Molecular mechanism regulating periplasmic proteolysis in bacterial pathogenesis

描述(由申请人提供)： 环状二聚体GMP(c-di-GMP)是细菌中广泛保守的第二信使，在转录、翻译和翻译后水平上控制着细菌的黏附、运动、生物膜的形成和细胞形态的形成。在我们理解这种核苷酸在细菌中的作用方面，一个关键的，最近的进展是识别具有特定输出的c-di-GMP受体。我们的研究已经建立了一个模型，其中控制黏附蛋白在细菌细胞表面的定位是由细胞质水平的c-di-GMP介导的。C-di-GMP水平由内膜定位的c-di-GMP效应器Lapd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。细胞内c-di-GMP的耗尽导致c-di-GMP从Lapd解离，进而将信号传递到Lapd的内膜和周质结构域。在细胞内高水平的c-di-GMP时，LAPD隔离周质蛋白LapG，阻止其处理其底物，即细胞表面的大黏附蛋白LapA，并将其从细胞表面释放，从而促进生物膜的形成。这项拟议的研究建立在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和机制基础。在目标1中，我们将检验这样的假设，即c-di-GMP与Lapd的细胞质结构域结合导致该效应器在包括重要病原体在内的广泛细菌中的结构重排。在目标2中，我们将检验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理工具干扰生物膜形成或毒力的关键。目标3将测试假设，即LapG识别LapA N-末端结构域的离散特征，使该蛋白酶能够特异性地靶向粘附素。这项工作确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系统控制细菌细胞黏附的调节原理对于从药理学上针对潜在的分子相互作用和机制将是非常有价值的。虽然这里描述的研究是基础性的，但它将对与病原体及其相关疾病有关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抗药性细菌的出现和新疗法的衰落之间日益扩大的差距。

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.