Table_1_Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum.xlsx

Plasmodium species cause malaria, and in the instance of Plasmodium falciparum is responsible for a societal burden of over 600,000 deaths annually. The symptoms and pathology of malaria are due to intraerythocytic parasites. Erythrocyte invasion is mediated by the parasite merozoite stage, and is accompanied by the formation of a parasitophorous vacuolar membrane (PVM), within which the parasite develops. The merozoite apical rhoptry organelle contains various proteins that contribute to erythrocyte attachment and invasion. RON3, a rhoptry bulb membrane protein, undergoes protein processing and is discharged into the PVM during invasion. RON3-deficient parasites fail to develop beyond the intraerythrocytic ring stage, and protein export into erythrocytes by the Plasmodium translocon of exported proteins (PTEX) apparatus is abrogated, as well as glucose uptake into parasites. It is known that truncated N- and C-terminal RON3 fragments are present in rhoptries, but it is unclear which RON3 fragments contribute to protein export by PTEX and glucose uptake through the PVM. To investigate and distinguish the roles of the RON3 C-terminal fragment at distinct developmental stages, we used a C-terminus tag for conditional and post-translational control. We demonstrated that RON3 is essential for blood-stage parasite survival, and knockdown of RON3 C-terminal fragment expression from the early schizont stage induces a defect in erythrocyte invasion and the subsequent development of ring stage parasites. Protein processing of full-length RON3 was partially inhibited in the schizont stage, and the RON3 C-terminal fragment was abolished in subsequent ring-stage parasites compared to the RON3 N-terminal fragment. Protein export and glucose uptake were abrogated specifically in the late ring stage. Plasmodial surface anion channel (PSAC) activity was partially retained, facilitating small molecule traffic across the erythrocyte membrane. The knockdown of the RON3 C-terminal fragment after erythrocyte invasion did not alter parasite growth. These data suggest that the RON3 C-terminal fragment participates in erythrocyte invasion and serves an essential role in the progression of ring-stage parasite growth by the establishment of the nutrient-permeable channel in the PVM, accompanying the transport of ring-stage parasite protein from the plasma membrane to the PVM.

疟原虫属（Plasmodium）物种可引发疟疾，其中恶性疟原虫（Plasmodium falciparum）每年造成超过60万例死亡，带来沉重的社会公共卫生负担。疟疾的症状与病理改变均由红细胞内寄生的疟原虫引发。疟原虫的红细胞入侵过程由裂殖子阶段介导，同时会形成纳虫空泡膜（parasitophorous vacuolar membrane, PVM），疟原虫即在该膜结构内发育增殖。裂殖子顶端的棒状体细胞器含有多种参与红细胞黏附与入侵的蛋白。棒状体球膜蛋白RON3会经历蛋白加工过程，并在入侵过程中被释放至纳虫空泡膜内。RON3基因缺陷的疟原虫无法突破红细胞内环状体阶段完成发育，同时其依赖疟原虫输出蛋白转位酶（Plasmodium translocon of exported proteins, PTEX）完成的红细胞内蛋白输出过程，以及疟原虫自身的葡萄糖摄取过程均被阻断。目前已知棒状体内存在截短的RON3 N端与C端片段，但尚不明确究竟是哪一段RON3片段参与PTEX介导的蛋白输出，以及通过PVM完成的葡萄糖摄取过程。为探究并明确RON3 C端片段在疟原虫不同发育阶段的功能，我们利用C端标签构建了条件性翻译后调控系统。本研究证实RON3对红内期疟原虫的存活至关重要；若从裂殖体早期开始敲低RON3 C端片段的表达，会导致红细胞入侵过程出现缺陷，进而影响环状体阶段疟原虫的后续发育。完整长度RON3的蛋白加工过程在裂殖体阶段受到部分抑制，且与RON3 N端片段相比，后续环状体阶段疟原虫内的RON3 C端片段已无法检测到。蛋白输出与葡萄糖摄取过程仅在晚期环状体阶段被完全阻断。疟原虫表面阴离子通道（Plasmodial surface anion channel, PSAC）的活性得到部分保留，仍可介导小分子物质跨红细胞膜转运。若在红细胞入侵完成后再敲低RON3 C端片段的表达，则不会对疟原虫的生长造成影响。上述实验结果表明，RON3 C端片段既参与红细胞入侵过程，又可通过在PVM中构建营养通透性通道，辅助环状体阶段疟原虫蛋白从质膜向PVM的转运，从而在环状体阶段疟原虫的生长进程中发挥核心作用。