A Feeding Tube Model for Bacterial Cell-Cell Communication

细菌细胞间通讯的饲管模型

基本信息

批准号：
8355616
负责人：
Amy Hitchcock Camp
金额：
$ 213.87万
依托单位：
MOUNT HOLYOKE COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (Provided by the applicant) Abstract: Contrary to long-standing assumptions, bacteria communicate extensively to coordinate sophisticated ""multi- cellular"" behaviors such as antibiotic production, toxin secretion, motility, biofilm formation, symbiosis, and de- velopment. The best understood examples of bacterial communication occur at a distance via diffusible signaling molecules. Recent evidence indicates that bacteria can also be more intimately connected, in some cases directly exchanging cytosolic contents; however, the extent, consequence, and molecular underpinnings of these intimate modes of bacterial cell-cell communication remain poorly understood. Here I propose an in- novative and multi-disciplinary line of investigation to test a paradigm-shifting ""feeding tube"" model for bacterial cell-cell communication in which direct intercellular metabolite exchange drives developmental gene expres- sion during spore formation by the model bacterium Bacillus subtilis. More specifically, the central hypothesis of this proposal is that the developing spore (the forespore) is programmed to lose its metabolic self-sufficiency at intermediate stages of sporulation and that a novel channel apparatus acts as a portal (a ""feeding tube"") through which the adjacent mother cell replenishes the small molecule resources generally required for late forespore developmental gene expression. To test this hypothesis, we will first comprehensively characterize the metabolic status of the forespore in the presence and absence of the channel using novel in vivo biosen- sors and global metabolite profiling. A reverse genetic screen will then be undertaken to identify the genetic circuit responsible for forespore metabolic shutdown. Second, we will develop and implement novel cytological assays to demonstrate the ability of small molecules to transit from the mother cell to the forespore in a feeding tube dependent manner. Finally, we will determine the structure of the feeding tube channel and key protein components thereof by cryo-electron microscopy and X-ray crystallography. In all, the proposed research is expected to have a profound impact on our understanding of this remarkable example of bacterial communica- tion as well as similarly intimate modes of bacterial cell-cell communication that have only recently come to light. This knowledge will be significant because it will address fundamental questions regarding bacterial inter- connectedness in nature, including during horizontal gene transfer, pathogenesis, and biofilm formation. This work also has high potential to identify novel antimicrobial targets in a broad range of bacteria and especially gram-positive, spore-forming pathogens related to B. subtilis such as B. anthracis, Clostridium botulinum, and C. difficile. Finally, this research plan is innovative not only because it will elucidate the molecular underpin nings of a remarkably intimate and unexpected mode of bacterial cell-cell communication and developmental gene regulation, but also because it will implement a unique combination of traditional and cutting-edge tech- nologies to accomplish this goal. Public Health Relevance: Recent evidence indicates that bacteria can be highly interconnected in nature, in some cases directly exchanging cytosolic contents; however, the extent, consequence, and molecular underpinnings of these intimate modes of bacterial cell-cell communication, including during horizontal gene transfer, pathogenesis, and biofilm formation, remain poorly understood. Here I propose an innovative and multi-disciplinary line of investigation to characterize a remarkable mode of bacterial cell-cell communication in which direct intercellular metabolite exchange drives developmental gene expression in the model bacterium Bacillus subtilis. This study will transform our understanding of intimate bacterial connections and has high potential to identify novel antimicrobial targets in a broad range of bacteria including gram-positive, spore-forming pathogens related to B. subtilis such as B. anthracis and Clostridium difficile.

描述（申请人提供）摘要：与长期的假设相反，细菌广泛通信以协调复杂的“多细胞”行为，例如抗生素产生，毒素分泌，运动性，生物膜形成，共生和降低。最好理解的细菌通信实例通过可扩散的信号分子出现在距离处。最近的证据表明，在某些情况下，细菌也可以更紧密地联系在一起，直接交换胞质含量。但是，这些亲密模式的细菌细胞通信方式的程度，后果和分子基础知之甚少。在这里，我提出了一项重要的和多学科的研究线，以测试细菌细胞 - 细胞 - 细胞传输的范式转移“喂食管”模型，其中直接细胞间代谢物交换驱动模型细菌性细菌枯草菌在孢子形成过程中孢子形成过程中的发育基因表达。 More specifically, the central hypothesis of this proposal is that the developing spore (the forespore) is programmed to lose its metabolic self-sufficiency at intermediate stages of sporulation and that a novel channel apparatus acts as a portal (a ""feeding tube"") through which the adjacent mother cell replenishes the small molecule resources generally required for late forespore developmental gene expression.为了检验这一假设，我们将首先全面地表征前孔的代谢状态，在存在和不存在通道的情况下，使用新颖的体内生物体和全局代谢物分析。然后将进行反向遗传筛选，以确定负责前孔代谢关闭的遗传回路。其次，我们将开发和实施新型的细胞学测定，以证明小分子以依赖的方式从母细胞转移到前孔的能力。最后，我们将通过冷冻电子显微镜和X射线晶体学确定进食管通道和关键蛋白质成分的结构。总的来说，拟议的研究预计将对我们对这一非凡的细菌通信的例子以及类似的细菌细胞 - 细胞通信模式产生深远的影响，这些模式直到最近才揭示。这些知识将是重要的，因为它将解决有关自然界细菌相互联系的基本问题，包括在水平基因转移，发病机理和生物膜形成期间。这项工作还具有很高的潜力，可以在广泛的细菌中鉴定新的抗菌靶标，尤其是革兰氏阳性的，孢子形成的病原体，与枯草芽孢杆菌有关，例如炭疽芽孢杆菌，肉毒杆菌梭菌和艰难梭菌。最后，该研究计划具有创新性，这不仅是因为它将阐明细菌细胞 - 细胞传播和发育基因调节的显着亲密和意外模式的分子基础，还因为它将实现传统和削减技术的独特组合，以实现这一目标。公共卫生相关性：最近的证据表明，在自然界中，细菌可以高度互连，在某些情况下直接交换胞质含量；然而，细菌细胞 - 细胞通信的这些亲密模式的程度，后果和分子基础，包括在水平基因转移期间，发病机理和生物膜形成期间，仍然知之甚少。在这里，我提出了一种创新和多学科的研究线，以表征一种显着的细菌细胞 - 细胞通信方式，其中直接细胞间代谢物交换驱动了枯草杆菌模型中的发育基因表达。这项研究将改变我们对亲密细菌连接的理解，并具有很高的潜力，可以在广泛的细菌中鉴定新的抗菌靶标，包括与枯草芽孢杆菌相关的革兰氏阳性，形成的孢子病原体，例如炭疽芽孢杆菌和艰难梭菌。