Dissecting the roles of superchaperones in type III secretion

描述（由申请人提供）：许多革兰氏阴性菌，包括许多NIAID生物防御优先病原体，利用III型分泌系统（t3ss）将数十种蛋白质输送到宿主细胞中。虽然这些复杂机器的许多组成部分是高度保守的，功能上是可互换的，但每个细菌都将自己独特的效应器传递到宿主细胞中。对于这种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传递到宿主细胞中，目前还不清楚。我的实验室最近开发了一种新的基于酵母的可视化实验来识别活细胞中的相互作用蛋白质，蛋白质相互作用平台实验或PIP。值得注意的是，我们发现一种伴侣蛋白Spa15与10种效应物相互作用，我们随后确定其中9种效应物需要Spa15才能有效分泌III型。此外，我们发现来自8种细菌的Spa15同源物在功能上与Spa15可互换。所有九种蛋白都识别一个确定的伴侣结合结构域序列。鉴于这8种伴侣蛋白在功能上的互换性，我们假设它们通过保守机制识别效应器并将效应器传递给分泌器官。该提案的目标是开始确定在III型分泌中这些重要步骤的分子机制。具体来说，我们建议(a)研究IB类伴侣蛋白如何区分效应蛋白和管家蛋白（目标1）和(b)确定伴侣蛋白/效应蛋白复合物如何被招募到III型分泌装置（目标2）。从这些研究中获得的见解可能会为开发治疗与革兰氏阴性细菌感染相关的各种疾病的新药提供新的方向。

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. PUBLIC HEALTH RELEVANCE: Many bacterial pathogens utilized a specialized secretion system to deliver virulence proteins directly into host cells. Our proposal is aimed at understanding the common strategies the bacteria use to recognize and deliver the secreted proteins to these specialized secretion systems. The results from our studies are likely to be valuable in the development of novel antimicrobials to treat common infections caused by these organisms.