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

Abstract

The amoeba-resistant bacterium <i>Legionella pneumophila</i> causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated <i>Legionella</i>-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-LD interactions in the genetically tractable amoeba <i>Dictyostelium discoideum</i>. Dually fluorescence-labeled <i>D. discoideum</i> producing LCV and LD markers revealed that Sey1 as well as the <i>L. pneumophila</i> T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δ<i>sey1</i> mutant <i>D. discoideum</i> indicated that Sey1 and GTP promote this process. Sey1 and the <i>L. pneumophila</i> fatty acid transporter FadL were 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 <i>L. pneumophila</i>.