BioID Reveals Novel Proteins of the Plasmodium Parasitophorous Vacuole Membrane.
BioID Reveals Novel Proteins of the Plasmodium Parasitophorous Vacuole Membrane.
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生物分子揭示了疟原虫寄生虫液泡膜的新型蛋白质。
DOI：
10.1128/msphere.00522-17
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发表时间：
2018-01
期刊：
影响因子：
4.8
通讯作者：
Burda PC
中科院分区：
文献类型：
--
作者：
Schnider CB;Bausch-Fluck D;Brühlmann F;Heussler VT;Burda PC
细胞内病原体通常被宿主细胞衍生的膜包围。这种膜被病原体根据自己的需要进行修饰，对它们的细胞内生活方式至关重要。在疟原虫寄生虫中，这种膜被称为PVM，到目前为止，只有有限数量的蛋白质是已知的。在这里，我们应用在啮齿动物P. berghei寄生虫称为BioID的方法，这是基于生物素化的近端和相互作用的蛋白质的混杂的生物素连接酶BirA*，并证明其在识别新的PVM蛋白的有用性。在脊椎动物宿主内发育期间，疟原虫寄生虫感染肝细胞和红细胞。在这些细胞内，寄生虫被寄生虫空泡膜（PVM）包围。PVM在寄生虫与其宿主细胞的相互作用中起着至关重要的作用;然而，迄今为止，只有有限数量的这种膜蛋白被鉴定出来。这部分是因为PVM的蛋白质含量的系统性蛋白质组学分析在过去是困难的，这是由于在试图将PVM与其他膜（例如寄生虫质膜）分离时遇到的困难。在这项研究中，我们适应的BioID技术在体外培养的伯氏疟原虫血液阶段的寄生虫，并利用混杂的生物素连接酶BirA* 融合PVM居民输出蛋白1生物素化蛋白的PVM。这些我们进一步处理的亲和纯化，液相色谱-串联质谱（LC-MS/MS），和无标记定量，导致61个已知的和候选PVM蛋白的列表。在血液和肝脏发育阶段进一步分析了7种蛋白质。这导致鉴定了三种新的PVM蛋白，它们是丝氨酸/苏氨酸蛋白磷酸酶UIS 2（PlasmoDB登录号PBANKA_1328000）和两种具有未知功能的保守疟原虫蛋白（PBANKA_0519300和PBANKA_0509000）。总之，我们的报告扩大了已知PVM蛋白的数量，并通过实验验证了BioID作为一种强大的方法来筛选伯氏疟原虫特定细胞区室的新成分。细胞内病原体通常被宿主细胞衍生的膜包围。这种膜被病原体根据自己的需要进行修饰，对它们的细胞内生活方式至关重要。在疟原虫寄生虫中，这种膜被称为PVM，到目前为止，只有有限数量的蛋白质是已知的。在这里，我们应用在啮齿动物P. berghei寄生虫称为BioID的方法，这是基于生物素化的近端和相互作用的蛋白质的混杂的生物素连接酶BirA*，并证明其在识别新的PVM蛋白的有用性。
Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P. berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins. During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro-cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P. berghei. IMPORTANCE Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P. berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.