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Legionella pneumophila CsrA 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, ?-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.

SUBMITTER: Hauslein I

PROVIDER: S-EPMC5717341 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Häuslein Ina I Sahr Tobias T Escoll Pedro P Klausner Nadine N Eisenreich Wolfgang W Buchrieser Carmen C

Open biology 20171101 11

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. ...[more]

PMID: 29093212

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Project description:Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.

Project description:UnlabelledCritical to microbial versatility is the capacity to express the cohort of genes that increase fitness in different environments. Legionella pneumophila occupies extensive ecological space that includes diverse protists, pond water, engineered water systems, and mammalian lung macrophages. One mechanism that equips this opportunistic pathogen to adapt to fluctuating conditions is a switch between replicative and transmissive cell types that is controlled by the broadly conserved regulatory protein CsrA. A striking feature of the legionellae surveyed is that each of 14 strains encodes 4 to 7 csrA-like genes, candidate regulators of distinct fitness traits. Here we focus on the one csrA paralog (lpg1593) that, like the canonical csrA, is conserved in all 14 strains surveyed. Phenotypic analysis revealed that long-term survival in tap water is promoted by the lpg1593 locus, which we name csrR (for "CsrA-similar protein for resilience"). As predicted by its GGA motif, csrR mRNA was bound directly by the canonical CsrA protein, as judged by electromobility shift and RNA-footprinting assays. Furthermore, CsrA repressed translation of csrR mRNA in vivo, as determined by analysis of csrR-gfp reporters, csrR mRNA stability in the presence and absence of csrA expression, and mutation of the CsrA binding site identified on the csrR mRNA. Thus, CsrA not only governs the transition from replication to transmission but also represses translation of its paralog csrR when nutrients are available. We propose that, during prolonged starvation, relief of CsrA repression permits CsrR protein to coordinate L. pneumophila's switch to a cell type that is resilient in water supplies.ImportancePersistence of L. pneumophila in water systems is a public health risk, and yet there is little understanding of the genetic determinants that equip this opportunistic pathogen to adapt to and survive in natural or engineered water systems. A potent regulator of this pathogen's intracellular life cycle is CsrA, a protein widely distributed among bacterial species that is understood quite well. Our finding that every sequenced L. pneumophila strain carries several csrA paralogs-including two common to all isolates--indicates that the legionellae exploit CsrA regulatory switches for multiple purposes. Our discovery that one paralog, CsrR, is a target of CsrA that enhances survival in water is an important step toward understanding colonization of the engineered environment by pathogenic L. pneumophila.

Project description:UnlabelledBacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA. Using bioinformatic analyses, we deduced that specific csrA paralogs are coinherited with particular lineages of the type IV secretion system that mediates horizontal spread of its ICE, suggesting a conserved regulatory interaction. As a first step to investigate the contribution of csrA regulators to this class of mobile genetic elements, we analyzed here the activity of the csrA paralog encoded on Legionella pneumophila ICE-?ox. Deletion of this gene, which we name csrT, had no observed effect under laboratory conditions. However, ectopic expression of csrT abrogated the protection to hydrogen peroxide and macrophage degradation that ICE-?ox confers to L. pneumophila. When ectopically expressed, csrT also repressed L. pneumophila flagellin production and motility, a function similar to the core genome's canonical csrA. Moreover, csrT restored the repression of motility to csrA mutants of Bacillus subtilis, a finding consistent with the predicted function of CsrT as an mRNA binding protein. Since all known ICEs of legionellae encode coinherited csrA-type IV secretion system pairs, we postulate that CsrA superfamily proteins regulate ICE activity to increase their horizontal spread, thereby expanding L. pneumophila versatility.ImportanceICEs are mobile DNA elements whose type IV secretion machineries mediate spread among bacterial populations. All surveyed ICEs within the Legionella genus also carry paralogs of the essential life cycle regulator csrA. It is striking that the csrA loci could be classified into distinct families based on either their sequence or the subtype of the adjacent type IV secretion system locus. To investigate whether ICE-encoded csrA paralogs are bona fide regulators, we analyzed ICE-?ox as a model system. When expressed ectopically, its csrA paralog inhibited multiple ICE-?ox phenotypes, as well as the motility of not only Legionella but also Bacillus subtilis. Accordingly, we predict that CsrA regulators equip legionellae ICEs to promote their spread via dedicated type IV secretion systems.

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

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<i>Legionella pneumophila</i> CsrA regulates a metabolic switch from amino acid to glycerolipid metabolism.

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