Malaria parasites increase their host erythrocyte’s permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell’s parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation.

中文翻译：

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活细胞FRET显示在整个曲折贩运过程中，疟疾营养通道蛋白CLAG3和RhopH2仍然保持关联。

疟原虫会增加其宿主红细胞对各种营养素的渗透性，从而加剧细胞内病原体的发育和复制。质子表面阴离子通道（PSAC）介导了这种摄取，并与由CLAG3，RhopH2和RhopH3组成的寄生虫编码RhopH复合物相连。虽然这些亚基之间的相互作用已得到很好的建立，但尚不清楚它们是否仍通过红细胞的入侵和转运到宿主膜而在合成裂殖子中保持其缔合状态。在这里，我们使用活细胞成像和Förster共振能量转移（FRET）实验探索了RhopH亚基之间的蛋白质相互作用。使用绿色荧光蛋白（GFP）衍生物mCerulean和mVenus，我们生成了用于荧光测量的单标签和双标签寄生虫品系。尽管CLAG3-mCerulean成为有效的FRET供体，用于rhoptry细胞器内的RhopH2-mVenus，但通过短信号序列靶向该细胞器的mCerulean产生的FRET可以忽略不计。裂殖子外出和入侵后，这些标记的RhopH亚基被沉积到新宿主细胞的寄生虫液泡中。然后将这些蛋白质输出并运输到红细胞膜，在那里CLAG3和RhopH2保持完全缔合。荧光强度测量确定了当红细胞被两种寄生虫感染时，输出的RhopH蛋白的化学计量增加。全细胞膜片钳显示了PSAC功能拷贝数的同时增加以及RhopH对离子和营养渗透性的贡献的剂量效应。这些研究建立了人类疟疾寄生虫的活细胞FRET成像，