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Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates.

ABSTRACT: Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-(13)C(3)]serine, [U-(13)C(6)]glucose, or [1,2-(13)C(2)]glucose. After growth, (13)C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-(13)C(3)]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [(13)C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-(13)C(2)]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Delta zwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.

SUBMITTER: Eylert E

PROVIDER: S-EPMC2903384 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates.

Eylert Eva E Herrmann Vroni V Jules Matthieu M Gillmaier Nadine N Lautner Monika M Buchrieser Carmen C Eisenreich Wolfgang W Heuner Klaus K

The Journal of biological chemistry 20100504 29

Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-(13)C(3)]serine, [U-(13)C(6)]glucose, or [1,2-(13)C(2)]glucose. After growth, (1 ...[more]

PMID: 20442401

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Project description:The Legionella pneumophila TF3/1 mutant of the Corby strain, which possesses a point mutation in the active site of the O-acetyltransferase, synthesized the polysaccharide chain with a reduced degree of substitution with O-acetyl groups. The mutant did not produce a high-molecular-weight lipopolysaccharide (LPS) fraction above 12 kDa. The disturbances in LPS synthesis have an effect on the composition of other macromolecules (lipids and proteins), as indicated by differences in the infrared absorption spectra between the L. pneumophila Corby strain and its TF3/1 mutant. The wild-type strain contained less N+-CH3 and C-N groups as well as more CH3 groups than the mutant. The fatty acid composition showed that the wild type strain synthesized more branched acyl residues (a15:0, i16:0, and a17:0), a less unsaturated acid (16:1), and a straight-chain acid (18:0) than the mutant. The mutant synthesized approximately twice more a long-chain fatty acid (20:0) than the wild type. The main differences in the phospholipids between both strains were found in the classes of phosphatidylcholines and phosphatidylglycerols (PG). Substantial differences in the cell surface topography of these bacteria and their nanomechanical properties were shown by atomic force microscopy (AFM). The wild type strain had no undulated surface and produced numerous vesicles. In the case of the mutant type, the vesicles were not numerous, but there were grooves on the cell surface. The average roughness of the cell surface of the mutant was approximately twofold higher than in the wild-type strain. In turn, the wild-type strain exhibited much better adhesive properties than the mutant. The kinetic study of the interaction between the L. pneumophila strains and Acanthamoeba castellanii monitored by Förster resonance energy transfer revealed a pronounced difference, i.e., almost instantaneous and highly efficient binding of the L. pneumophila Corby strain to the amoeba surface, followed by penetration into the amoeba cells. This process was clearly not as efficient in the case of the mutant. The results point to LPS and, in particular, to the length of the polysaccharide fraction as an important L. pneumophila determinant involved in the process of adhesion to the host cell.

Dataset Information

Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates.

Publications

Isotopologue profiling of Legionella pneumophila: role of serine and glucose as carbon substrates.

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