Molecular mechanism regulating periplasmic proteolysis in bacterial pathogenesis

描述（由申请人提供）： 分子环状二聚GMP（c-di-GMP）已经作为细菌中广泛保守的第二信使出现，控制不同细菌物种中的粘附、运动性、生物膜形成和细胞形态发生，同时在转录、翻译和翻译后水平施加控制。我们对这种核苷酸在细菌中的作用的理解的一个关键的最新进展是鉴定出具有确定输出的c-di-GMP受体。我们的研究已经建立了一个模型，其中控制的粘附蛋白定位在细菌细胞表面是介导的c-di-GMP的细胞质水平。c-di-GMP水平通过LapD（一种内膜定位的c-di-GMP效应物）监测。LapD在c-di-GMP结合的开启状态和无核苷酸的关闭状态之间切换。细胞c-di-GMP的消耗导致c-di-GMP从LapD解离，其继而将信号传播穿过内膜并到达LapD的周质域。在高细胞溶质c-di-GMP水平下，LapD螯合周质蛋白酶LapG，防止其处理其底物，即细胞表面的大粘附蛋白LapA，并防止其从细胞表面释放，从而促进生物膜形成。拟议的研究建立在O'Toole和Sondermann实验室正在进行的合作研究的基础上，以探索LapD/LapG信号转导系统调节细菌表面蛋白定位的保守性和机制基础。在目的1中，我们将测试的假设，c-di-GMP结合到LapD的胞质结构域导致结构重排的这种效应在广泛的细菌，包括重要的病原体。在目标2中，我们将测试LapD介导的LapG控制依赖于保守的、直接的蛋白质-蛋白质相互作用的假设。这种相互作用是开发药物工具干扰生物膜形成或毒力的关键点。目的3将测试LapG识别LapA的N-末端结构域的离散特征的假设，所述特征允许该蛋白酶特异性靶向粘附素。这项工作建立的核心原则将适用于广泛的细菌细胞表面受体。此外，阐明通过LapDGA系统控制细菌细胞粘附的调节原理对于靶向潜在的分子相互作用和机制将是非常宝贵的。虽然这里描述的研究是基础性的，但它将对病原体及其相关疾病的感染生物学研究产生影响，如铜绿假单胞菌（囊性纤维化，医院获得性感染），霍乱弧菌（霍乱）和嗜肺军团菌（军团病）。最终，我们希望我们的努力将有助于抵消耐药细菌的出现和新型治疗方法的下降之间日益扩大的差距。 公共卫生相关性： 细菌第二信使c-di-GMP在控制被称为生物膜的细菌群落的形成中起关键作用。这些生物膜在临床环境中具有深远的负面影响，导致慢性感染，延长住院时间并提高医疗成本。我们已经确定了一种监管系统，该系统控制在广泛的微生物中制造生物膜所需的关键粘附功能。在这个建议中，我们将研究关键分子， 这一信号系统的机制，这可能有助于新的抗生物膜和抗感染疗法。

DESCRIPTION (provided by applicant): The molecule cyclic dimeric GMP (c-di-GMP) has emerged as a broadly conserved second messenger in bacteria, controlling adhesion, motility, biofilm formation and cell morphogenesis in diverse bacterial species, while exerting control at transcriptional, translational and post-translational levels. A key, recent advance in our understanding of this nucleotide's role in bacteria has been the identification of c-di-GMP receptors with defined outputs. Our studies have established a model wherein control of adhesion protein localization on the bacterial cell surface is mediated by cytoplasmic levels of c-di-GMP. c-di-GMP levels are monitored by LapD, an inner membrane-localized c-di-GMP effector. LapD switches between a c-di- GMP-bound on-state and a nucleotide-free off-state. Depletion of cellular c-di-GMP results in the dissociatio of c-di-GMP from LapD, which in turn propagates a signal across the inner membrane and to the periplasmic domain of LapD. At high cytosolic c-di-GMP levels, LapD sequesters the periplasmic protease LapG, preventing it from processing its substrate, a large adhesion protein LapA at the cell surface, and from releasing it from the cell surface, and thus promoting biofilm formation. The proposed research builds upon ongoing collaborative studies in the O'Toole and Sondermann labs to explore the conservation and mechanistic basis whereby the LapD/LapG signal transduction system regulates the localization of bacterial surface proteins. In Aim 1, we will test the hypothesis that c-di-GMP binding to the cytoplasmic domain of LapD causes structural rearrangements of this effector in a wide range of bacteria including important pathogens. In Aim 2, we will test the hypothesis that LapD-mediated control of LapG is dependent on a conserved, direct protein-protein interaction. This interaction is a pivotal point for the development for pharmacological tools to interfere with biofilm formation or virulence. Aim 3 will test the hypothesis that LapG recognizes discrete features of the N-terminal domain of LapA that allows this protease to specifically target the adhesin. Core principles established by this work will be applicable to a wide range of bacterial cell surface receptors. In addition, elucidating the regulatory principles that control bacterial cell adhesion via the LapDGA system will be invaluable for targeting the underlying molecular interactions and mechanisms pharmacologically. While the research described here is basic in nature, it will have ramifications for infection biology research pertaining to pathogens and their associated diseases such as Pseudomonas aeruginosa (cystic fibrosis, hospital-acquired infections), Vibrio cholerae (cholera) and Legionella pneumophila (Legionnaires' disease). Ultimately, we hope that our efforts will help to counteract the increasing disparity between the emergence of drug-resistant bacteria and the decline in novel therapeutics. PUBLIC HEALTH RELEVANCE: The bacterial second messenger c-di-GMP plays a critical role in the control of the formation of bacterial communities known as biofilms. These biofilms have a profound negative impact in clinical settings, causing chronic infections that extend hospital stays and raise healthcare costs We have identified a regulatory system that controls the function of a key adhesion required to make biofilms in a wide range of microbes. In this proposal, we will investigate critical molecular mechanisms of this signaling system, which may contribute to new anti-biofilm and anti-infective therapies.