CAREER: The Molecular Basis of Persister Cell and Biofilm Formation by the E. Coli Protein MqsR

知识价值：对生物膜的研究已经进行了很多年，生物膜是每个人都熟悉的细菌群落，因为它们在晚上覆盖在我们的牙齿上。然而，即使在今天，唯一确定的去除生物膜的方法是通过机械力，即牙刷。虽然刷牙是例行公事，但从船舶、管道和医疗设备以及其他表面去除生物膜要困难得多，也要昂贵得多。生物膜的形成是细菌利用的主要防御和生存机制之一。然而，对生物膜如何在分子水平上组装和调节的详细了解只是初步了解。细菌持久性的形成是生物膜形成的核心，细菌持久性是一种基因相同的代谢静止细胞亚群，它们表达蛋白质毒素并表现出多药耐受性。然而，持久性也是细菌在环境压力下生存的最不为人所知的机制之一。最近，大肠杆菌蛋白MqsR （B3022, YgiU）被确定为一个关键的持久性因子，因为它是持久性基因中表达最高的基因。由于其序列与任何特征蛋白的序列都不相似，因此其分子功能，即如何在分子水平上进行调节，特别是如何驱动持久性表型的形成，目前尚不清楚。因此，这个CAREER项目的长期目标是阐明导致持久性状态的分子机制，重点是了解导致持久性形成的蛋白质活性。此外，对于这些活性在正常和应激条件下是如何被调节的，以及它们如何被阻断以开发抑制持久性细胞表型形成的新药物，将会有一个全面的了解。具体来说，将回答以下问题：1)MqsR毒素的三维结构是什么，因此功能是什么，以及它如何通过与MqsA （B3021, YgiT）的相互作用减轻毒性？2) MqsA和MqsR:MqsA复合物如何差异调控大肠杆菌基因转录？3) MqsR毒性如何导致生物膜和持久性细胞的形成？综上所述，这些研究将定义MqsR的分子机制，并为MqsR如何控制细菌持久性和生物膜形成提供重要的新见解。更广泛的影响：生物膜是一种复杂的细菌群落，对抗菌素具有高度耐药性，每年给世界经济造成数十亿美元的损失。对在持久性中起关键作用的蛋白质（如MqsR）的功能和调控的分子理解，将为开发针对生物膜的新化学制剂提供所需的新靶标。在这个CAREER项目中，将整合包括结构生物学、生物化学和遗传学在内的多个研究学科，为本科生和研究生提供揭示科学研究跨学科性质的项目。该教育计划的基石是一个多组分蛋白质科学研讨会（PSW），它将为罗德岛社区学院（CCRI）的学生提供扩展他们研究经验的机会。它由布朗大学和RI-EPSCoR设施的讲座和实验室组成，将使CCRI的学生接触到最先进的研究和技术。此外，每年将邀请一名CCRI学生进行自己的独立研究项目。与CCRI合作的长期目标是，从经常被遗忘的成年学生中吸引和培养新一代科学家，这些学生重返校园，以获得知识和技能，使他们能够接受高等教育和/或进入生物技术行业。这是罗德岛州首个此类合作。

Intellectual Merit:Research on biofilms, bacterial communities familiar to everyone as they coat our teeth at night, has been ongoing for many years. However, even today, the only certain way to remove biofilms is by mechanical force, i.e. a toothbrush. While brushing our teeth is routine, removal of biofilms from ships, pipes and medical devices, and other surfaces, is much more difficult and expensive. The formation of biofilms is one of the major defense and survival mechanisms utilized by bacteria. However, a detailed understanding of how biofilms assemble and are regulated at a molecular level is only rudimentarily understood. The formation of bacterial persisters, a genetically identical sub-population of metabolically quiescent cells that express protein toxins and exhibit multidrug tolerance, is at the core of biofilm formation. However, persistence is also one of the most poorly understood mechanisms used by bacteria to survive environmental stress. Recently, the Escherichia coli protein MqsR (B3022, YgiU) was identified as a key persistence factor, as it is the most highly upregulated gene in persisters. Because its sequence does not resemble that of any characterized protein, its molecular function, i.e. how it is regulated at a molecular level and especially how it drives the formation of the persister phenotype, is currently unknown. Accordingly, the long-term objective of this CAREER project is to elucidate the molecular mechanisms that lead to the persister state, with a focus on understanding the protein activities that lead to persister formation. In addition, a thorough understanding of how these activities are regulated under normal and stressful conditions and how they can be blocked for the development of novel agents that inhibit the formation of the persister cell phenotype will be determined. Specifically, the following questions will be answered: 1) What is the 3-dimensional structure, and thus the function, of the MqsR toxin and how is its toxicity mitigated by its interaction with MqsA (B3021, YgiT)? 2) How do MqsA and the MqsR:MqsA complex differentially regulate E. coli gene transcription? and 3) How does MqsR toxicity lead to biofilm and persister cell formation? Taken together, these studies will define the molecular mechanism of MqsR and provide essential new insights into how MqsR controls bacterial persistence and biofilm formation.Broader impacts:Biofilms, complex communities of bacteria that are highly resistant to antimicrobials and cost the world economy billions of dollars every year, are extraordinarily enriched in persister cells. A molecular understanding of the function and regulation of the proteins that play a key role in persistence, like MqsR, will provide novel targets needed for development of new chemical agents that target biofilms. In this CAREER project, multiple research disciplines, including structural biology, biochemistry, and genetics, will be integrated to provide projects for both undergraduate and graduate students that reveal the interdisciplinary nature of scientific research. The cornerstone of the educational program is a multi-component Protein Science Workshop (PSW) that will provide Community College of Rhode Island (CCRI) students with the opportunity to expand their research experiences. It is composed of lectures and laboratories at Brown University and RI-EPSCoR facilities and will expose the CCRI students to state-of-the-art research and technologies. In addition, each year, one CCRI student will be invited to carry-out their own independent research project. The long-term goal of the collaboration with CCRI, the first of its kind in Rhode Island, is to attract and develop a new generation of scientists from the often forgotten pool of adult students who are returning to school in order to obtain the knowledge and skills that will allow them to pursue higher education and/or entry into the biotechnology workforce.