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

Abstract

AbstractThe facultative intracellular bacteriumLegionella pneumophilaemploys the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, theLegionella-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 inDictyostelium discoideumamoeba. Comparative proteomics of LDs isolated from theD. discoideumparental strain Ax3 or ⊗sey1revealed 144 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or ⊗sey1, 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 theL. pneumophilaIcm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions.In vitroreconstitution of the LCV-LD interactions using purified LCVs and LDs fromD. discoideumAx3 or ⊗sey1revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired for ⊗sey1-derived LDs, suggesting that Sey1 regulates LD composition. Palmitate promoted the growth of (i)L. pneumophilawild-type inD. discoideumAx3 but not in ⊗sey1mutant amoeba and (ii)L. pneumophilawild-type but not ⊗fadLmutant bacteria lacking a homologue of theE. colifatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellularL. pneumophilametabolizes13C-palmitate, and its catabolism was reduced inD. discoideum⊗sey1andL. pneumophila⊗fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellularL. pneumophila.