Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking
Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking
复制标题
DOI：
10.1128/mbio.01354-20
复制
发表时间：
2020-09-01
期刊：
影响因子：
6.4
通讯作者：
Desaia, Sanjay A.
中科院分区：
文献类型：
--
作者：
Ahmad, Moaz;Manzella-Lapeira, Javier;Desaia, Sanjay A.
疟疾寄生虫增加其宿主红细胞对各种营养物质的通透性，促进细胞内病原体的发展和复制。质膜表面阴离子通道(PSAC)介导这种摄取，并与寄生虫编码的RHopH复合体相连，该复合体由CLAG3、Rhop H2和Rhop H3组成。虽然这些亚基之间的相互作用已经建立得很好，但目前还不清楚它们是否仍然与它们的合成有关，通过红细胞入侵和运输到宿主膜来发育裂殖子。在这里，我们使用活细胞成像和Forster共振能量转移(FRET)实验来探索Rhop H亚基之间的蛋白质-蛋白质相互作用。使用绿色荧光蛋白(GFP)衍生物mCerulean和mVenus，我们产生了用于荧光测量的单标记和双标记寄生虫线。当CLAG3-mCerulean作为Rhop H2-mVenus在杆状细胞器内的有效FRET供体时，mCerulean通过短信号序列靶向该细胞器产生的FRET可以忽略不计。在裂殖子离开和重新入侵时，这些标记的Rhop H亚基被沉积到新宿主细胞的寄生虫空泡中；然后这些蛋白被输出并运输到红细胞膜，在那里CLAG3和Rhop H2仍然完全结合。荧光强度测量发现，当红细胞感染两种寄生虫时，输出的Rhop H蛋白的化学计量增加；全细胞膜片钳显示PSAC功能拷贝数随之增加，并对Rhop H对离子和营养通透性的贡献存在剂量效应。这些研究在人类疟疾寄生虫中建立了活细胞FRET成像，揭示了Rhop H亚基不解离地运输到其宿主膜目的地，并提出了对PSAC形成的定量贡献。重要的是疟疾寄生虫生长在循环中的红细胞内，并通过其宿主膜上的一个孔吸收营养。在这里，我们使用基因编辑技术，用荧光标记物标记CLAG3和RHopH2，这是两种与营养孔相连的蛋白质，并在活的感染细胞中跟踪这些蛋白质。在成熟寄生虫体内合成后，成像显示这两种蛋白都被包装成膜结合的棒状病毒。当寄生虫破坏宿主细胞并入侵新的红细胞时，这些蛋白质在迁移并插入宿主细胞表膜之前，在寄生虫周围的空泡中被检测到。通过同时标记CLAG3和Rhop H2，我们确定这些蛋白质在迁移过程中和表面膜插入后紧密相互作用。感染了两种寄生虫的红细胞表面的蛋白质增加了一倍，营养毛孔的数量也相应增加。我们的工作表明，这些蛋白质直接促进寄生虫从人类血浆中摄取营养。
Malaria parasites increase their host erythrocyte's permeability to vari-ous 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 Forster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated singleand 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.IMPORTANCE Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma.