Mammalian cell collaboration with bacterial type III secretion

描述(申请人提供)：大量细菌病原体通过将蛋白质输送到宿主细胞中来引起疾病，目的是解除免疫防御或在组织中建立微生物复制场所。其中最重要的递送系统是多亚单位III型分泌系统。虽然将这些错误调节因子转移到宿主细胞所涉及的细菌决定因素是已知的，但关于真核细胞如何促进细菌蛋白质跨宿主细胞膜的移动，或者宿主如何促进与细胞的靶标识别，几乎没有信息。这项应用旨在解决这一差距，因为它代表了第一次系统地尝试识别所有热细胞蛋白质，这些蛋白质相互协作，确保细菌蛋白质有效地跨质膜转移并移动到它们预期的目标。拟议的研究重点是假结核耶尔森菌的蛋白质传递，这是一种肠道致病微生物，从肠道传播后会导致全身疾病。他们利用了在这些研究过程中开发的一种新策略，其中使用荧光共振能量转移(FRET)报告程序来测量在细菌接触过程中传递蛋白质YopE后单细胞中的活动量。分离了含有干扰YopE活性的shRNAs的细胞，并鉴定了一些参与宿主细胞质膜中细菌通道组装的人类蛋白。在筛选中发现的基因指向一个模型，在该模型中，易位通道的插入需要来自趋化因子受体的信号，据预测，趋化因子受体将组织宿主细胞骨架蛋白来促进通道的组装。该应用的目的是确定细菌转位系统功能所需的一整套真核蛋白，目的是这些蛋白可以被证明参与以下三个过程之一：1)通道的组装；2)退出通道的底物的折叠；或3)将转位的细菌蛋白靶向其细胞内靶点。特别令人感兴趣的是确定趋化因子受体是否作为通道的识别位置，或者它们是否作为信号分子来允许其他宿主蛋白支持通道组装。最后，将检验一个假设，即宿主细胞中易位通道的组装需要多个细胞表面信号来放大细胞骨架反应。通过确定细菌蛋白质和它们的宿主细胞伙伴之间的重要合作关系，人们希望能够确定针对微生物感染的治疗干预地点。此外，所获得的数据旨在挑选协作的人类蛋白质，这些蛋白质可能允许识别对传染病具有不同敏感性的个体。

DESCRIPTION (provided by applicant): A large number of bacterial pathogens cause disease by delivering proteins into host cells for the purposes of disarming immune defenses or establishing sites for microbial replication in tissues. Among the most important delivery systems is the multisubunit type III secretion system. Although the bacterial determinants involved in moving these misregulators into host cells are known, there is almost no information regarding how the eukaryotic cell contributes to bacterial protein movement across the host cell membrane, nor how the host facilitates recognition of targets with the cell. This application is intended to address this gap, in that it represents the first systematic attempt to identify all hot cell proteins that collaborate to ensure efficient translocation of bacterial proteins across the plasma membrane and movement to their intended targets. The proposed studies focus on protein delivery by Yersinia pseudotuberculosis, an enteropathogenic organism that causes systemic disease after spread from the intestine. They take advantage of a novel strategy developed in the course of these studies, in which a Fluorescence Resonance Energy Transfer (FRET) reporter is used to measure the amount of activity in single cells after delivery of the protein YopE during bacterial encounter. Cells having shRNAs that interfere with YopE activity were isolated, and a number of human proteins involved in assembly of a bacterial channel in the host cell plasma membranes were identified. The genes identified in the screen point to a model in which insertion of the translocation channel requires signaling from chemokine receptors, which are predicted to organize host cytoskeletal proteins to promote assembly of the channel. The Aims of the application are directed toward identifying the complete set of eukaryotic proteins necessary for function of the bacterial translocation system, with the goal that these proteins can be shown to be involved in one of three processes: 1) assembly of the channel; 2) folding of substrates exiting the channel; or 3) targeting of the translocated bacteria proteins to their intracellular targets. Of particular interest is determining whether chemokine receptors act as recognition sites for the channel, or whether they act as signaling molecules to allow other host proteins to support channel assembly. Finally, a hypothesis will be tested that assembly of the translocation channel in host cells requires multiple cell surface signals to amplify a cytoskeletal response. By identifying important collaborative relationships between bacterial proteins and their host cell partners, it is hoped that sites for therapeutic interventio against microbial infections can be identified. Furthermore, the data obtained are intended to single out collaborative human proteins that may allow the identification of individuals with variant sensitivities to infectious diseases.