Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner.
Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner.
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DOI：
10.3389/fcimb.2017.00397
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发表时间：
2017
影响因子：
5.7
通讯作者：
Neunuebel MR
中科院分区：
文献类型：
--
作者：
Allgood SC;Romero Dueñas BP;Noll RR;Pike C;Lein S;Neunuebel MR
兼性细胞内细菌嗜肺军团菌在阿米巴原虫和人类肺泡巨噬细胞内增殖，它是军团病（一种危及生命的肺炎）的病原体。在宿主细胞内，嗜肺军团菌通过将大量效应蛋白递送到宿主细胞质中来建立复制避难所，其中许多效应蛋白通过操纵 Rab GTPases 的功能来靶向膜运输。军团菌效应器 AnkX 是一种磷酸胆碱转移酶，可共价修饰宿主 Rab1 和 Rab35。然而，对 Rab GTP 酶磷酸胆酸化的生物学后果的详细了解仍然难以捉摸。在这里，我们通过提出三个证据证明 AnkX 干扰宿主内吞循环，扩大了对 AnkX 功能的理解。首先，使用免疫金透射电子显微镜，我们确定哺乳动物细胞中异位产生的 GFP 标记的 AnkX 定位于质膜和管状膜区室，这些位点与靶向内吞回收途径一致。此外，AnkX 的 C 端区域负责与质膜结合，我们确定该区域也能够在体外结合磷酸肌醇脂质 PI(3)P 和 PI(4)P。其次，我们观察到 mCherry-AnkX 与 Rab35（一种回收内吞作用的调节剂）和主要组织相容性 I 类蛋白（MHC-I）共定位，MHC-I 是一种关键的免疫调节蛋白，其从质膜的回收和返回到质膜的回收是 Rab35 依赖性的。第三，我们报告在巨噬细胞感染期间，AnkX 负责破坏转铁蛋白的内吞循环，而 AnkX 的磷酸胆酸化活性对此功能至关重要。这些结果支持 AnkX 在宿主细胞感染期间以内吞循环为目标的假设。最后，我们证明 AnkX 的磷酸胆酸活性对于抑制含军团菌的液泡 (LCV) 与溶酶体的融合也至关重要。
The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3)P and PI(4)P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella-containing vacuole (LCV) with lysosomes.