Evolution of bacterial communication systems

项目摘要/摘要 群体感应(Quorum Sensing，QS)是细菌通讯的一种重要形式，用于协调基因表达和 群体行为以依赖于细胞密度的方式进行。许多细菌使用一种QS形式，在这种形式中信号合成酶 产生一种膜可穿透的小分子，成对的胞质受体对其产生反应。数以百计 具有不同性质的QS系统已被鉴定，其中许多对致病力很重要 病原菌。这种多样性是如何演变的，这是一个对该领域至关重要的问题。在我早年 来自铜绿假单胞菌的模型QS系统LASI-LASR的博士后工作，我观察到 受体LasR尚未进化到最高信号灵敏度，而LasI和LasR的变体往往 没有野生型那么挑剔。此外，QS系统通常受到严格监管，以确保活动仅在 比细胞密度。基于这些观察，我假设QS系统进化为平衡信号 敏感度、选择性和适合性。这项建议使用LASI-LASR模型系统来检验这一假设。目标1 将研究使用高灵敏度和低灵敏度LasR变体的信号灵敏度的成本和优势 之前确认过的。我将测量这些突变体中QS活动的时间和水平，以及 突变体对信号的干扰，并最终决定突变体在单一培养和竞争中的适应性 野生型铜绿假单胞菌。AIM 2将评估铜绿假单胞菌对LASI-LasR信号的压力反应 选择性。我将使用我最近发现的非选择性LASI和LasR变体作为体外试验的起点 新信号选择性的演变。这一目标将阐明QS选择性的其他决定因素，并将 为未来研究QS信号选择性演变的轨迹生成工具。把这些目标放在一起 将加深我们对细菌通信系统进化的认识，有望为 未来对这些系统在多菌群落中的作用的研究，将带来新的途径 了解和治疗感染。这项研究提案将成为我申请 独立教员的职位和我所获得的成绩有望帮助我成功地竞争未来 资金问题。这些实验将在奖励的K99阶段启动，并将包括 细菌共培养、体外进化和生物统计学方法。此外，这份提案还包括职业生涯 发展和培训计划，以补充我以前的经验。我已经收集了一份多学科的建议 帮助我实现目标的团队，我的导师格林伯格博士是细菌方面的先驱和专家 与长期成功的导师关系进行沟通。由该组织提供的培训和支持 获奖将使我能够实现我的长期目标，即建立一个专注于 关于复杂环境中的细菌信号和生理学。

PROJECT SUMMARY/ ABSTRACT Quorum sensing (QS) is an important form of bacterial communication used to coordinate gene expression and group behaviors in a cell density-dependent manner. Many bacteria use a form of QS in which a signal synthase produces a membrane-permeable small molecule to which a paired cytosolic receptor responds. Hundreds of QS systems with diverse properties have been identified, many of which are important for the virulence of pathogenic bacteria. How this diversity evolved is a question of fundamental importance to the field. In my early postdoctoral work on the model QS system LasI-LasR from Pseudomonas aeruginosa, I observed that the receptor, LasR, has not evolved to maximal signal sensitivity and that variants of both LasI and LasR tend to be less selective than wildtype. Further, QS systems are often tightly regulated to ensure activity only occurs at a specific cell density. Based on these observations, I hypothesize that QS systems evolve to balance signal sensitivity, selectivity, and fitness. This proposal tests that hypothesis using the LasI-LasR model system. Aim 1 will investigate the costs and benefits of signal sensitivity using hyper- and hypo-sensitive LasR variants I identified previously. I will measure the timing and level of QS activity in these mutants, the susceptibility of the mutants to signal interference, and ultimately the fitness of the mutants in monoculture and in competition with wildtype P. aeruginosa. Aim 2 will evaluate how P. aeruginosa responds to pressure on LasI-LasR signal selectivity. I will use my recently identified non-selective LasI and LasR variants as a starting point for the in vitro evolution of new signal selectivity. This aim will illuminate additional determinants of QS selectivity and will generate tools for future research on the trajectory by which QS signal selectivity evolves. Together these aims will deepen our understanding of the evolution of bacterial communication systems, are expected to facilitate future research into the roles of these systems in polymicrobial communities, and will lead to new avenues for understanding and treating infections. This research proposal will form the foundation of my applications for an independent faculty position and the results I obtain are expected to help me successfully compete for future funding. These experiments will be initiated during the K99 phase of the award and will include training in bacterial co-culture, in vitro evolution, and biostatistical methods. Additionally, this proposal includes a career development and training plan to complement my prior experience. I have assembled a multidisciplinary advisory team to help me achieve my goals and my mentor, Dr. Greenberg, is a pioneer and expert in bacterial communication with a long track record of successful mentorship. The training and support provided by this award will enable me to achieve my long-term goal of establishing an independent research program focused on bacterial signaling and physiology in complex environments.