Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii
Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii
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DOI：
10.1074/jbc.m504158200
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发表时间：
2005-10-07
影响因子：
4.8
通讯作者：
Boothroyd, JC
中科院分区：
文献类型：
--
作者：
Bradley, PJ;Ward, C;Boothroyd, JC
棒状体是特殊的分泌细胞器，独特地存在于顶复门的原生动物寄生虫中。这些专性细胞内寄生虫包括一些人类和动物最重要的寄生虫，包括疟疾（疟原虫属）和鸡球虫病（艾美耳球虫属）的病原体。在侵入宿主细胞期间，棒状体的内容物被释放到新生的寄生液泡中，所产生的蛋白质通常代表宿主和病原体之间的实际界面。我们开发了一种从研究最充分的顶复门弓形虫中高效纯化菱形体的方法，并使用质谱法进行了详细的蛋白质组分析，鉴定出了 38 种新蛋白质。为了确认它们的棒状体起源，针对合成肽和/或重组蛋白产生了抗体。其中 12 个抗体中的 11 个产生了抗体，通过免疫荧光显示棒状体颈和/或其球根状基部内有强烈的棒状体染色。血凝素表位标记证实了另一种来自菱形球的新蛋白质。以前从弓形虫和疟原虫中鉴定出的棒状体蛋白对于一种或另一种生物体是独特的，但我们对弓形虫棒状体蛋白质组的阐明揭示了两者共有的同源物。这项研究还鉴定了第一个编码棒状体颈蛋白的弓形虫基因，我们将其命名为 RON，证明了弓形虫蛋白和 Rab11 是棒状体蛋白，并鉴定了新的激酶、磷酸酶和蛋白酶，它们可能在寄生虫侵入和选择宿主细胞以实现自身生存和生长的能力中发挥关键作用。
Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria ( Plasmodium spp.) and chicken coccidiosis ( Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.