Showing papers by "Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto published in 2023"

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

Abstract: Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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-LD 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-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila 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 L. pneumophila. Editor's evaluation This important study advances our understanding of host-derived lipid droplets (LDs) interaction with intracellular pathogens. The use of amoeba species Dictyostelium discoideum as a host for Legionella pneumophila infection is compelling and goes beyond the current state of the art. The data were collected and analyzed using convincing methodology and this paper will interest cell biologists and microbiologists working on the interaction of microbes with host cells. https://doi.org/10.7554/eLife.85142.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The causative agent of Legionnaires’ disease, Legionella pneumophila, is a facultative intracellular bacterium, which adopts a similar mechanism to replicate in free-living protozoa and lung macrophages (Newton et al., 2010; Boamah et al., 2017; Mondino et al., 2020). To govern the interactions with eukaryotic host cells, Legionella spp. employ the genus-conserved Icm/Dot type IV secretion system (T4SS), which in L. pneumophila translocates more than 300 different ‘effector’ proteins (Qiu and Luo, 2017; Hilbi and Buchrieser, 2022; Lockwood et al., 2022). The effector proteins subvert pivotal processes and establish a unique replication niche, the Legionella-containing vacuole (LCV), which communicates with the endosomal, secretory and retrograde vesicle trafficking pathways, but restricts fusion with lysosomes (Isberg et al., 2009; Asrat et al., 2014; Finsel and Hilbi, 2015; Personnic et al., 2016; Sherwood and Roy, 2016; Bärlocher et al., 2017; Steiner et al., 2018a; Swart and Hilbi, 2020b). Given that many cellular pathways and effector protein targets are conserved, the genetically tractable amoeba Dictyostelium discoideum is a versatile and powerful model to analyze pathogen-phagocyte interactions (Cardenal-Muñoz et al., 2017; Swart et al., 2018). During LCV formation, the phosphoinositide (PI) lipid phosphatidylinositol 3-phosphate (PtdIns(3)P) is converted to PtdIns(4)P (Weber et al., 2006; Weber et al., 2014b; Steiner et al., 2018a; Weber et al., 2018; Swart and Hilbi, 2020b). Additionally, the LCV intercepts and fuses with endoplasmic reticulum (ER)-derived vesicles (Kagan and Roy, 2002; Robinson and Roy, 2006; Arasaki et al., 2012), and the LCV itself tightly associates with the ER (Swanson and Isberg, 1995; Robinson and Roy, 2006). The LCV-ER association persists even upon isolation and purification of intact LCVs (Urwyler et al., 2009; Hoffmann et al., 2014; Schmölders et al., 2017). The tight LCV-ER association has recently been confirmed by showing that LCVs form extended membrane contact sites (MCS) with the ER (Vormittag et al., 2023). The ER is a highly dynamic organelle (Shibata et al., 2006; Hu and Rapoport, 2016; Nixon-Abell et al., 2016), and its morphology and dynamics are largely controlled by the reticulon family of membrane tubule-forming proteins (Voeltz et al., 2006; Hu et al., 2008) and the atlastin family of trans-membrane large fusion GTPases (Hu et al., 2009; Orso et al., 2009). Atlastins are conserved from yeast to plants and mammals (Anwar et al., 2012; Zhang et al., 2013), mediate the homotypic fusion of ER tubules and share a similar domain organization, which comprises an N-terminal GTPase domain linked through a helical bundle (HB) domain to two adjacent transmembrane segments and a C-terminal tail that contains an amphipathic helix (Hu and Rapoport, 2016). Structural and biochemical studies revealed that upon GTP binding, the GTPase and HB domains of atlastins on two distinct ER tubules dimerize, and the trans-homodimers pull together opposing membranes thus facilitating their fusion (Bian et al., 2011; Byrnes and Sondermann, 2011; Liu et al., 2012; Byrnes et al., 2013; Liu et al., 2015). D. discoideum produces a single orthologue of human atlastin-1–3 (Atl1-3) termed Sey1, which shares the same domain organization as the mammalian atlastins (Steiner et al., 2017). Initially, Sey1, Atl3, and reticulon-4 (Rtn4) were identified by proteomics in intact LCVs purified from L. pneumophila-infected D. discoideum or macrophages (Hoffmann et al., 2014), and the localization of Sey1/Atl3 and Rtn4 to the ER surrounding LCVs was validated by fluorescence microscopy (Haenssler et al., 2015; Steiner et al., 2017). Sey1 is not implicated in the formation of the PtdIns(4)P-positive LCV membrane and not essential for the recruitment of ER, but promotes pathogen vacuole expansion and enhances intracellular replication of L. pneumophila (Steiner et al., 2017; Steiner et al., 2018b). The production of a catalytically inactive, dominant-negative Sey1_K154A mutant protein, or the depletion of mammalian Atl3, restricts L. pneumophila replication and impairs LCV maturation. D. discoideum ∆sey1 mutant amoeba are enlarged but grow and develop similarly to the parental strain (Hüsler et al., 2021). The mutant strain shows pleiotropic defects, including aberrant ER architecture and dynamics, inability to cope with prolonged ER stress, defective intracellular proteolysis, cell motility and growth on bacterial lawns (Hüsler et al., 2021). In the ∆sey1 mutant amoeba LCV-ER interactions, LCV expansion and intracellular L. pneumophila replication are impaired, similar to what was observed with D. discoideum producing dominant negative Sey1 (Steiner et al., 2017). Taken together, Sey1/Atl3 controls circumferential ER remodeling during LCV maturation and intracellular replication of L. pneumophila (Steiner et al., 2017; Steiner et al., 2018b; Hüsler et al., 2021). In addition to promoting ER dynamics, atlastins contribute to a number of other cellular processes including the biogenesis of ER-derived lipid droplets (LDs; Klemm et al., 2013). LDs are the major cellular storage compartments of neutral lipids; however, they are also involved in many other cellular processes such as energy homeostasis, lipid metabolism, generation of membrane lipids and signaling molecules as well as retention of harmful proteins and lipids (Walther and Farese, 2012; Hashemi and Goodman, 2015; Welte, 2015; Kimmel and Sztalryd, 2016; Welte and Gould, 2017). In D. discoideum, LDs accumulate upon feeding the cells with fatty acids (in particular palmitate) or bacteria (Du et al., 2013). Proteomics and lipidomics analysis of D. discoideum LDs revealed that the lipid constituents are similar to mammalian LDs and comprise mainly triacylglycerol (57%), free fatty acids (22 %) and sterol esters (4%). LDs are coated by a polar phospholipid monolayer and distinct proteins (Du et al., 2013), such as perilipin (Miura et al., 2002), and small GTPases, as well as ER proteins (reticulon C, RtnlC; protein disulfide isomerase, PDI; lipid droplet membrane protein, LdpA; and 15 lipid metabolism enzymes), the latter reflecting their cellular organelle origin (Du et al., 2013). LDs are transported along microtubules and actin filaments or moved by actin polymerization (Welte, 2004; Welte, 2009; Pfisterer et al., 2017; Welte and Gould, 2017; Kilwein and Welte, 2019), and they form contact sites with various cell organelles (Kumar et al., 2018; Benador et al., 2019; Yeshaw et al., 2019; Herker et al., 2021). Only after the replication-permissive LCV has been formed, L. pneumophila engages in intracellular replication. The bacteria employ a biphasic lifestyle comprising a transmissive (motile, virulent) and a replicative phase (Molofsky and Swanson, 2004). L. pneumophila is an obligate aerobe bacterium, which previously has been thought to rely on certain amino acids as carbon and energy source (Abu Kwaik and Bumann, 2013; Manske and Hilbi, 2014). Indeed, isotopologue profiling studies with stable 13C-isotopes indicated that serine is a major carbon and energy source for L. pneumophila and readily metabolized by the bacteria (Eylert et al., 2010). More recent physiological and isotopologue profiling studies established that glucose, inositol, and glycerol are also metabolized by L. pneumophila under extracellular and intracellular conditions (Eylert et al., 2010; Harada et al., 2010; Häuslein et al., 2016; Manske et al., 2016). Finally, isotopologue profiling studies indicated that extracellular L. pneumophila efficiently catabolizes exogenous [1,2,3,4-13C4]palmitic acid, yielding 13C2-acetyl-CoA, which is used to synthesize the storage compound polyhydroxybutyrate (PHB; Häuslein et al., 2017). It is unknown how fatty acids are taken up by L. pneumophila. In E. coli, the long-chain fatty acid transporter FadL localizes to the outer membrane, where the monomeric protein adopts a 14-stranded, anti-parallel β barrel structure (van den Berg et al., 2004). The N-terminal 42 amino acid residues of FadL form a small ‘hatch’ domain that plugs the barrel, and the hydrophobic substrate leaves the transporter by lateral diffusion into the outer membrane (Hearn et al., 2009). L. pneumophila encodes a homolog of E. coli FadL, Lpg1810, which was identified as a surface-associated protein by fluorescence-labeling and subsequent mass spectrometry (MS), confirming its presence in the bacterial outer membrane (Khemiri et al., 2008). Given the role of LDs as lipid storage organelles regulated by atlastins, we set out to analyze the contribution of LDs, Sey1 and FadL for intracellular replication and palmitate catabolism of L. pneumophila in D. discoideum. We found that Sey1 regulates LD protein composition and promotes Icm/Dot-dependent LCV-LD interactions as well as FadL-dependent fatty acid metabolism of intracellular L. pneumophila. Results Palmitate-induced LDs interact with LCVs in D. discoideum To initially explore whether fatty acids and/or LDs play a role for intracellular replication of L. pneumophila, we fed D. discoideum strain Ax3 with palmitate, and assessed intracellular replication of the L. pneumophila wild-type strain JR32 or the mutant strain ΔicmT, which lacks a functional T4SS and is defective for effector protein secretion. Feeding with 200 µM palmitate overnight significantly promoted the intracellular growth of L. pneumophila, while higher concentrations of palmitate had a negative effect on growth (Figure 1A). This growth reduction was not owing to fatty acid toxicity for D. discoideum, as up to 800 µM palmitate were not toxic for the amoeba (Figure 1—figure supplement 1). Figure 1 with 1 supplement see all Download asset Open asset Palmitate-induced lipid droplets interact with LCVs in D. discoideum. (A) D. discoideum Ax3, untreated (LoFlo medium) or treated with increasing concentrations of sodium palmitate (100–800 µM, 3 hr), were infected (MOI 10) with GFP-producing L. pneumophila wild-type JR32 or ΔicmT (pNT28). The GFP-fluorescence was measured with a microtiter plate reader at 1 hr, 24 hr, and 48 hr p.i. Data show the relative fluorescence increase between 1 hr and 24 hr or 48 hr p.i. (JR32: black/grey bar; ΔicmT: white/dotted bar). Data represent means ± SD of three independent experiments (*p<0.05). (B) Representative cryotomograms of D. discoideum Ax3, fed (3 hr) with 200 µM sodium palmitate and infected (MOI 100) with L. pneumophila JR32 for 30 min (top) or 3 hr (bottom). Intimate LCV-LD interactions are clearly visible (red arrows). Reconstruction of LCV-LD interaction observed at 30 min p.i. (top; right). OM, outer membrane; IM, inner membrane; LCV, Legionella-containing vacuole (limiting membrane); ER, endoplasmic reticulum; LD, lipid droplet; cyt, L. pneumophila cytoplasm; Hcyt, host cell cytoplasm. Scale bars: 100 nm. To assess whether the growth-promoting effect of palmitate might involve LDs, we thought to visualize the possible interactions between LCVs and LDs. To this end, D. discoideum strain Ax3 was fed with palmitate, infected with the L. pneumophila wild-type strain JR32 and subjected to cryo-electron tomography (cryoET). The obtained cryotomograms clearly show an intensive interaction between LCVs and LDs (Figure 1B). Upon contact with the LCV, the LDs tightly interact with the limiting membrane of the pathogen vacuole and even appear to integrate into the LCV limiting membrane. We did not observe tethering of LDs to the LCV with the LD lipid monolayer and the LCV lipid bilayer spanning a discrete, short distance in the nm range. Hence, LDs undergo robust and intimate interactions with LCVs in infected D. discoideum. Sey1 promotes LD recruitment to intact LCVs in D. discoideum Given that large GTPases of the atlastin family are implicated in LD formation in mammalian cells (Klemm et al., 2013), we next assessed whether Sey1 affects early LCV-LD interactions in D. discoideum. To this end, we used dually fluorescence-labelled D. discoideum producing the LCV marker P4C-GFP and the LD marker mCherry-Plin. The D. discoideum parental strain Ax3 or Δsey1 mutant amoeba were fed overnight with 200 µM palmitate, stained with LipidTOX Deep Red and infected with mCerulean-producing L. pneumophila JR32. Within the first hour of infection, the dynamic interactions of single LCVs with LDs were recorded for 60 s each at different time points (Figure 2A, Figure 2—figure supplement 1). As the LCVs matured over the course of 1 hr post infection (p.i.), the overall LCV-LD contact time gradually increased in D. discoideum Ax3, while it remained lower in Δsey1 mutant amoeba (Figure 2B). Moreover, the retention time of individual LDs on LCVs was also signficantly higher in strain Ax3 than in Δsey1 mutant amoeba (Figure 2C). Taken together, these real-time data indicate that Sey1 promotes the dynamics of LCV-LD interactions during the course of LCV maturation. Figure 2 with 1 supplement see all Download asset Open asset Sey1 promotes LDs recruitment to intact LCVs in D. discoideum. (A) Representative fluorescence micrographs of D. discoideum Ax3 or Δsey1 producing P4C-GFP (pWS034) and mCherry-Plin (pHK102), fed overnight with 200 µM sodium palmitate, stained with LipidTOX Deep Red and infected (MOI 5) with mCerulean-producing L. pneumophila JR32 (pNP99). Infected cells were recorded for 60 s each at the times indicated. Examples are shown for contact between LDs and the LCV membrane (white arrowheads). Scale bars: 0.5 µm. (B) Quantification of (A), total contact time of LDs with the LCV recorded for 60 s at the indicated time points p.i. (nLCV-LD contacts > 30). Data represent means ± SD of three independent experiments (*p<0.05). (C) Quantification of (A), retention time of single LDs with the LCV recorded for 60 s at the indicated time points p.i. (nLCV-LD contacts > 30). Data represent means ± SD of three independent experiments (*p<0.05, **p<0.01; ***p<0.001). (D) Representative fluorescence micrographs of D. discoideum Ax3 or Δsey1 producing P4C-GFP (pWS034) and cytosolic mCherry (pDM1042), fed overnight with 200 µM sodium palmitate and infected (MOI 10, 1 hr) with mCerulean-producing L. pneumophila JR32 (pNP99), fixed with PFA and stained with LipidTOX Deep Red. Examples are shown for contact between LDs and the LCV membrane (white arrowheads). Scale bars: overview (2 µm), inset (1 µm). (E) Quantification of (D), mean number of LDs contacting a single LCV (nLCVs >102). Data represent means ± SD of three independent experiments (***p<0.001). To assess the integrity of the LCVs during their interaction with LDs, we used D. discoideum Ax3 or Δsey1 producing P4C-GFP and cytoplasmic mCherry (Figure 2D). The production of cytoplasmic mCherry allows to assess the integrity of pathogen vacuoles in the course of D. discoideum infection (Koliwer-Brandl et al., 2019). The D. discoideum strains were fed overnight with 200 µM palmitate, infected with mCerulean-producing L. pneumophila JR32, and LDs were stained with LipidTOX Deep Red. This approach revealed that 1 hr post infection, all LCVs formed in either D. discoideum Ax3 or Δsey1 mutant amoeba were impermeable to cytoplasmic mCherry, and therefore, LCV membrane integrity was not compromised (Figure 2D). In addition, these experiments confirmed that LCVs in D. discoideum Ax3 are decorated with approximately twice as many LDs as LCVs in the Δsey1 strain (Figure 2E). The L. pneumophila T4SS promotes Sey1-dependent LCV-LD interactions Next, we sought to validate that Sey1 promotes LCV-LD interactions in palmitate-fed, fixed D. discoideum and to test if the process also depends on the L. pneumophila Icm/Dot T4SS. To this end, we used D. discoideum producing mCherry-Plin and AmtA-GFP, a probe localizing to vacuoles containing either wild-type strain JR32 or ΔicmT mutant bacteria (Figure 3A). This approach indicated that the mean number of LDs localizing to LCVs harboring strain JR32 was more than twice as high in D. discoideum Ax3 as compared to the Δsey1 mutant amoeba, and the effect was of similar magnitude, when the number of LDs per LCV area was calculated (Figure 3B). Contrarily, Sey1 did not promote the interaction of vacuoles harboring ΔicmT mutant bacteria with LDs, and overall, significantly fewer LDs associated with these vacuoles (Figure 3B). Taken together, these studies using fixed D. discoideum amoeba reveal that Sey1 promotes LCV-LD interactions and the Icm/Dot T4SS is required for LD accumulation on LCVs. Figure 3 with 1 supplement see all Download asset Open asset The L. pneumophila T4SS promotes Sey1-dependent LCV-LD interactions. (A) Representative fluorescence micrographs of D. discoideum Ax3 or Δsey1 producing AmtA-GFP (pHK121) and mCherry-Plin (pHK102), fed overnight with 200 µM sodium palmitate and infected (MOI 10, 1 hr) with mCerulean-producing L. pneumophila JR32 (top) or ΔicmT (bottom) (pNP99), fixed with PFA and stained with LipidTOX Deep Red. Examples are shown for contact between LDs and the LCV membrane (white arrowheads) or no contact (red arrowhead). Scale bars: overview (2 µm), inset (1 µm). (B) Quantification of (A), mean number of LDs contacting a single LCV (left) and ratio of LD number contacting one LCV divided by the LCV area (right) (nLCVs = 30). Data represent means ± SD of three independent experiments. (n.s., not significant; *p<0.05; **p<0.01). Atlastins regulate the number and size of LDs in mammalian cells (Klemm et al., 2013). Hence, we assessed the role of Sey1 for the number and size of LDs in D. discoideum. D. discoideum producing the LD marker mCherry-Plin and the phagosome marker AmtA-GFP were left untreated or fed with 200 µM palmitate overnight, fixed and stained with LipidTOX Deep Red, and the number and size of LDs were quantified. D. discoideum Ax3 or ∆sey1 amoeba were found to harbor approximately 10 LDs per cell, regardless of whether they were uninfected or infected with L. pneumophila wild-type JR32 or ∆icmT (Figure 3—figure supplement 1). Palmitate feeding did not significantly change the size of the LDs. Upon feeding D. discoideum with 200 µM palmitate overnight, uninfected or ∆icmT-infected amoeba contained significantly more LDs (ca. eightfold), and amoeba infected with L. pneumophila JR32 contained only ca. fourfold more LDs (Figure 3—figure supplement 1). Taken together, feeding with palmitate increased the number but not the size of LDs in D. discoideum, and infection with wild-type L. pneumophila reduced the LDs number compared to uninfected or ∆icmT-infected amoeba. However, in apparent contrast to mammalian cells, Sey1 did not seem to affect the number and size of LDs in D. discoideum. We also quantified the ratio of LDs per LCV and LDs per LCV area in D. discoideum Ax3 or ∆sey1 mutant amoeba, which were unstimulated or fed with 200 µM palmitate overnight (Figure 3—figure supplement 1). In unstimulated as well as in palmitate-fed D. discoideum, the number of LDs per LCV and the number of LDs per LCV area was significantly larger in the parental D. discoideum strain Ax3 as compared to Δsey1 mutant amoeba. Therefore, feeding with palmitate does not affect the stimulation of LCV-LD interactions by Sey1. However, in agreement with an increased overall number of LDs per cell, the overall number of LDs per LCV or LDs per LCV area increased upon feeding the amoeba with palmitate (Figure 3—figure supplement 1). Taken together, these results indicate that while feeding D. discoideum with palmitate increases the total number of LDs in amoeba and on LCVs, palmitate feeding does not change the positive effect of Sey1 on LCV-LD interactions also seen in unstimulated amoeba. Proteomics analysis of purified LDs identifies RanA GTPase and RanBP1 To gain further insights into the possible role of Sey1 for LD composition, we performed a comparative proteomics analysis of LDs from D. discoideum. To this end, LDs were harvested from palmitate-fed mCherry-Plin-producing D. discoideum Ax3 or Δsey1 mutant amoeba, purified by sucrose gradient centrifugation (Figure 4—figure supplement 1), and subjected to tandem mass spectrometry. This approach revealed 144 differentially produced proteins (log2 fold change > |0.8|), including some enzymes implicated in lipid metabolism (phospholipase PldA, phosphatidylinositol phosphate kinase Pik6/PIPkinA, sterol methyl transferase SmtA, acetoacetyl-CoA hydrolase) (Supplementary file 1, Figure 4—figure supplement 1). Among the differentially produced proteins, 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or Δsey1, respectively. Sey1 was identified on LDs isolated from strain Ax3, but as expected not on LDs isolated from Δsey1 mutant amoeba. Contrarily, the phospholipase PldA, the ER protein calnexin (CnxA) and the protein SCFD1/SLY1 implicated in ER to Golgi transport were identified only on LDs isolated from the Δsey1 strain (Supplementary file 1). The 50 most highly abundant proteins, which were not significantly different on LDs isolated from Ax3 or Δsey1 mutant amoeba, included perilipin (Plin, PlnA), which is involved in the formation and maintenance of LDs (Du et al., 2013), as well as – to our surprise – the small GTPase RanA (Du et al., 2013) and its effector RanBP1 (Supplementary file 1). Intriguingly, RanA is activated in L. pneumophila-infected cells and implicated in microtubule stabilization and LCV motility (Rothmeier et al., 2013; Swart et al., 2020c). To assess the localization of Sey1 with regard to LDs, we used D. discoideum Ax3 producing GFP-Sey1 as well as the LD marker mCherry-Plin, and further stained LDs with LipidTOX Deep Red (Figure 4—figure supplement 1). Under the conditions used, GFP-Sey1 accumulated in the vicinity of LDs in intact cells as well as in cell homogenates, but apparently did not co-localize with LDs. This staining pattern suggests that Sey1 localizes only in very low amounts to LDs, or that localization of Sey1 to LDs is impaired due to the fluorescent protein tag. To assess the localization of RanA and RanBP1 to LDs, we used D. discoideum Ax3 producing either RanA-mCherry or RanBP1-GFP and GFP-Plin or mCherry-Plin and further stained LDs with LipidTOX Deep Red (Figure 4—figure supplement 1). Under these conditions, ectopically produced RanA-mCherry or RanBP1-GFP localized to membranous structures in the cell, including to Plin- and LipidTOX Deep Red-positive LDs. In summary, comparative proteomics of LDs isolated from D. discoideum Ax3 or Δsey1 revealed that the phospholipase PldA is present exclusively in the mutant amoeba, and Plin, RanA as well as RanBP1 are detected in LDs from both D. discoideum strains. L. pneumophila LegG1 promotes Sey1-dependent LCV-LD interactions Next, we sought to identify L. pneumophila effector proteins, which possibly determine LCV-LD interactions. The RCC1 repeat domain effector LegG1 activates the small GTPase RanA, which in its active, GTP-bound form interacts with RanBP1 and promotes microtubule stabilization (Rothmeier et al., 2013; Swart et al., 2020c). Since we found that LDs harbor RanA and RanBP1 (Supplementary file 1), we tested the hypothesis that LegG1 is implicated in LCV-LD dynamics. To this end, we infected palmitate-fed D. discoideum Ax3 or Δsey1 producing P4C-GFP and mCherry-Plin with mCerulean-producing L. pneumophila JR32 or ΔlegG1 and additionally stained LDs with LipidTOX Deep Red (Figure 4A, Videos 1–4). At 1 hr or 2 hr p.i., the overall LCV-LD contact time was lowered by ca. 50% upon infection with ΔlegG1 (compared to JR32) or in Δsey1 mutant D. discoideum (compared to strain Ax3) (Figure 4B). Intriguingly, the overall LCV-LD contact time was further significantly reduced upon infection of Δsey1 mutant amoeba with ΔlegG1 mutant bacteria (Figure 4B). Similar results were obtained by quantifying the retention time of individual LDs on LCVs (Figure 4B). The defects of the ΔlegG1 mutant strain regarding the duration of LCV-LD contacts were complemented by providing the legG1 gene on a plasmid (Figure 4C). In summary, these results indicate that the host large GTPase Sey1, as well as the L. pneumophila Ran GTPase activator LegG1 promote and additively affect the dynamics of LCV-LD interactions. Figure 4 with 2 supplements see all Download asset Open asset L. pneumophila LegG1 promotes Sey1-dependent LCV-LD interactions. (A) Representative fluorescence micrographs of D. discoideum Ax3 or Δsey1 producing P4C-GFP (pWS034) and mCherry-Plin (pHK102), fed overnight with 200 µM sodium palmitate, stained with LipidTOX Deep Red and infected (MOI 5) with L. pneumophila JR32 or ΔlegG1 producing mCerulean (pNP99). Infected cells were recorded for 60 s each at the times indicated. Examples are shown for contact between LDs and the LCV membrane (white arrowheads) or no contact (red arrowheads). Scale bars: 0.5 µm. (B) Quantification of (A), total contact time of LDs with the LCV (left) or retention time of single LDs with the LCV (right) recorded for 60 s at the indicated time points p.i. (nLCV-LD contacts > 30). Data represent means ± SD of three independent experiments (n.s., not significant; *p<0.05; ***p<0.001). (C) Quantification of total contact time of LDs with the LCV (left) or retention time of single LDs with the LCV (right) recorded for 60 s at 120 min p.i. (nLCV-LD contacts > 30). D. discoideum Ax3 or Δsey1 producing P4C-GFP (pWS034) and mCherry-Plin (pHK102), fed overnight with 200 µM sodium palmitate, stained with LipidTOX Deep Red and infected (MOI 5) with L. pneumophila JR32 or ΔlegG1 producing DsRed (pSW001), or ΔlegG1 producing DsRed and M45-LegG1 (pER005; ΔlegG1::legG1) were analysed. Data represent means ± SD of three independent experiments (n.s., not significant; *p<0.05; **p<0.01; ***p<0.001). (D) Representative fluorescence micrographs of D. discoideum Ax3 or Δsey1 producing GFP-tubulin A (pLS110) and P4C-mCherry (pWS032), fed overnight with 200 µM sodium palmitate and infected (MOI 10, 1 hr) with L. pneumophila JR32, ΔicmT or ΔlegG1 producing DsRed (pSW001), or ΔlegG1 producing DsRed and M45-LegG1 (pER005; ΔlegG1::legG1), fixed with PFA and stained with LipidTOX Deep Red. Scale bars: 2 µm. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg D. discoideum Ax3 infected with L. pneumophila JR32. Representative movie of D. discoideum Ax3 producing P4C-GFP (pWS034) and mCherry-Plin (pHK102), fed overnight with 200 µM sodium palmitate, stained with LipidTOX Deep Red and infected (MOI 5) with mCerulean-producing L. pneumophila JR32 (pNP99). Infected cells were recorded for 60 s each at the times indicated. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg D. discoideum Ax3 infected with L. pneumophila ΔlegG1. Representative movie of D. discoideum Ax3 producing P4C-GFP (pWS034) and mCherry-Plin (pHK102), fed overnight with 200 µM sodium palmitate, stained with LipidTOX Deep Red and infected (MOI 5) with mCerulean-producing L. pneumophila ΔlegG1 (pNP99). Infected cells were recorded for 60 s each at the times indicated. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg D. discoideum Δsey1 infected with L. pneumophila JR32. Representative movie of D. discoideum Δsey1 producing P4C-GFP (pWS034) and mCherry-Plin (pHK102), fed overnigh