Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands
Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands
复制标题
DOI：
10.1128/msphere.00325-20
复制
发表时间：
2020-07-01
期刊：
影响因子：
4.8
通讯作者：
Ishino, Tomoko
中科院分区：
文献类型：
--
作者：
Nozaki, Mamoru;Baba, Minami;Ishino, Tomoko
在疟原虫生命周期中，寄生虫的两个感染阶段，即分生孢子和子孢子，共享杆状和微线形顶端结构。裂殖子侵入红细胞的一个关键步骤是一些杆状体颈蛋白以复合体的形式排入宿主细胞膜，然后在寄生虫膜上形成一个涉及寄生虫分泌蛋白AMA1的移动连接。裂殖子棒状颈蛋白复合体的组成成分也在子孢子中表达，即RON2、RON4和RON5，这表明入侵机制元件在这些感染阶段之间可能是保守的。最近，我们在伯氏疟原虫的啮齿动物寄生虫模型中，利用子孢子阶段特异的基因敲除策略，证明了子孢子入侵蚊子唾液腺细胞和哺乳动物肝细胞需要RON2。在这里，我们使用免疫共沉淀法和卵囊衍生的子孢子提取物来证明RON2、RON4和RON5在子孢子中也形成了一个复合体。子孢子阶段特异性基因敲除策略表明，RON4和RON5在子孢子侵入唾液腺的过程中都起着至关重要的作用，包括滑行开始所需的附着能力显著降低。进一步的分析表明，在发育的子孢子中，RON2和RON4相互影响运输到棒状体，而RON5是独立运输的。这些发现表明，RON2和RON4之间的相互作用除了参与子孢子的附着外，还有助于它们的稳定性和向棒虫的运输。重要的子孢子是介导疟疾原虫从蚊子传播到哺乳动物宿主的运动性感染阶段。这项研究解决了棒状颈蛋白复合体是否在子孢子中的形式和功能，以及它在裂殖子中的作用的问题。通过免疫共沉淀和子孢子阶段特异性基因敲除实验，证明了RON2、RON4和RON5形成了一个复合体，并通过它们的附着能力参与了子孢子对唾液腺的侵袭。这些发现阐明了在顶端复合体感染阶段之间的共同侵袭机制。此外，子孢子阶段特异性基因敲除系统首次在疟原虫中揭示了RON2和RON4的相互作用相互影响它们的稳定性和运输到棒状体。我们的研究提出了RON复合体在子孢子成熟以及向靶细胞迁移和入侵过程中发挥作用的可能性。
In the Plasmodium life cycle, two infectious stages of parasites, merozo-ites and sporozoites, share rhoptry and microneme apical structures. A crucial step during merozoite invasion of erythrocytes is the discharge to the host cell mem-brane of some rhoptry neck proteins as a complex, followed by the formation of a moving junction involving the parasite-secreted protein AMA1 on the parasite mem-brane. Components of the merozoite rhoptry neck protein complex are also ex-pressed in sporozoites, namely, RON2, RON4, and RON5, suggesting that invasion mechanism elements might be conserved between these infective stages. Recently, we demonstrated that RON2 is required for sporozoite invasion of mosquito salivary gland cells and mammalian hepatocytes, using a sporozoite stage-specific gene knockdown strategy in the rodent malaria parasite model, Plasmodium berghei. Here, we use a coimmunoprecipitation assay and oocyst-derived sporozoite extracts to demonstrate that RON2, RON4, and RON5 also form a complex in sporozoites. The sporozoite stage-specific gene knockdown strategy revealed that both RON4 and RON5 have crucial roles during sporozoite invasion of salivary glands, including a significantly reduced attachment ability required for the onset of gliding. Further analyses indicated that RON2 and RON4 reciprocally affect trafficking to rhoptries in developing sporozoites, while RON5 is independently transported. These findings in-dicate that the interaction between RON2 and RON4 contributes to their stability and trafficking to rhoptries, in addition to involvement in sporozoite attachment.IMPORTANCE Sporozoites are the motile infectious stage that mediates malaria par-asite transmission from mosquitoes to the mammalian host. This study addresses the question whether the rhoptry neck protein complex forms and functions in sporozoites, in addition to its role in merozoites. By applying coimmunoprecipitation and sporozoite stage-specific gene knockdown assays, it was demonstrated that RON2, RON4, and RON5 form a complex and are involved in sporozoite invasion of salivary glands via their attachment ability. These findings shed light on the con-served invasion mechanisms among apicomplexan infective stages. In addition, the sporozoite stage-specific gene knockdown system has revealed for the first time in Plasmodium that the RON2 and RON4 interaction reciprocally affects their stability and trafficking to rhoptries. Our study raises the possibility that the RON complex functions during sporozoite maturation as well as migration toward and invasion of target cells.