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

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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.