Dissecting the roles of superchaperones in type III secretion

描述(由申请人提供)：许多革兰氏阴性细菌，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。虽然这些复杂机器的许多组件高度保守，功能可互换，但每种细菌都向宿主细胞提供了一组独特的效应器。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输送到宿主细胞，目前尚不清楚。我的实验室最近开发了一种新的基于酵母的可视化分析方法，用于识别活细胞中相互作用的蛋白质，即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Spa15与10个效应器相互作用，其中9个效应器需要Spa15才能有效地分泌III型细胞。此外，我们发现来自8个细菌物种的Spa15同源物在功能上可以与Spa15互换。所有九种蛋白质都识别一个已定义的伴侣结合域序列。鉴于这八种伴侣蛋白的功能可互换，我们假设它们识别效应器，并通过保守的机制将效应器输送到分泌器。该提案的目标是开始确定III型分泌中这些重要步骤背后的分子机制。具体地说，我们建议(A)研究IB类伴侣如何区分效应器和管家蛋白(目标1)，以及(B)确定伴侣/效应器复合体如何招募到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物提供新的方向。

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.