The PTEX Pore Component EXP2 Is Important for Intrahepatic Development during the Plasmodium Liver Stage.
The PTEX Pore Component EXP2 Is Important for Intrahepatic Development during the Plasmodium Liver Stage.
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在脊椎动物感染期间，专性细胞内疟原虫在寄生虫空泡内发育，该空泡构成寄生虫与其肝细胞或红细胞宿主细胞之间的界面。为了穿过这一屏障，疟原虫属（Plasmodium spp.）利用由EXP 2形成的双功能孔进行营养转运，并且在PTEX易位子的情况下，效应蛋白穿过液泡膜输出。虽然对血液阶段的存活至关重要，但对肝脏阶段的EXP 2/PTEX功能知之甚少，尽管肝内空泡中PTEX解折叠酶HSP 101的缺乏表明了输出机制的主要差异。在这里，我们采用葡萄糖胺激活glmS核酶研究在伯氏疟原虫肝脏阶段的发展过程中，在肝癌细胞的EXP 2的作用。将glmS序列插入EXP 2 3 '非翻译区（UTR）能够在肝细胞侵袭后依赖于葡萄糖胺耗尽EXP 2，从而将肝内发育期间的EXP 2功能与最近报道的在肝细胞侵袭中的作用分开。侵袭后EXP 2敲低降低了寄生虫的大小，并在很大程度上消除了中期至晚期肝脏阶段标志物LISP 2的表达。作为监测发育的正交方法，将EXP 2-glmS寄生虫和对照工程化以表达纳米荧光素酶。激活glmS入侵后大大降低发光肝癌单层和培养上清液中对应于裂体脱离，这标志着高潮的肝脏阶段的发展在以后的时间点。总的来说，我们的研究结果扩展了glmS核酶的实用性，以研究蛋白质在肝脏阶段的功能，并揭示了EXP 2是重要的肝内寄生虫的发展，表明PTEX组件也在肝细胞寄生虫接口的功能。
During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3′ untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface.
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