Linking c-di-GMP signalling and the Gac/Rsm signal transduction pathway

铜绿假单胞菌是一种革兰氏阴性细菌，在包括土壤、潮湿表面或植物在内的多种环境中茁壮成长。它也是一种机会性的人类病原体，导致与高发病率和高死亡率相关的感染。流行病学研究表明，临床或环境分离株的毒力潜力没有显著差异，这表明现有的毒力因子武器库也可能有助于对抗环境中可能遇到的真核捕食者(如原生动物和后生动物)。因此，铜绿假单胞菌是一种多才多艺的有机体，它对波动的环境线索的适应能力依赖于众多而复杂的信号通路。这些途径决定了铜绿假单胞菌是采取移动/有毒还是固着/生物膜的生活方式。这些生活方式与急性或慢性感染模式直接相关。在生活方式中做出选择的决策过程受到主要调控途径的支配，这些途径是对抗性的，但也通过正负反馈环路相互作用。这些涉及群体感应，或第二信使分子c-AMP，这两个分子都是促进细菌毒力因子(如毒素)产生的主要调节因素。相比之下，所谓的Gac/RSM途径，涉及探测环境条件的分子传感器，以及第二信使分子c-di-GMP是控制生物膜形成的主要参与者。生物膜是附着在表面的细菌种群，并由多糖、DNA或黏附蛋白组成的粘性细胞外基质结合在一起。嵌入在这种基质中的细菌形成了一个非常稳定的种群，在环境中可以抵抗各种压力，例如附着在岩石上的河流水流、紫外线照射、脱水或营养匮乏。对于病原体，一旦细菌在一个特定的组织或器官中建立起生物膜，例如在囊性纤维化患者的肺部，就基本上不可能根除。该人群对免疫系统的攻击或抗生素疗法具有高度抵抗力。在我们的项目中，我们将研究铜绿假单胞菌生物膜的形成/扩散与Gac/RSM和c-di-GMP两条调控通路之间可能存在的密切联系。我们希望在Gac/RSM通路的特性以及我们最近证明这条通路与c-di-GMP信号通路相交的基础上，建立我们在Gac/RSM通路表征方面的专业和开创性工作。我们想在这两个主要角色之间建立分子联系，并了解它如何作用于生物膜的形成，以及除此之外，它如何对毒素等毒力因子的产生产生拮抗作用。事实上，这些调控途径处于医生Jekyll和先生Hyde的游戏的交叉点，在游戏中，细菌犹豫不决，要么转向侵略性和有毒的生活方式，要么转向更安静和隐蔽的生物膜形式的生活方式。总而言之，我们将调查的调控网络并不是人类病原体所特有的，而是对c-di-GMP的大多数革兰氏阴性细菌和对Gac/RSM的大多数假单胞菌都是共同的，包括植物生长促进剂(恶臭假单胞菌和荧光假单胞菌)或植物病原体(丁香假单胞菌)。我们相信，对这一电路的精确分子理解将使我们能够开发出旨在操纵细菌命运的小分子，颠覆和影响它们的决策过程，以造福于我们自己。这项研究将在几个领域产生重大影响，包括生态学和农业(生态位/根生殖化)、医学和治疗方法以及基础科学和基础科学。

Pseudomonas aeruginosa is a gram-negative bacterium that thrives in a multitude of environmental niches including soil, moist surfaces or plants. It is also an opportunistic human pathogen causing infections associated with high levels of morbidity and mortality. Epidemiological studies have shown that no significant differences in the virulence potential of clinical or environmental isolates could be observed, which suggests that the available arsenal of virulence factors might also be useful to fight eukaryotic predators that may be encountered in the environment (e.g. protozoans and metazoans). As such, P. aeruginosa is a versatile organism and its adaptive potential to fluctuating environmental cues relies on numerous and complex signalling pathways. These pathways determine whether P. aeruginosa adopts a motile/toxic or sessile/biofilm lifestyle. These lifestyles directly relate to acute or chronic infection mode.The decision making process for choosing in between lifestyles is governed by major regulatory pathways which are antagonistic but also cross-talk via positive and negative feedback loops. These involve Quorum Sensing, or the second messenger molecule c-AMP, which are both master regulators for promoting production of bacterial virulence factors such as toxins. In contrast the so-called, Gac/Rsm pathway, which involves molecular sensors for probing environmental conditions, and the second messenger molecule c-di-GMP are main players to control the formation of biofilm.A biofilm is a bacterial population attached on a surface and held together by a sticky extracellular matrix made of polysaccharide, DNA or adhesive proteins. Bacteria embedded in such matrix form a very stable population which in the environment can resist various stresses such as the current of a river flow when attached on rocks, UV irradiation, dehydration or nutrient starvation for example. For pathogens, once the bacteria establish as a biofilm in one particular tissue or organ, such as in the lungs of cystic fibrosis patients in case of P. aeruginosa, it is basically impossible to eradicate. The population became highly resistant to attack by the immune system or to antibiotic therapy. This way a chronic and persistent infection establishes and morbidity reaches a very high level.In our project we will investigate the intimate connections that may exist in between the formation/dispersion of a P. aeruginosa biofilm and the two regulatory pathways Gac/Rsm on the one hand and c-di-GMP on the other hand. We want to build on our expertise and pioneering work in the characterization of the Gac/Rsm pathway and on our recent demonstration that this route intersects with the c-di-GMP signaling pathway. We want to establish the molecular links between these two main players and understand how it may then act on the formation of the biofilm and beyond this how it exerts an antagonistic action on the production of virulence factors such as toxins. Indeed, these regulatory pathways are at the intersection of a Doctor Jekyll and Mister Hyde game, in which bacteria hesitate to balance either towards an aggressive and toxic lifestyle or towards a more silent and insidious lifestyle under the form of a biofilm.In conclusion, the regulatory network we will investigate is not unique to the human pathogen P. aeruginosa but common to most gram-negative bacteria for c-di-GMP and to most Pseudomonas species for Gac/Rsm including plant growth promoters (Pseudomonas putida and Pseudomonas fluorescens) or plant pathogens (Pseudomonas syringae). We believe that a precise molecular understanding of this circuitry will allow to develop small molecules designed for manipulating the fate of bacteria, subverting and influencing their decision making process to our own benefit. This study will have strong implications in several areas including, Ecology and Agriculture (niche/root colonization), Medical and Therapeutic approaches and Fundamental and Basic Sciences.