A systems level analysis of spore coat assembly in Bacillus subtilis

描述（由申请人提供）：几种致病性芽孢形成细菌，包括炭疽杆菌的病原体，以孢子的形式被吸入或摄入，并在发芽后在宿主体内恢复生长和毒素产生。包裹孢子的孢子蛋白外壳是感染周期中的重要因素，因为它具有有效的保护功能，并且通过控制萌芽体接触位于孢子内膜的受体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 种不同的孢子形成蛋白组成，这些蛋白聚集在新生孢子周围。在本提案中，我们将使用综合系统生物学方法来绘制蛋白质相互作用网络，并确定使孢子外壳组装成核的关键调控中心——这可以作为孢子检测的标记和/或作为在反恐应用中控制孢子萌发的目标。除了与公共卫生问题相关之外，我们预计我们的项目还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蛋白质定位屏幕来表征模型生物枯草芽孢杆菌中控制孢子衣组装的蛋白质相互作用网络。具体来说，我们将使用枯草芽孢杆菌中所有外壳蛋白的荧光蛋白融合文库，来定义孢子周围孢子外壳蛋白沉积的时空层次结构，以确定招募所有孢子外壳蛋白所需的形态发生蛋白，并了解外壳组装调节背后的转录和翻译后机制。外壳蛋白对之间建立的相互作用的性质将使用大规模酵母两种杂交测定和各种重点生化方法来证明。最后，我们将使用计算工具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，该网络将作为研究其他孢子形成细菌，特别是炭疽芽孢杆菌的模板。 公共卫生相关性我们的研究将为芽孢杆菌孢子的抗性特性以及控制孢子萌发的机制得出新的假设和机制。我们预计我们的结果将对检测和消除致病性芽孢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.