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

Abstract

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.

Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth

“…pneumophila also utilize fatty acids and/or complex lipids for intracellular growth, as demonstrated by fluorescent labelling of bacteria when infected macrophages were fed fluorophore-conjugated fatty acids ( Fig 4E ) and, in the amoebae Dictyostelium discoideum , incorporation of 13 C into L . pneumophila when infected amoeba were supplemented with [ 13 C]palmitate [ 45 ]. Additionally, Hilbi and colleagues demonstrated that a L .…”

“…pneumophila mutant lacking a homolog of fadL , which encodes a fatty acid import protein, accumulated less 13 C-containing molecules and grew more poorly in D . discoideum [ 45 ]. Finally, Legionella gormanii membranes were recently shown to surprisingly contain the sphingolipid ceramide, implicating host-derived fatty acid-containing molecules in performing a structural role in the bacterium as well [ 46 ].…”

“…In contrast to oleate-induced lipid droplets, palmitate-induced lipid droplets were more likely to associate with LCVs and were consumed during infection of macrophages ( Fig 5 ), and have been shown to colocalize with the LCV in D . discoideum [ 45 ], a process mediated by the L . pneumophila effector LegG1 [ 45 ].…”

“…discoideum [ 45 ], a process mediated by the L . pneumophila effector LegG1 [ 45 ]. However, while palmitate-induced lipid droplets enhance L .…”

“…pneumophila replication in D . discoideum [ 45 ], this did not occur in macrophages ( Fig 2A ), although they were consumed in both host cell types. The difference may be due to the effects of palmitate on the mammalian immune system, which includes the induction of M1 polarization, activation of TLR signaling and production of proinflammatory cytokines [ 55 – 57 ].…”