A systems level analysis of spore coat assembly in Bacillus subtilis

描述(申请人提供)：几种致病芽胞形成细菌，包括炭疽芽孢杆菌的病原体，作为孢子被吸入或摄入，并在萌发后在宿主中恢复生长和毒素产生。包裹着孢子的孢子蛋白外壳是侵染循环中的一个重要因素，因为它具有有效的保护功能，并通过控制萌发菌对位于孢子内膜上的受体的访问而在萌发过程中发挥调节作用。孢子衣是一种形态复杂的结构，由大约60种不同的产孢蛋白组成，它们聚集在新生孢子周围。在这项提案中，我们将使用一种综合的系统生物学方法来绘制蛋白质相互作用网络图，并确定使孢子壳组装成核的关键调控中心-这可能作为孢子检测的标记和/或在对抗生物恐怖主义的应用中控制孢子萌发的目标。除了与公共卫生问题相关外，我们预计我们的项目将说明系统生物学方法在研究复杂结构组装方面的价值。我们建议使用高通量的蛋白质定位筛选来表征在模式生物枯草芽孢杆菌中控制孢子壳组装的蛋白质相互作用网络。具体地说，我们将使用一个与枯草杆菌所有外壳蛋白融合的荧光蛋白文库，来定义孢子周围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壳蛋白招募所必需的，并了解调控外壳组装的转录和翻译后机制。将使用大规模酵母双杂交试验和各种重点生物化学方法来证明在一对外壳蛋白之间建立的相互作用的本质。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，并获得孢子壳蛋白相互作用网络的全面表示，这将作为研究其他芽胞形成细菌，特别是炭疽杆菌的模板。与公共健康相关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得出新的假说和机制。我们预计，我们的结果将对检测和消除病原性芽胞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.