Legionella pneumophila CsrA regulates a metabolic switch from amino acid to glycerolipid metabolism

Legionella pneumophila CsrA plays a crucial role in the life-stage-specific expression of virulence phenotypes and metabolic activity. However, its exact role is only partly known. To elucidate how CsrA impacts L. pneumophila metabolism we analysed the CsrA depended regulation of metabolic functions by comparative 13C-isotopologue profiling and oxygen consumption experiments of a L. pneumophila wild-type (wt) strain and its isogenic csrA- mutant. We show that a csrA- mutant has significantly lower respiration rates when serine, alanine, pyruvate, α-ketoglutarate or palmitate is the sole carbon source. By contrast, when grown in glucose or glycerol, no differences in respiration were detected. Isotopologue profiling uncovered that the transfer of label from [U-13C3]serine via pyruvate into the citrate cycle and gluconeogenesis was lower in the mutant as judged from the labelling patterns of protein-derived amino acids, cell-wall-derived diaminopimelate, sugars and amino sugars and 3-hydroxybutyrate derived from polyhydroxybutyrate (PHB). Similarly, the incorporation of [U-13C6]glucose via the glycolysis/Entner-Doudoroff (ED) pathway but not via the pentose phosphate pathway was repressed in the csrA- mutant. On the other hand, fluxes due to [U-13C3]glycerol utilization were increased in the csrA- mutant. In addition, we showed that exogenous [1,2,3,4-13C4]palmitic acid is efficiently used for PHB synthesis via 13C2-acetyl-CoA. Taken together, CsrA induces serine catabolism via the tricarboxylic acid cycle and glucose degradation via the ED pathway, but represses glycerol metabolism, fatty acid degradation and PHB biosynthesis, in particular during exponential growth. Thus, CsrA has a determining role in substrate usage and carbon partitioning during the L. pneumophila life cycle and regulates a switch from amino acid usage in replicative phase to glycerolipid usage during transmissive growth.