In the Plasmodium life cycle, two infectious stages of parasites, merozoites and sporozoites, share rhoptry and microneme apical structures. A crucial step during merozoite invasion of erythrocytes is the discharge to the host cell membrane 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 membrane. Components of the merozoite rhoptry neck protein complex are also expressed 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 indicate that the interaction between RON2 and RON4 contributes to their stability and trafficking to rhoptries, in addition to involvement in sporozoite attachment.

中文翻译：

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疟原虫子孢子中菱形颈蛋白复合物的检测及其对唾液腺子孢子入侵的贡献。

在疟原虫中在生命周期中，寄生虫的两个感染阶段，裂殖子和子孢子，共享菱形和微线体顶端结构。裂殖子侵入红细胞过程中的一个关键步骤是将一些菱形颈蛋白作为复合物排放到宿主细胞膜，然后在寄生虫膜上形成涉及寄生虫分泌蛋白 AMA1 的移动连接。裂殖子菱形颈蛋白复合物的成分也在子孢子中表达，即 RON2、RON4 和 RON5，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，我们在啮齿动物疟疾寄生虫模型中使用子孢子阶段特异性基因敲除策略证明了 RON2 是子孢子侵入蚊子唾液腺细胞和哺乳动物肝细胞所必需的，伯氏疟原虫。在这里，我们使用免疫共沉淀试验和卵囊衍生的子孢子提取物来证明 RON2、RON4 和 RON5 也在子孢子中形成复合物。子孢子阶段特异性基因敲低策略表明，RON4 和 RON5 在子孢子侵入唾液腺过程中都具有关键作用，包括滑动开始所需的附着能力显着降低。进一步的分析表明，RON2 和 RON4 在子孢子发育过程中相互影响向菱形体的运输，而 RON5 是独立运输的。这些发现表明 RON2 和 RON4 之间的相互作用有助于它们的稳定性和向菱形体的运输，此外还参与子孢子附着。