YscE/YscG chaperone-dependent secretion of Yersinia pestis YscF

描述(申请人提供)：鼠疫耶尔森氏菌是鼠疫的病原体，使用III型分泌系统(T3SS)将效应蛋白注入真核细胞。耶尔森氏菌T3S装置的组装和功能依赖于21种耶尔森氏菌必需分泌物(YSC)蛋白。在组装了一个功能碱基结构后，T3S装置只分泌起组装杆状(YscI)和针状(YscF)结构功能的早期底物(YscF、YscI和YscP)；然而，人们对YscF和其他能够选择性识别和分泌它们的早期底物的独特特性知之甚少。在这项提案中，我们将描述识别YscF为早期T3S底物的独特分子特征，以及识别这些特征的T3S装置的组件。重要的是，细胞质YscF与其异源二聚体YscE/YscG伴侣形成1：1：1的复合体，这种相互作用是防止YscF蛋白降解所必需的。我们还提供了实验证据，证明YscE/YscG伴侣对YscF的分泌是必不可少的，而不是独立于其稳定YscF的作用。首先，我们将研究YscF N端分泌信号在YscF分级分泌中的作用。此外，结合和稳定YscF，但不分泌YscF的YscE和YscG点突变体将被鉴定。总之，这些研究将确定YscF和YscE/YscG伴侣的独特分子特征，这是识别YscEFG复合体和选择性分泌YscF所必需的。最后，我们将使用表征YscKYscQYscL分选平台和YscN ATPase在识别天然和突变的YscEFG复合体中的作用。这些研究将定义识别事件，使T3S装置能够区分早期和晚期的T3S底物，这一过程对于建立分泌层次至关重要。 与公共卫生相关：该项目的总体目标是了解病原菌如何组装专门的蛋白质分泌系统，使它们能够直接将毒素注入人类细胞。干扰这些组装活动的药物有望解除这些病原体的武装，并允许自然防御机制消除细菌。

DESCRIPTION (provided by applicant): Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. The assembly and function of the Yersinia T3S apparatus is dependent upon 21 essential Yersinia secretion (Ysc) proteins. Following assembly of a functional base structure, the T3S apparatus secretes only early substrates (YscF, YscI and YscP) that function to assemble the rod (YscI) and needle (YscF) structures; however, little is known about the unique features of YscF and other early substrates that enable their selective recognition and secretion. In this proposal, we will characterize the unique molecular features that identify YscF as an early T3S substrate; as well as the components of the T3S apparatus that recognize these features. Importantly, cytoplasmic YscF is found exclusively in a 1:1:1 complex with its heterodimeric YscE/YscG chaperone, an interaction that is essential to prevent proteolytic degradation of YscF. We also provide experimental evidence that the YscE/YscG chaperone is essential for YscF secretion independent of its role in stabilizing YscF. Initially, we will investigate the role of the YscF N-terminal secretion signal in the hierarchical secretion of YscF. In addition, YscE and YscG point mutants that bind and stabilize YscF, but do not secrete YscF will be identified. Together, these studies will define the unique molecular features of YscF and the YscE/YscG chaperone that are required for recognition of the YscEFG complex and selective secretion of YscF. Finally, we will use characterize the role of a YscKYscQYscL sorting platform and YscN ATPase in the recognition of native and mutant YscEFG complexes. These studies will define the recognition events that allow the T3S apparatus to differentiate early and late T3S substrates, a process that is essential for establishing a secretion hierarchy. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to understand how pathogenic bacteria assemble specialized protein secretion systems that allow them to inject toxins directly into human cells. Drugs that interfere with these assembly events would be expected to disarm these pathogens and allow natural defense mechanisms to eliminate the bacteria.