A systems level analysis of spore coat assembly in Bacillus subtilis

描述（由申请方提供）：几种致病性孢子形成细菌，包括炭疽芽孢杆菌的病原体，以孢子形式吸入或摄入，并在萌发后在宿主体内恢复生长和毒素产生。包裹孢子的孢子蛋白质外壳是感染循环中的重要因素，因为其有效的保护功能和其在萌发过程中通过控制萌发剂进入位于孢子内膜中的受体而起的调节作用。孢子衣是一种形态复杂的结构，由大约60种不同的孢子形成蛋白组成，它们聚集在新生孢子周围。在这项提案中，我们将使用一个综合的系统生物学方法来映射蛋白质相互作用网络，并确定关键的调控中心，成核孢子外壳组件-这可能作为孢子检测的标记和/或作为控制孢子萌发的目标，在生物恐怖主义的应用。除了与公共卫生问题的相关性外，我们设想我们的项目将说明系统生物学方法在调查复杂结构组装方面的价值。我们建议使用高通量的蛋白定位屏幕的特点蛋白质相互作用网络，管理孢子外壳组装在模式生物枯草芽孢杆菌。具体地说，我们将使用荧光蛋白融合物库与B中的所有外壳蛋白融合。枯草芽孢杆菌，以定义孢子周围的孢子外壳蛋白沉积的时空层次，以确定哪些形态发生蛋白是所有孢子外壳蛋白的募集所需的，并了解外壳组装调控的转录和翻译后机制。将使用大规模酵母双杂交测定和各种集中的生物化学方法来证明外壳蛋白对之间建立的相互作用的性质。最后，我们将使用计算工具来整合从这些实验中收集的数据，并获得孢子外壳蛋白相互作用网络的全面表示，这将作为其他孢子形成细菌，特别是B的研究模板。炭疽病 我们的研究将为芽孢杆菌孢子的抗性特性和控制孢子萌发的机制提出新的假设和机制。我们预计，我们的研究结果将对检测和消除致病性孢子形成细菌的治疗方法的设计产生影响。

DESCRIPTION (provided by applicant): Several pathogenic spore-forming bacteria, including the causative agent of anthrax Bacillus anthracis, are inhaled or ingested as spores and resume growth and toxin production in the host after germination. The spore protein coat, which encapsulates the spore, is an important factor in the infectious cycle, because of its effective protective function and its regulatory role in the germination process by controlling access of germinants to receptors located in the inner membrane of the spore. The spore coat is a morphologically complex structure composed of approximately 60 different sporulation proteins that assemble around the nascent spore. In this proposal, we will use an integrated systems biology approach to map protein interaction networks and identify key regulatory hubs that nucleate spore coat assembly- which may serve as markers for spore detection and/or as targets for control of spore germination in applications against bioterrorism. In addition to its relevance to public health issues, we envision that our project will illustrate the value of systems biology approaches for investigating the assembly of complex structures. We propose to use high throughput protein localization screens to characterize the protein interaction networks that govern spore coat assembly in the model organism Bacillus subtilis. Specifically, we will use a library of fluorescent protein fusions to all of the coat proteins in B. subtilis, to define the spatiotemporal hierarchy of deposition of spore coat proteins around the spore, to identify which morphogenetic proteins are required for the recruitment of all spore coat proteins and learn the transcriptional and post-translational mechanisms underlying the regulation of coat assembly. The nature of the interactions established between pairs of coat proteins will be demonstrated using a large scale yeast two hybrid assay and various focused biochemical approaches. Finally, we will use computational tools to integrate the data gathered from these experiments and obtain a comprehensive representation of the spore coat protein interaction network that will serve as a template for the study of other spore-forming bacteria, particularly B. anthracis. PUBLIC HEALTH RELEVANCE Our research will derive new hypotheses and mechanisms for the resistance properties of Bacillus spores and the mechanisms that control spore germination. We anticipate that our results will have an impact on the design of therapeutic approaches to detect and eliminate pathogenic spore-forming bacteria.