Dissecting the roles of superchaperones in type III secretion

描述（由申请人提供）：包括许多NIAID生物防御优先病原体在内的许多革兰氏阴性细菌利用III型分泌系统（T3 SS）将数十种蛋白质递送到宿主细胞中。虽然这些复杂机器的许多组件是高度保守的，并且在功能上是可互换的，但每种细菌都将其独特的效应子传递到宿主细胞中。关于细菌如何确保它们编码的数千种蛋白质中只有一小部分被特异性地递送到宿主细胞中的完整理解仍然不清楚。我的实验室最近开发了一种新的基于酵母的可视化分析，以识别活细胞中相互作用的蛋白质，蛋白质相互作用平台分析或PIP。值得注意的是，我们发现一个伴侣Spa 15与10个效应子相互作用，其中9个效应子随后建立需要Spa 15来进行有效的III型分泌。此外，我们发现，Spa 15同源物从8个细菌物种是功能上可互换的Spa 15。所有九种蛋白质都识别一个确定的伴侣结合结构域序列。鉴于这八个分子伴侣的功能互换，我们假设它们通过保守的机制识别效应子并将效应子递送到分泌装置。该提案的目标是开始确定III型分泌中这些重要步骤的分子机制。具体而言，我们建议（a）研究IB类分子伴侣如何区分效应子和管家蛋白（目标1）和（B）确定分子伴侣/效应子复合物如何被募集到III型分泌装置（目标2）。从这些研究中获得的见解可能会产生新的方向，用于开发新的药物，用于治疗与革兰氏阴性细菌感染相关的各种疾病。

DESCRIPTION (provided by applicant): Numerous Gram-negative bacteria including many NIAID Biodefense priority pathogens utilize type III secretion systems (T3SSs) to deliver tens of proteins into host cells. While many of the components of these complex machines are highly conserved and functionally interchangeable, each bacterium delivers its own unique set of effectors into host cells. A complete understanding of how the bacteria ensure that only small subsets of the thousands of proteins they encode are specifically delivered into host cells remains unclear. My laboratory recently developed a novel yeast-based visualization assay to identify interacting proteins in living cells, the Protein Interaction Platform assay or PIP. Remarkably, we found that one chaperone, Spa15, interacted with 10 effectors, nine of which we subsequently established require Spa15 for their efficient type III secretion. Furthermore, we find that the Spa15 homologs from eight bacterial species are functionally interchangeable with Spa15. All nine proteins recognize a defined chaperone-binding domain sequence. Given the functional interchangeable of these eight chaperones, we hypothesize that they recognize effectors and deliver effectors to the secretion apparatus by conserved mechanisms. The goals of the proposal are to begin to determine the molecular mechanisms underlying these important steps in type III secretion. Specifically we propose to (a) investigate how class IB chaperones differentiate between effectors and housekeeping proteins (Aim 1) and (b) determine how chaperone/effector complexes are recruited to the type III secretion apparatus (Aim 2). Insights gained from these studies will likely result in generating new directions for the development of novel agents for the treatment of a variety of diseases associated with Gram-negative bacterial infections.