Tuning of metabolic fluxes in Pseudomonas aeruginosa by the second messenger c-di-GMP

铜绿假单胞菌是一种机会性人类病原体，可引起一系列危及生命的急性和慢性感染，最近被世界卫生组织列为最优先的传染性威胁之一。P. aeruginosa具有高度通用性和严格调控的代谢，能够适应各种环境条件，感染广泛的生物，并成功地在不同的宿主组织中生存。然而，对宿主体内中心碳代谢及其复杂调控的作用知之甚少。我最近发现甘油醛3-磷酸脱氢酶（GAPDH）是中心碳代谢的关键酶，是细菌全球第二信使c-二- gmp （cdG）的潜在效应物。铜绿假单胞菌具有GAPDH、GapA、GapB和GapC三个同源基因。初步实验不仅证实了GapC与cdG结合，而且表明这三种同源物在碳代谢中都有特定的作用。虽然GapA似乎具有双功能，但GapB和GapC分别只催化糖酵解和糖异生反应。这表明GapC及其可能受到cdG的调控是铜绿假单胞菌碳通量调控的核心。重要的是，虽然cdG在调节重要细胞过程（如运动性、表面粘附性或毒力）中的作用已得到证实，但其对细菌代谢的干扰却完全未被探索。本文拟从结构和生化水平研究cdG对GapC的依赖调控，揭示配体相互作用的原子细节及其对催化的影响。这一信息将为研究GapC的生理作用以及cdG在铜绿假单胞菌体外生长和最终在代宿主系统中的控制作用提供基础。碳通量分析将在实验室菌株和不同生长条件下从慢性感染患者分离的菌株中进行，以阐明cdG和GapC的作用。此外，将开发特定的突变体和细胞标记来检查体内和单细胞水平上的碳通量控制。这些研究将探讨碳代谢是如何被全球第二信使调节的，并由此提供对最重要的人类病原体之一的中心代谢过程控制的详细见解。长期目标是利用这些信息来评估人类患者体内重要的细菌代谢过程。

Pseudomonas aeruginosa is an opportunistic human pathogen causing a wide array of life-threatening acute and chronic infections and has recently been listed as one of the highest priority infectious threats by the World Health Organization. With its highly versatile and tightly regulated metabolism, P. aeruginosa can adapt to various environmental conditions, infect a wide range of organisms and successfully dwell on different host tissues. However, little is known about the role of the central carbon metabolism and its complex regulation within the host. I have recently identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of the central carbon metabolism, as a potential effector of the global bacterial second messenger c-di-GMP (cdG). P. aeruginosa possess three orthologs of GAPDH, GapA, GapB, and GapC. Preliminary experiments not only demonstrated that GapC binds cdG but also indicated that all three orthologs have specific roles in carbon metabolism. While GapA appears to be bi-functional, GapB and GapC only catalyze glycolytic and gluconeogenic reactions, respectively. This indicated that GapC and its potential regulation by cdG is at the center of carbon flux regulation in P. aeruginosa. Importantly, while the role of cdG in regulating important cellular processes like motility, surface adherence, or virulence is well established, its interference with bacterial metabolism is entirely unexplored.Here, I propose to study the cdG dependent regulation of GapC on the structural and biochemical level to unveil the atomic details of ligand interaction and its effect on catalysis. This information will provide the basis for the examination of the physiological role of GapC and its control by cdG during P. aeruginosa growth in vitro and eventually in surrogate host systems. Carbon flux analysis will be carried out in laboratory strains and in strains isolated from chronically infected patients under different growth conditions to clarify the role of cdG and of GapC. In addition, specific mutants and cellular markers will be developed to examine carbon flux control in vivo and at single cell level. These studies will address how carbon metabolism is adjusted by a global second messenger and by that provide detailed insight into the control of central metabolic processes in one of the most important human pathogens. The long-term goal is to use this information to assess important bacterial metabolic processes in the human patient.