrating malaria parasite egress.
鸟苷酸环化酶（GCs）合成环鸟苷酸（cGMP），与环核苷酸磷酸二酯酶一起，负责调节这种细胞内信使的水平，这种信使介导真核生物的众多功能。在疟原虫（）及其孢子虫和纤毛虫亲属中，GCs与一种独特的双功能构型中的 P4-ATPase 样结构域相关联。P4-ATPases 通过将磷脂从外叶向内叶转运来产生膜双层脂质不对称性。在这里，我们研究了鸟苷酸环化酶α（GCα）及其相关的 P4-ATPase 模块的作用，表明缺乏环化酶和 P4-ATPase 结构域的无性血期寄生虫无法从宿主红细胞中逸出。GCα 缺失的寄生虫无法合成 cGMP 或动员钙，这是出芽所必需的 cGMP 依赖性蛋白激酶（PKG）驱动的要求。我们使用 cGMP 类似物的化学互补和 P4-ATPase 结构域中关键保守残基的定点突变，表明 P4-ATPase 活性是 cGMP 合成的上游，并与之相关联。总的来说，我们的结果表明 GCα 是 PKG 的关键调节剂，其相关的 P4-ATPase 结构域在产生出芽所需的 cGMP 方面起着主要作用。疟疾的临床表现是由于寄生虫在红细胞内的连续复制。一旦成熟，子孢子从受感染的红细胞中释放出来，以一种称为出芽的紧密调节过程侵入新的细胞。先前的研究表明，环鸟苷酸（cGMP）信号的激活对于启动出芽至关重要。在这里，我们证明 GCα，一种独特的双功能酶，是无性血期唯一负责产生 cGMP 的酶，并且是导致子孢子出芽的细胞事件所必需的。我们进一步证明，除了 GC 结构域外，GCα 的附加 ATPase 样结构域也参与了 cGMP 的产生。我们的结果强调了 GCα 在 cGMP 信号传导中的关键作用，这对于协调疟原虫出芽是必需的。
