The Pathometabolism of Legionella Studied by Isotopologue Profiling

Heuner, Klaus; Kunze, Mareike; Fan, Chen; Eisenreich, Wolfgang

doi:10.1007/978-1-4939-9048-1_2

Cited by 3 publications

(1 citation statement)

References 29 publications

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“…To label L. pneumophila intracellularly growing in D. discoideum with 13 C-substrates, we used previously published protocols with minor modifications ( Heuner and Eisenreich, 2013 ; Heuner et al, 2019 ). In brief, D. discoideum Ax3 or Δ sey1 mutant producing calnexin-GFP (CnxA-GFP) were cultivated in eight T75 cell culture flasks per bacterial strain.…”

Section: Methodsmentioning

confidence: 99%

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

Hüsler

Stauffer

Keller

et al. 2023

eLife

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The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LDs interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LDs interactions. In vitro reconstitution using purified LCVs and LDs from parental or Dsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL are implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

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Section: Methodsmentioning

confidence: 99%

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

Hüsler

Stauffer

Keller

et al. 2023

eLife

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Metabolic adaption of Legionella pneumophila during intracellular growth in Acanthamoeba castellanii

Kunze

Steiner

Chen

et al. 2021

International Journal of Medical Microbiology

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The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism ofLegionella pneumophila

Hüsler

Stauffer

Keller

et al. 2022

Preprint

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The facultative intracellular bacterium Legionella pneumophila employs the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, the Legionella containing vacuole (LCV). The endoplasmic reticulum (ER)-resident large fusion GTPase Sey1/atlastin promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we show that LCVs intimately interact with palmitate-induced LDs in Dictyostelium discoideum amoeba. Comparative proteomics of LDs isolated from the D. discoideum parental strain Ax3 or Δsey1 revealed 144 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or Δsey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C GFP or AmtA-GFP and the LD marker mCherry-perilipin, we discovered that Sey1 and the L. pneumophila Icm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions. In vitro reconstitution of the LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or Δsey1 revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired for Δsey1-derived LDs, suggesting that Sey1 regulates LD composition. Palmitate promoted the growth of (i) L. pneumophila wild-type in D. discoideum Ax3 but not in Δsey1 mutant amoeba and (ii) L. pneumophila wild-type but not ΔfadL mutant bacteria lacking a homologue of the E. coli fatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellular L. pneumophila metabolizes 13C-palmitate, and its catabolism was reduced in D. discoideum Δsey1 and L. pneumophila ΔfadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

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The Pathometabolism of Legionella Studied by Isotopologue Profiling

Cited by 3 publications

References 29 publications

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

Metabolic adaption of Legionella pneumophila during intracellular growth in Acanthamoeba castellanii

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism ofLegionella pneumophila

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