A bacterial c-di-GMP responsive enzyme modulates LPS structure and triggers immune evasion

细菌在多种环境中茁壮成长，细菌病原体在它们的宿主中定居时会遇到压力和恶劣的条件。为了应对营养限制、免疫系统的攻击和许多其他环境波动，大多数细菌已经进化出复杂的调节网络。它们可以放置专门的探测器，通过这些探测器，它们可以感知特定生态位中遇到的情况，并调节它们的遗传程序，从而激活/抑制一组特定的基因，从而重组细胞的生理机能，使其得到最佳适应。在许多细菌中，最初的检测可能是所谓的双组分调节系统的责任，该系统检测刺激并将该信息传递给调节器，该调节器将通过直接控制基因表达来调节基因表达。在许多情况下，这种信息的传递可能不是直接的，而是利用细胞内的第二信使，这是一种传递信息的小分子。在目前的提议中，我们将研究一种称为c-di-GMP的信使。我们知道，这种分子是最可怕的革兰氏阴性细菌病原体之一铜绿假单胞菌的生活方式的核心，它不仅是一种多重耐药生物，而且由于细胞包膜渗透性差，外排能力高，因此将药物保留在体外，因此对标准的抗生物疗法也一直具有极强的耐药性。此外，铜绿假单胞菌可以采取非常不同的感染策略和生活方式，这可能导致急性感染或慢性感染。在后一种情况下，细菌建立了一个被称为生物膜的微生物城市，这个细菌群落对抗生素治疗和免疫系统的根除变得更加耐药。然而，c-二gmp是一个非常简单的分子，它是生活方式改变和适应的绝对核心，通过提高c-二gmp的细胞内水平，细菌立即进入生物膜模式，通过减缓运动，增加细胞外基质的生产，将所有细胞粘合在一起，激活分子武器，这将有助于对抗驻地细菌或免疫系统。对于一个分子来说，这是很多任务，但机制的美妙之处在于它是多重的。c-di-GMP可以被大量的酶合成或破坏（环化酶制造或磷酸二酯酶破坏），并可以结合大量的蛋白质，从而改变它们的活性。例如，c-di-GMP可以结合一个被激活的调节因子，从而驱动基因表达。它可以结合一些特定的蛋白质，改变它们的构象，然后与鞭毛马达等相互作用，从而停止旋转，阻止运动，进入生物膜模式。在之前的工作中，我们已经发现了一种非常新颖的c-di-GMP信号通路，并发现由环化酶SadC合成的分子直接转移到蛋白质上，我们称之为直接蛋白质-蛋白质相互作用。我们已经证明了这种叫做WarA的蛋白质具有酶活性，也就是甲基转移酶，它通过改变LPS的结构来修饰细菌的表面。众所周知，在革兰氏阴性细菌中，脂多糖是触发免疫系统的一个关键因素（也称为病原体相关基序模式或PAMP）。我们已经证明，缺乏这种酶WarA的细菌株，会立即被免疫系统识别，正如我们在透明斑马鱼模型中展示的那样，免疫细胞在感染部位的招募。这是一个非常令人兴奋的发现，目前的建议旨在了解分子事件的级联反应，我们将能够操纵这一途径，使铜绿假单胞菌容易被免疫系统检测到，并更容易获得抗生素治疗。

Bacteria thrive in a multitude of environments and bacterial pathogens encounter stressful and harsh conditions when they colonize their host. In order to cope with nutrient limitations, aggression by the immune system and many other environmental fluctuations, most bacteria have evolved sophisticated regulatory networks. They can put in place specialized detectors by which they sense the conditions encounter in a specific niche and modulate their genetic program so that a specific set of genes are activated/repressed in order to reorganize the physiology of the cell so that it is optimally adapted.In many bacteria the initial detection could be the responsibility of so called two component regulatory systems, which detect a stimulus and transfer this information to a regulator that will modulate gene expression through direct control of gene expression. In many cases the transmission of this information might not be direct but make use of an intracellular second messenger, which is a small molecule that will relay this information.I the present proposal we will study one such messenger that is called c-di-GMP. We know that this molecule is central to the lifestyle of one of the most dreadful gram-negative bacterial pathogen, Pseudomonas aeruginosa, which not only emerges as a multi-resistant organisms but has always been extremely resistant even to standard anti-biotherapy due to a poorly permeable cell envelope and high efflux capacity thus retaining drugs outside. Furthermore P. aeruginosa can adopt very different infection strategies and lifestyles, and this can lead to acute infection or chronic infection. In the latter case the bacterium build a city of microbe that is known as biofilm, and this bacterial community becomes even more resistant to antibiotic treatment and eradication by the immune system.Whereas c-di-GMP is a very simple molecule it is absolutely central to the lifestyle changes and adaptation and by elevating the intracellular levels of c-di-GMP bacteria turns immediately into the biofilm mode by slowing down motility, increasing the production of an extracellular matrix which glue all cells together, activating molecular weapon which will help fight against resident bacteria or the immune system. This is a lot of tasks for one single molecule but the beauty of the mechanisms is that it is many-fold. The c-di-GMP can be made or broken by a large number of enzymes (cyclase to make or phosphodiesterase to break) and can bind a large number of proteins, which will in turn modify their activity. For example, c-di-GMP can bind a regulator that is then activated and that will drive gene expression. It can bind some specific proteins, which will modify their conformation, which will then interact for example with the flagellar motor, thus stopping rotation and arresting movement to enter the biofilm mode.In previous work we have identified an very novel c-di-GMP signalling pathway and found that the molecule which is synthesized by the cyclase SadC is directly transferred onto a protein that we called through a direct protein-protein interaction. We have shown that this protein, that we called WarA has an enzymatic activity, namely methyltransferase, and modifies the surface of the bacterium by changing the structure of the LPS. The LPS is well known in gram-negative bacteria to be one key element (also called Pathogen associated Motif Pattern or PAMP) that triggers the immune system. We have shown that a bacterial strain devoid of this enzyme WarA, becomes immediately recognized by the immune system, as we demonstrate the recruitment of immune cells on the infection site in the transparent zebra fish model.This is a very exciting discovery and the present proposal is aiming at understanding the cascade of molecular events in a way that we will be able to manipulate this pathway to make P. aeruginosa readily detected by the immune system and more accessible to antibiotic treatment.