NMR Studies of Bacterial Regulatory Proteins

细菌调节蛋白的核磁共振研究

基本信息

批准号：
7728150
负责人：
John Cavanagh
金额：
$ 28.92万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7728150
关键词：
Adjuvant Therapy Antibiotics Area Bacteria Binding Cell Death Cells Comparative Study Complex Consensus Consensus Sequence DNA DNA Sequence Decision Making Development Docking Effectiveness Ensure Environment Exposure to Funding Gastroenteritis Gel Genes Goals Health Hostility Human Infection Length Marines Mass Spectrum Analysis Microbial Biofilms Mind Modeling Multi-Drug Resistance Mutagenesis Pathway interactions Pattern Performance Porifera Process Production Promoter Regions Proteins Resistance Sensory Septicemia Shapes Signal Transduction Signaling Protein Solutions Stress Structure Surface Plasmon Resonance System Techniques Testing Therapeutic Time Toxicology Vibrio Virulence Work Wound Infection analog antimicrobial bacterial resistance base biological adaptation to stress chemical synthesis combat genetic regulatory protein methicillin resistant Staphylococcus aureus novel pathogenic bacteria public health relevance research study response small molecule small molecule libraries success therapeutic target trait

项目摘要

DESCRIPTION (provided by applicant): Bacteria occupy countless niches. They respond to innumerable environmental situations to ensure their survival and to allow them to become pathogenic. In times of change/hostility bacteria encode genes normally unexpressed. Certainly, there is not a single type of response - each situation requires a specific strategy. Bacteria sense signals in the environment, recognize its composition and initiate the correct survival approach. With this in mind, and using a variety of techniques including NMR, mass spectrometry, mutagenesis and chemical synthesis, our work focuses in three areas: (i) transition state regulator proteins (TSRs); (ii) two-component signaling proteins; and (iii) small molecules that overcome the bacterial stress response, including biofilm formation. The transition state is a "cellular holding pattern" during which bacteria decide which protective strategy is most appropriate. Incorrect signaling during this time results in cell death. TSRs control numerous pathways by their ability to bind to a diverse array of gene promoter regions which have no consensus DNA sequence. The mechanism by which they target their DNA targets is novel and very different than the classical "consensus sequence recognition" model. We will perform detailed comparative studies on three structurally homologous TSRs (AbrB, Abh and SpoVT) to better understand how DNA targets are recognized by this new class of proteins. We will also study the newly discovered protein AbbA which may influence the transition state by a completely novel mechanism. When the cell decides which response is necessary in order to survive, ubiquitous signaling modules known as the two-component system/phosphorelay are called upon. These modules are responsible for the majority of all sensory-response functions in bacteria. Understanding their mechanism of action is essential since they are significant targets for antimicrobial therapeutics. We will provide the very first characterization of a two-domain response regulator in solution (Spo0A) and elucidate the structure, dynamics, interactions and recognition determinants of multiple proteins from a complex phosphorelay that controls biofilm development in all human pathogenic Vibrio species. Finally, we have recently discovered a class of synthetically accessible, non-toxic small molecules that act as anti-biofilm agents with unprecedented effectiveness. We have recently discovered that these compounds also completely re-sensitize bacteria to conventional antibiotics and have demonstrated their effectiveness against multi-drug resistant bacterial strains including MRSA. We will develop further analogues of our compounds and will detail their effects, as adjuvant therapies, on the performance of many current antibiotics against multiple bacterial strains. We will also develop new toxicology screens and initiate differential array work to begin determining the mode of action of our compounds. Our overall goals are: (a) to better understand how bacteria are able to adapt and reach their pathogenic potential and (b) develop approaches to stop pathogenic bacteria becoming infectious. PUBLIC HEALTH RELEVANCE: Bacteria survive and reach their pathogenic potential towards humans by their ability to invoke the correct protective strategies at the appropriate times. In order to better define anti-microbial therapeutic targets, we will elucidate the mechanism of action for protein signaling cascades involved in the transition state and in biofilm formation. In addition we will further develop powerful new, non-toxic, small molecules that have been shown to both eradicate insidious pathogenic biofilms and to re-sensitize resistant bacteria to conventional antibiotics.

描述（由申请人提供）：细菌占据了无数的壁ni。他们应对无数环境情况，以确保其生存并允许它们成为致病性。在变化/敌对的时期，细菌编码通常未表达的基因。当然，没有一种类型的响应 - 每种情况都需要特定的策略。细菌在环境中的感知信号，识别其组成并启动正确的生存方法。考虑到这一点，并使用多种技术，包括NMR，质谱，诱变和化学合成，我们的工作集中在三个领域：（i）过渡状态调节蛋白（TSRS）；（ii）两个组分信号蛋白；（iii）克服细菌应激反应的小分子，包括生物膜形成。过渡状态是一种“细胞保持模式”，在此过程中，细菌决定哪种保护策略最合适。在此期间，信号不正确导致细胞死亡。 TSR通过与没有共识DNA序列的各种基因启动子区域结合的能力来控制众多途径。他们靶向其DNA靶标的机制是新颖的，与经典的“共识序列识别”模型非常不同。我们将对三种新的蛋白质识别DNA靶标的三种结构同源TSR（ABRB，ABH和SPOVT）进行详细的比较研究。我们还将研究新发现的蛋白质ABBA，该蛋白可能会通过一种完全新颖的机制影响过渡状态。当细胞确定为了生存的必要响应时，请调用称为两组分组/磷层的普遍信号传导模块。这些模块负责细菌中所有感觉响应函数的大多数。了解它们的作用机理至关重要，因为它们是抗菌治疗剂的重要靶标。我们将提供溶液中两域反应调节剂的首次表征（SPO0A），并阐明从复杂的磷酸层中多种蛋白质的结构，动力学，相互作用和识别决定因素，从而控制了所有人类致病性颤动物种中生物膜发育。最后，我们最近发现了一类可访问的，无毒的小分子，它们充当了具有前所未有的有效性的抗生物胶片药。我们最近发现，这些化合物还完全将细菌重新敏感为常规抗生素，并证明了它们针对包括MRSA在内的多药耐药细菌菌株的有效性。我们将开发出我们化合物的进一步类似物，并将其详细介绍其对辅助疗法对许多当前抗生素对多种细菌菌株的性能的作用。我们还将开发新的毒理学筛选，并启动差分阵列工作，以开始确定化合物的作用方式。我们的总体目标是：（a）更好地了解细菌如何能够适应并发挥其致病潜力，并且（b）开发方法以阻止致病细菌变得感染。公共卫生相关性：细菌在适当的时候援引正确的保护策略的能力生存并发挥其对人类的致病潜力。为了更好地定义抗微生物治疗靶标，我们将阐明参与过渡态和生物膜形成的蛋白质信号传导级联反应机理。此外，我们还将进一步开发出强大的新型，无毒的小分子，这些分子既可以消除阴险的致病生物膜，又可以将抗性细菌重新敏感到常规的抗生素。