Key Molecular Mechanisms of c-di-GMP Signaling in Bacterial Biofilm Formation

细菌生物膜在不同的表面上定植，对抗生素或免疫系统具有高度抗性。在这些生物膜中，细胞被嵌入由分泌蛋白、淀粉样纤维（如curli）、外多糖（如纤维素）甚至DNA组成的自产基质中。这些基质成分决定了这些生物膜复杂的微结构和形态。在大肠杆菌中，卷曲纤维和纤维素的合成受固定相sigma因子RpoS和第二信使c-di-GMP的控制。后者由二胍酸环化酶（DGC，具有GGDEF结构域）产生，并被特定的磷酸二酯酶（PDE，具有EAL结构域）降解。许多这些差异表达的DGCs和pde是膜相关的，并通过n端感觉结构域控制活性。许多细菌拥有多个GGDEF/EAL结构域蛋白（大肠杆菌K-12中有29个），这导致了基于直接和高度特异性蛋白-蛋白相互作用的局部作用c-di-GMP控制模块的概念。我们最近对YciR（细菌第二信使信号的第一和典型触发酶，也是卷曲蛋白和纤维素生产的关键开关装置）的分析已经证明了这是如何工作的：YciR同时是(i)通过直接相互作用抑制两种靶蛋白的调节剂，（ii） PDE, (iii) c-di-GMP效应组分，因为其与c-di-GMP的结合和降解释放其与靶蛋白（DGC和产生curli和纤维素所需的转录因子）的直接相互作用。这里提出的项目将重点关注c-di-GMP信号传导的两个关键方面的分子机制：1。涉及c-di-GMP相关触发酶的特定蛋白-蛋白相互作用的局部c-di-GMP信号传导：在这里，YciR也与另外三种DGCs一起作用的潜在作用，以及另外两种pde和推定的触发酶的分子功能，分别参与纤维素生物合成和基因表达的直接调节，将被表征。c-di-GMP信号的感觉输入：在这里，目标将是识别进入生物膜形成的新的环境输入信号，并将特定的信号感知分配给不同的DGCs和pde。DGCs和PDEs的特定n端感觉域信号处理的分子机制将被表征，特别关注氧化还原响应的PDEs和多域DGCs和PDEs的多信号集成。最后，将阐明这些分子机制在产生生物膜功能的大型调控网络中的整合。由于c-di-GMP信号几乎无处不在地被细菌用于控制生物膜的形成，本项目产生的见解将为抗生物膜策略和药物开辟新的视角。

Bacterial biofilms colonize diverse surfaces and are highly resistant against antibiotics or the immune system. In these biofilms, cells are embedded in a self-produced matrix consisting of secreted proteins, amyloid fibres (e.g. curli), exopolysaccharides (e.g. cellulose) and even DNA. These matrix components determine the complex microarchitecture and morphology of these biofilms. In E. coli, synthesis of curli fibres and cellulose is under control of the stationary phase sigma factor RpoS and the second messenger c-di-GMP. The latter is produced by diguanylate cyclases (DGC, with GGDEF domains) and is degraded by specific phosphodiesterases (PDE, with EAL domains). Many of these differentially expressed DGCs and PDEs are membrane-associated and activity-controlled via N-terminal sensory domains. Many bacteria possess multiple GGDEF/EAL domain proteins (29 in E. coli K-12), which has led to the concept of locally acting c-di-GMP control modules based on direct and highly specific protein-protein interactions. Our recent analysis of YciR, the first and paradigmatic trigger enzyme in bacterial second messenger signaling and a key switching device for the production of curli and cellulose, has demonstrated how this can work: YciR is at the same time (i) a regulator that inhibits two target proteins by direct interaction, (ii) a PDE, and (iii) a c-di-GMP effector component, because its binding and degradation of c-di-GMP releases its direct interaction with the target proteins (a DGC and a transcription factor required to produce curli and cellulose).The project proposed here will focus on the molecular mechanisms that underly two key aspects of c-di-GMP signaling:1. Local c-di-GMP signaling that involves specific protein-protein interactions of c-di-GMP-related trigger enzymes: here, the potential role of YciR acting also in conjunction with three additional DGCs, as well as the molecular functions of two additional PDEs and putative trigger enzymes that are involved in cellulose biosynthesis and direct regulation of gene expression, respectively, will be characterized.2. Sensory input into c-di-GMP signaling: here, the goal will be to identify novel environmental input signals into biofilm formation and to assign specific signal perception to distinct DGCs and PDEs. The molecular mechanisms of signal processing by specific N-terminal sensory domains of DGCs and PDEs will be characterized with a particular focus on redox-responsive PDEs and multiple signal integration by multi-domain DGCs and PDEs.Finally, the integration of these molecular mechanisms within the large regulatory networks that generate functionality of a biofilm will be elucidated. Since c-di-GMP signaling is almost ubiquitously used by bacteria to control biofilm formation, insights generated by this project will open new perspectives on anti-biofilm strategies and drugs.