Malaria Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
PLoS Pathog. 2010 Feb 5;6(2):e1000746. doi: 10.1371/journal.ppat.1000746.
The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.
疟原虫裂殖子入侵红细胞需要宿主受体与寄生虫配体之间的特异性相互作用。在入侵过程中与红细胞受体结合的寄生虫蛋白定位于称为微线体和棒状体的顶端细胞器中。微线体和棒状体蛋白的受控分泌到裂殖子表面以实现受体结合是入侵过程中的关键步骤。这些分泌事件的顺序以及触发释放的外部信号尚不清楚。我们使用延时视频显微镜研究了在红细胞入侵过程中恶性疟原虫裂殖子细胞内钙离子水平的变化。此外，我们开发了基于流式细胞术的方法来测量裂殖子细胞中细胞质钙离子的相对水平，并研究不同外部信号对恶性疟原虫裂殖子表面顶端细胞器蛋白表达的影响。我们证明，暴露于低钾离子浓度（如血浆中发现的）会通过磷脂酶 C 介导的途径导致细胞质钙离子水平升高。细胞质钙离子的升高触发微线体蛋白（如 175 kD 红细胞结合抗原（EBA175）和顶端膜抗原-1（AMA-1））分泌到裂殖子表面。随后，EBA175 与红细胞上的受体糖蛋白 A（glyA）相互作用，恢复基础细胞质钙离子水平并触发棒状体蛋白的释放。我们的结果首次确定了在红细胞入侵过程中微线体和棒状体蛋白顺序释放的外部信号，并为剖析这些关键顶端细胞器的调节性胞吐作用涉及的信号转导途径提供了起点。参与顶端细胞器排出的信号通路成分可能成为新药开发的新靶点，因为抑制微线体和棒状体的分泌可以阻断入侵并限制血期寄生虫的生长。
