Legionella pneumophilaCsrA regulates a metabolic switch from amino acid to glycerolipid metabolism

Abstract

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.

The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism

“…Indeed, using 13 C-isotopologue profiling and carbon-flux analyses of a wild-type and a csrA mutant strain confirmed that CsrA plays a major role in regulating the carbon flux between the PPP and the glycolysis (Häuslein et al, 2017 ). Furthermore, this study highlighted the impact of CsrA on the bipartite metabolism of L. pneumophila , as the absence of CsrA induces a reduction of the carbon flux from serine via gluconeogenesis into the PPP.…”

“…Similar to glucose, the carbon from glycerol is mainly shuffled into gluconeogenesis and PPP for histidine and mannose production, but only low flux rates of carbon from glycerol into the TCA cycle were reported (Häuslein et al, 2016 ). In contrast, saturated lipids like palmitate, another carbon source shown to be predominantly used after amino acid depletion, is used for energy production and PHB synthesis (Häuslein et al, 2017 ). Taken together, the results from 13 C- isotopologue profiling and flux analyses suggested that the life cycle switch of Legionella is also reflected by a metabolic shift from amino acids usage during replication to glycerolipids and glucose when entering transmissive phase (Häuslein et al, 2017 ; Figure 2 ).…”

“…Although less is known about the intracellular metabolism in the late phase of growth, it is likely that L. pneumophila gets access to carbohydrate sources such as glycogen, glucose and other polymers from the host upon LCV lysis (Lang and Flieger, 2011 ). In addition, the transmissive form of L. pneumophila contains high amounts of PHB, which serves as energy and carbon storage for the maintenance of the intracellular growth cycle (Gillmaier et al, 2016 ; Häuslein et al, 2017 ). Moreover, intracellular L. pneumophila metabolizes myo -inositol, which was reported to promote infection of A. castellanii and macrophages (Manske et al, 2016 ), and engages the translocated protein MavN, which once integrated in the host-derived LCV membrane, facilitates the acquisition of iron into its vacuole (Isaac et al, 2015 ).…”

“…By contrast, CsrA has a negative impact on the incorporation and the metabolism of glycerol and glucose. As such, the absence of CsrA results in the increase of the carbon flux from glucose into the PPP and ED pathways and the carbon flux from glycerol into the PPP and the gluconeogenesis (Häuslein et al, 2017 ). These studies also showed the important influence of CsrA on the production of the storage molecule PHB suggesting that CsrA is a major player in the utilization of the different carbon sources during the biphasic life cycle of L. pneumophila (Häuslein et al, 2017 ).…”

“…As such, the absence of CsrA results in the increase of the carbon flux from glucose into the PPP and ED pathways and the carbon flux from glycerol into the PPP and the gluconeogenesis (Häuslein et al, 2017 ). These studies also showed the important influence of CsrA on the production of the storage molecule PHB suggesting that CsrA is a major player in the utilization of the different carbon sources during the biphasic life cycle of L. pneumophila (Häuslein et al, 2017 ). The biphasic life cycle of L. pneumophila within the host supports the usage of amino acids as main carbon and energy source during multiplication due to the expression of CsrA that is simultaneously repressing the usage of alternative carbon sources, such as glycerol.…”