In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.
In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.
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DOI：
10.1128/mbio.00808-16
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发表时间：
2016-08-02
期刊：
影响因子：
6.4
通讯作者：
Bradley PJ
中科院分区：
文献类型：
--
作者：
Nadipuram SM;Kim EW;Vashisht AA;Lin AH;Bell HN;Coppens I;Wohlschlegel JA;Bradley PJ
弓形虫是一种专性的细胞内寄生虫，它入侵宿主细胞，并在一个独特的寄生空泡内复制。为了维持这种细胞内的生态位，寄生虫将一系列致密颗粒蛋白(GRAS)分泌到新生的寄生虫液泡中。当寄生虫在宿主细胞内复制时，这些GRAs被认为在空泡重塑、营养吸收和免疫逃避中发挥关键作用。尽管GRAS在弓形虫的生命周期中起着核心作用，但目前只鉴定了这些蛋白中的一小部分，并且它们的许多作用还没有完全阐明。在这篇报道中，我们利用混杂的生物素连接酶Bira*来生物素化分泌到液泡中的GRA蛋白，然后通过亲和纯化和质谱分析鉴定这些蛋白。以GRA-BIRA*融合蛋白为诱饵，我们鉴定了大量已知和候选的GRA蛋白，并通过内源基因标记验证了13个新的GRA蛋白的定位。我们接着用基因敲除的方法对该组中三个相关的GRAA(GRA38、GRA39和GRA40)进行了功能鉴定。虽然ΔGra38和ΔGra40寄生虫没有表现出改变的表型，但破坏GRA39会导致寄生虫生长缓慢，在寄生虫的液泡中含有显著的脂质沉积，这表明在对寄生虫生长至关重要的脂质调节中发挥了作用。此外，缺乏GRA39的寄生虫在体内显示出极大的降低毒力和较低的组织包囊负荷。综上所述，这项工作的发现揭示了弓形虫的部分空泡蛋白质组，并确定了一种在寄生虫复制和致病过程中发挥关键作用的新的GRA。大多数细胞内病原体驻留在其宿主细胞内的一个膜结合的空泡内，该空泡被病原体广泛修饰，以优化细胞内的生长并避免宿主防御。在弓形虫中，这个液泡被一系列分泌型GRA蛋白修饰，其中许多蛋白仍未被识别。在这里，我们证明了在体内使用混杂生物素连接酶BIRA*对近端和相互作用的蛋白进行生物素化是快速鉴定空泡GRA蛋白的有效方法。我们进一步证明，这种方法确定的一个因子，GRA39，在寄生虫在其宿主细胞内复制并导致疾病的能力中发挥重要作用。
Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease.