Differential impacts on host transcription by ROP and GRA effectors from the intracellular parasite Toxoplasma gondii. Differential impacts on host transcription by ROP and GRA effectors from the intracellular parasite Toxoplasma gondii

The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. Overall design: A total of 8395 cells were sequenced and a total of 5354 cells passed quality control and were sequenced. Samples were treated for three timepoints. For each treatment, we have measured a negative control sample treated with mock infection.

刚地弓形虫（Toxoplasma gondii）作为一种细胞内寄生原虫，可通过分泌源自棒状体（rhoptry）与致密颗粒（dense granule）细胞器的大量效应蛋白，调控宿主细胞的生物学过程；这类效应蛋白分别被称为ROP与GRA。为探究ROP与GRA对宿主基因表达的独立影响，我们开发了一套稳定可靠的新型实验方案，用于富集超高纯度、天然存在且可重复的宿主细胞群体——未感染但已注射（uninfected-injected, U-I）细胞，这类细胞会被刚地弓形虫注入ROP蛋白，但后续无法发生入侵过程。随后，我们对感染后1-3小时的U-I细胞（同时设置未感染对照组与感染对照组）开展单细胞转录组分析，这些细胞分别来自野生型弓形虫感染，或是缺失MYR1蛋白的弓形虫感染；MYR1蛋白是可溶性GRA跨越纳虫空泡膜（parasitophorous vacuole membrane, PVM）并进入宿主细胞胞质所必需的功能因子。通过对比感染细胞与U-I细胞的转录组特征，宿主对感染的早期应答主要由注入的ROP蛋白介导，这类蛋白可诱导免疫应答与细胞应激通路。上述依赖ROP的促炎转录特征，可被至少部分依赖MYR1的GRA所拮抗，而嵌入纳虫空泡膜的非MYR1依赖型GRA则可能增强该特征。此外，对感染单层细胞中的未感染旁观者细胞进行分析发现，依赖MYR1的旁分泌效应同样可拮抗依赖ROP的炎症反应过程。实验整体设计：本次研究共对8395个细胞进行测序，最终5354个细胞通过质量质控并纳入后续分析。样本设置3个时间梯度，每种处理条件均设置模拟感染的阴性对照样本。