Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN No. 2508, Ciudad de México C.P. 07360, Mexico.
Unidad de Microscopía Electrónica, LaNSE, CINVESTAV-IPN, Mexico.
J Proteomics. 2019 Sep 30;208:103490. doi: 10.1016/j.jprot.2019.103490. Epub 2019 Aug 18.
After the cell invasion, the parasite Toxoplasma gondii locates within a parasitophorous vacuole to proliferate. It continuously modifies the composition of the parasitophorous vacuole by the secretion of GRA and ROP proteins, some of which become inserted into the vacuole membrane, remain as soluble proteins or involved in the intravacuolar network. In this report, we analyze the excretion/secretion products and the vesicles released by extracellular tachyzoites, this structures were morphologically analyzed by electron microscopy and characterized by mass spectrometry. The structural analysis showed parasites secreting in vitro individual vesicles with similarities to ectosomes and exosomes and which characterized to self-assembly in vitro forming vesicle-tubular structures morphologically similar to the intravacuolar network from infected cells. The vesicle-tubular structures were recognized with antibodies against ROP2 and GRA2. In addition, analysis by Western blot evidenced proteins from the secretory organelles. A detailed proteomic analysis of exosomes, ectosomes and soluble proteins released in vitro is here reported. Presence of GRA proteins in secretions from resting extracellular parasites indicates that these molecules are not exclusively secreted within the parasitophorous vacuole of the infected cell as reported but they are constitutively excreted/secreted even in an extracellular condition. Data are available via ProteomeXchange with identifier PXD013767. SIGNIFICANCE: Extracellular tachyzoites constitutively secrete components that previously were considered be secreted only within the parasitophorous vacuole, suggesting that in the infected host these molecules are in direct interaction with cells and molecules of the host cell including those of the immune response.
细胞入侵后，寄生虫刚地弓形虫定位于一个寄生空泡内进行增殖。它通过分泌 GRA 和 ROP 蛋白不断改变寄生空泡的组成，其中一些蛋白插入空泡膜，保持可溶性蛋白状态或参与腔内网络。在本报告中，我们分析了细胞外速殖子的分泌/排出产物和释放的囊泡，通过电子显微镜对这些结构进行形态分析，并通过质谱进行特征分析。结构分析显示，寄生虫在体外分泌具有类似外泌体和细胞外囊泡的个体囊泡，并且能够在体外自组装形成囊泡管状结构，其形态与感染细胞内的腔内网络相似。用针对 ROP2 和 GRA2 的抗体识别出了囊泡管状结构。此外，Western blot 分析证实了分泌细胞器中的蛋白质。本文还报道了体外释放的外泌体、细胞外体和可溶性蛋白的详细蛋白质组学分析。静止的细胞外寄生虫分泌体中的 GRA 蛋白表明，这些分子并非像以前报道的那样仅在感染细胞的寄生空泡中分泌，而是即使在细胞外条件下也持续分泌/排出。数据可通过 ProteomeXchange 以标识符 PXD013767 获得。意义：细胞外速殖子持续分泌以前被认为仅在寄生空泡内分泌的成分，这表明在感染宿主中，这些分子与宿主细胞的细胞和分子直接相互作用，包括免疫反应的分子。
