Pathway analysis using ¹³C‐glycerol and other carbon tracers reveals a bipartite metabolism of Legionella pneumophila

Abstract

Amino acids represent the prime carbon and energy source for Legionella pneumophila, a facultative intracellular pathogen, which can cause a life-threatening pneumonia termed Legionnaires' disease. Genome, transcriptome and proteome studies indicate that L. pneumophila also utilizes carbon substrates other than amino acids. We show here that glycerol promotes intracellular replication of L. pneumophila in amoeba or macrophages (but not extracellular growth) dependent on glycerol-3-phosphate dehydrogenase, GlpD. An L. pneumophila mutant strain lacking glpD was outcompeted by wild-type bacteria upon co-infection of amoeba, indicating an important role of glycerol during infection. Isotopologue profiling studies using (13) C-labelled substrates were performed in a novel minimal defined medium, MDM, comprising essential amino acids, proline and phenylalanine. In MDM, L. pneumophila utilized (13) C-labelled glycerol or glucose predominantly for gluconeogenesis and the pentose phosphate pathway, while the amino acid serine was used for energy generation via the citrate cycle. Similar results were obtained for L. pneumophila growing intracellularly in amoeba fed with (13) C-labelled glycerol, glucose or serine. Collectively, these results reveal a bipartite metabolism of L. pneumophila, where glycerol and carbohydrates like glucose are mainly fed into anabolic processes, while serine serves as major energy supply.

Metabolism of myo -Inositol by Legionella pneumophila Promotes Infection of Amoebae and Macrophages

“…Furthermore, during exponential growth, SpoT acts as a ppGpp hydrolase, which keeps the concentration of the alarmone low (67). In a previous study, we showed that the metabolism of serine results in a high rate of fatty acid biosynthesis (28), and thus, the SpoT-dependent cellular levels of ppGpp are likely decreased under these conditions.…”

“…This is substantiated by the following findings: (i) inositol (and not a "secondary product") is metabolized, as intracellular growth promotion by the carbohydrate is dependent on iolT or iolG, (ii) inositol reaches the LCV after phagosome closure and pathogen vacuole formation, since the compound promotes growth when added several hours after infection, (iii) the fluorescent glucose analogue 2-NBDG accumulates in vacuoles harboring strain JR32 but not icmT mutant bacteria, and (iv) glycerol and glucose also reach LCVs during intracellular growth of L. pneumophila in A. castellanii (28). Noteworthy, compared to LCVs, 50% fewer vacuoles harboring icmT (or rpoS) mutant bacteria accumulate 2-NBDG, indicating that vacuole remodelling by a functional Icm/Dot T4SS is required for carbohydrate transport.…”

“…L. pneumophila strains (Table 1) were cultured under aerobic conditions at 37°C in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) broth or grown on charcoal yeast extract (CYE) agar plates supplemented with chloramphenicol (Cm; 5 g ml Ϫ1 ) or kanamycin (Km; 50 g ml Ϫ1 in broth or 10 g ml Ϫ1 in agar plates), if necessary. Alternatively, L. pneumophila was cultivated at 37°C in chemically defined medium (CDM) (22), modified from Ristroph et al (19), or minimal defined medium (MDM) (28). CDM and MDM were prepared by dissolving components in 950 ml of double-distilled water (ddH 2 O).…”

“…More recent physiological and isotopologue profiling studies established that glucose and glycerol are metabolized by L. pneumophila under extracellular and intracellular conditions (22,27,28). L. pneumophila employs a bipartite metabolism, where amino acids, such as serine, are preferentially catabolized and serve as a major supplier of energy, while glycerol and carbohydrates, like glucose, are predominantly utilized for anabolic processes (28).…”

“…More recent physiological and isotopologue profiling studies established that glucose and glycerol are metabolized by L. pneumophila under extracellular and intracellular conditions (22,27,28). L. pneumophila employs a bipartite metabolism, where amino acids, such as serine, are preferentially catabolized and serve as a major supplier of energy, while glycerol and carbohydrates, like glucose, are predominantly utilized for anabolic processes (28). A bipartite metabolic strategy is also employed by other intracellular pathogens, like Listeria monocytogenes (29) or Mycobacterium tuberculosis (30,31), and might be an adaptation to an intracellular lifestyle providing the bacteria with a variety of different carbon sources.…”