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

Abstract

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.

Zooming into lipid droplet biology through the lens of electron microscopy

“…Less‐well studied intracellular interactions, such as those of LDs with peroxisomes [33,119], pathogens [120–123], and the cytoskeleton [124,125], have been supported by EM observations. Recent investigations into LD–LD contact sites support a model where a potentially phase‐separated Cidec‐condensate facilitates the flux of neutral lipids along an internal pressure gradient from smaller to larger LDs [47,113, 126,150].…”

Tapping lipid droplets: A rich fat diet of intracellular bacterial pathogens

“…The ER‐residing large fusion GTPase Sey1/atlastin 3 promotes LCV expansion and intracellular replication of L. pneumophila (Hüsler et al, 2021; Steiner et al, 2017; Steiner, Weber, Kaech, et al, 2018). Dictyostelium discoideum ∆ sey1 mutant amoeba show pleiotropic defects, including aberrant ER architecture and dynamics, impaired LCV–ER interactions, and intracellular bacterial replication (Hüsler et al, 2021), as well as reduced LCV–LD interactions (Hüsler et al, 2023).…”

“…A recent study revealed that LCVs intimately interact with palmitate‐induced LDs in D. discoideum , and the large GTPase Sey1 as well as the L. pneumophila Icm/Dot T4SS and the effector LegG1 promote LCV–LD interactions (Hüsler et al, 2023). In vitro reconstitution of LCV–LD interactions using purified LCVs and LDs from D. discoideum Ax3 or Δ sey1 indicated that Sey1 and GTP promote this process.…”

“…Dictyostelium discoideum ∆sey1 mutant amoeba show pleiotropic defects, including aberrant ER architecture and dynamics, impaired LCV-ER interactions, and intracellular bacterial replication (Hüsler et al, 2021), as well as reduced LCV-LD interactions (Hüsler et al, 2023).…”