The small GTPase Ran and the Icm/Dot substrate LegG1 localize to LCVs

The small GTPase Ran and its effector RanBP1 have been identified on purified LCVs by proteomic analysis [7], [39]. To directly investigate the presence of these proteins on LCVs by fluorescence microscopy, Dictyostelium discoideum producing the corresponding GFP fusion proteins was infected with red fluorescent L. pneumophila. Ran was found to localize to the LCV membrane in D. discoideum infected with L. pneumophila wild-type or ΔlegG1 but not with ΔicmT mutant bacteria (Figure 1A). Moreover, RanBP1 localized to LCVs harboring wild-type L. pneumophila (see below). These results confirm the proteomic data and show that Ran and RanBP1 localize to LCVs in an Icm/Dot-dependent manner.

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Figure 1. The small GTPase Ran and the Icm/Dot substrate LegG1 localize to LCVs.

(A) Ran accumulates on LCVs. D. discoideum producing RanA-GFP was infected (MOI 50, 1 h) with DsRed-producing L. pneumophila wild-type, ΔlegG1 or ΔicmT harboring pSW001 and immuno-stained for the LCV membrane marker SidC. LCVs in lysates of infected cells are shown. (B) Depletion of Ran or RanBP1 inhibits intracellular growth of L. pneumophila. A549 lung epithelial carcinoma cells were treated with AllStars siRNA (negative control) or with siRNA oligonucleotides targeting Ran, RanBP1 or Arf1 (positive control) for 2 d, and intracellular replication of GFP-producing L. pneumophila harboring pNT28 was quantified by fluorescence measurements after 24 h. Data represent mean and standard deviation of three independent experiments considering the 3 most effective out of 4 different oligonucleotides (Fig. S1B). Student's t-test; ***, p<0.001. (C) Icm/Dot-dependent localization of M45-LegG1 on LCVs in cell homogenates. D. discoideum producing calnexin-GFP was infected (MOI 50) with L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pSU19 (M45-LegG1) or with ΔlegG1 harboring pCR033 (vector), homogenized and immuno-stained with an anti-M45 antibody and with DAPI. The percentage of M45-LegG1-positive LCVs (n = 100/strain, 3 independent experiments) was scored in lysates of infected cells (*, p<0.05). Bars (A, C), 0.5 µm.

https://doi.org/10.1371/journal.ppat.1003598.g001

To assess whether Ran or RanBP1 play a role for intracellular replication of L. pneumophila we depleted the proteins by RNA interference. To this end, A549 lung epithelial cells were treated with siRNA oligonucleotides targeting Ran, RanBP1 or, as a positive control, Arf1, and intracellular replication of L. pneumophila was monitored over 24 h (Figure 1B). Upon depletion of either Ran or RanBP1 the number of intracellular L. pneumophila was reduced two-fold, indicating that the small GTPase as well as its effector RanBP1 are required for efficient intracellular growth of L. pneumophila. As expected, the depletion of Arf1 also reduced intracellular growth of L. pneumophila, albeit less efficiently than depletion of Ran or RanBP1. The treatment with siRNA oligonucleotides efficiently depleted Ran or RanBP1, yet had no effect on A549 cell viability (Figure S1). Therefore, the depletion of Ran or RanBP1 impedes the intracellular replication of L. pneumophila without dramatically affecting the host cell physiology.

The legG1 gene is conserved among the L. pneumophila strains sequenced to date (Philadelphia-1, Paris, Lens, Corby, Alcoy, 130b/AA100, Lorraine, HL06041035), but apparently not present in other Legionella spp. LegG1 is translocated by the Icm/Dot T4SS as a TEM-β-lactamase fusion protein into J774 or RAW264.7 macrophages ([36]; Figure S2), or as an adenylate cyclase fusion protein into CHO cells [37]. Upon infection of D. discoideum producing the ER/LCV marker calnexin-GFP with red fluorescent L. pneumophila producing M45-tagged LegG1, the effector protein accumulated in an Icm/Dot-dependent manner on the LCV membrane (Figure 1C). Under these conditions, approximately 60–70% of the LCVs scored positive for M45-LegG1. In summary, Ran, RanBP1 and LegG1 all accumulate on the LCV membrane in an Icm/Dot-dependent manner.

L. pneumophila wild-type but not ΔlegG1 activates Ran on LCVs

To analyze the function of LegG1 genetically, an L. pneumophila strain lacking legG1 (ΔlegG1, Table S1) was constructed by deleting the gene from the chromosome by double homologous recombination. LCVs harboring ΔlegG1 mutant bacteria stained for the GTPase Ran faintly but to the same extent as wild-type L. pneumophila (Figure 1A), suggesting that LegG1 is dispensable for the recruitment of the small GTPase to the pathogen vacuole. In contrast, however, significantly fewer LCVs containing ΔlegG1 acquired detectable levels of the Ran effector RanBP1 compared to wild-type L. pneumophila (Figure 2A). Less than 50% of LCVs containing ΔlegG1 stained positive for RanBP1 compared to wild-type LCVs, and the phenotype of the ΔlegG1 mutant strain was fully complemented by expressing plasmid-encoded M45-legG1. This finding indicates that LegG1 promotes the accumulation of RanBP1 on LCVs and thus activates Ran on LCV membranes.

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Figure 2. L. pneumophila wild-type but not ΔlegG1 activates Ran on LCVs.

(A) LegG1 promotes RanBP1 accumulation on LCVs. D. discoideum producing RanBP1-GFP (green) was infected (MOI 50, 1 h) with DsRed-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR077 (red), or with ΔlegG1/pER5 (M45-LegG1) and immuno-stained for SidC (blue). The percentage of RanBP1-GFP-positive LCVs (n = 100/strain, 5 independent experiments) was scored in lysates of infected cells (**, p<0.01; ***, p<0.001). (B) Production of Ran(GTP) in infected macrophages. RAW264.7 macrophages were infected (MOI 25, 1 h) with L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR033 (vector) or with ΔlegG1/pSU19 (M45-LegG1). The infected macrophages were lysed, activated Ran was immuno-precipitated with an antibody specifically recognizing Ran(GTP) and visualized by Western blot using an anti-Ran antibody. Lysates of uninfected cells incubated with GTP or GDP in presence of EDTA were used as positive or negative controls for endogenous GEF activity. Loading control: Western blot of Ran in samples before immuno-precipitation. (C) Production of Ran(GTP) in cell lysates. A549 epithelial cells were lysed and incubated with purified His₆-LegG1 (native or heat-inactivated, h. i.) in presence of excess GTP (100 µM) or GDP (1 mM). Activated Ran was immuno-precipitated with an antibody specifically recognizing Ran(GTP) and visualized by Western blot using an anti-Ran antibody. Loading control: Western blot of Ran in samples before immuno-precipitation. The relative amount of Ran(GTP) was determined by densitometry; means and standard deviations of 4 (B, C) independent experiments are shown (*, p<0.05; **, p<0.01).

https://doi.org/10.1371/journal.ppat.1003598.g002

To determine more directly whether L. pneumophila activates Ran and whether LegG1 plays a role in this process, we assayed the production of activated Ran in pulldown experiments using an antibody specifically recognizing Ran(GTP), as detailed in the Materials and Methods section. To this end, RAW264.7 macrophages were infected with L. pneumophila wild-type, ΔicmT or ΔlegG1, or with ΔlegG1 expressing M45-legG1. The infected macrophages were lysed and Ran(GTP) was immuno-precipitated and visualized by Western blot using an anti-Ran antibody. Ran(GTP) was detected following infection with L. pneumophila wild-type but not with ΔicmT or ΔlegG1, and the phenotype of the ΔlegG1 mutant strain was complemented by providing legG1 on a plasmid (Figure 2B). Therefore, LegG1 is required to catalyze the activation of Ran by L. pneumophila.

A similar pulldown experiment was performed by adding purified His₆-LegG1 to lysates of A549 cells (Figure 2C). Under these conditions, Ran(GTP) was produced in cell lysates upon addition of native (but not heat-inactivated) LegG1 in presence of GTP (but not GDP). Moreover, purified LegG1-His₆ but not the mutant protein LegG1_N223A-His₆ also activated Ran in cell lysates (Fig. S3C).

Using purified N- or C-terminally His-tagged LegG1 fusion constructs, we also attempted to directly measure Ran GEF activity in vitro with fluorescent mantGDP (2′/3′-O-(N-methyl-anthraniloyl)-guanosine-5′-diphosphate). As a positive control, the human Ran GEF RCC1 significantly stimulated mantGDP-release from Ran(mantGDP) in the presence of excess GTP as indicated by a rapid decrease in mantGDP-fluorescence. However, under various conditions tested the purified LegG1 fusion constructs did not show Ran GEF activity in vitro even at elevated concentrations (Fig. S3; data not shown). In summary, these results demonstrate that L. pneumophila activates Ran in infected protozoan and mammalian host cells by means of a translocated effector and that LegG1 is required to activate Ran.

LegG1 promotes intracellular replication of L. pneumophila

L. pneumophila lacking legG1 grew in broth at the same rate as the isogenic wild-type strain (data not shown). Yet, the ΔlegG1 strain was slightly impaired for intracellular replication in RAW264.7 macrophages (Figure 3A) and Acanthamoeba castellanii (Figure S4A), but not in D. discoideum (Figure S4B). Upon co-infection of ΔlegG1 with wild-type L. pneumophila at a 1∶1 ratio, the mutant strain was efficiently out-competed by wild-type bacteria and eradicated within 6 days (Figure 3B). Thus the Ran activator LegG1 is essential for competition against wild-type L. pneumophila upon co-infection of amoebae. In contrast, the Rab1 GEF SidM was not required for competition under the same conditions, since a mutant strain lacking sidM did not show a competition defect (Figure S5).

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Figure 3. LegG1 promotes intracellular replication of L. pneumophila.

(A) Intracellular replication in RAW264.7 macrophages infected (MOI 0.1) with L. pneumophila wild-type strain JR32, ΔicmT or ΔlegG1. The infected cells were lysed and CFU were determined. Data shows means and standard deviations of triplicates and is representative of three independent experiments (*, p<0.05; ***, p<0.001). (B) For competition assays A. castellanii amoebae were co-infected with wild-type L. pneumophila and the ΔlegG1 mutant strain at a 1∶1 ratio (MOI of 0.01 each) and grown at 37°C during 21 d. Every third day the supernatant and lysed amoebae were diluted 1∶5000, fresh amoebae were infected, and CFU determined on CYE agar plates containing kanamycin or not. The data shown are means and standard deviations of triplicates and representative of 3 independent experiments. For toxicity assays RAW264.7 macrophages were infected (MOI 10, 4 h) with L. pneumophila wild-type, ΔicmT, or ΔlegG1 harboring pCR033, or with ΔlegG1/pSU19 (M45-LegG1), and (C) analyzed by transmission electron microscopy (TEM) after embedding the sample in epoxy resin, or (D) analyzed by flow cytometry after detaching the cells by scraping and staining with the membrane integrity marker propidium iodide (1 µg/ml). (E) For TEM analysis of Golgi stacks, RAW264.7 macrophages were infected with L. pneumophila wild-type, ΔicmT or ΔlegG1 (MOI 10) and embedded in epoxy resin. The total length of Golgi cisternae and the ratio of the total number of Golgi cisternae relative to the total cytoplasmic area were quantified by stereology in thin sections. Bars, 1 µm. (F) Single round replication assay in RAW264.7 macrophages infected (MOI 20) with GFP-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pNT28, in presence or absence of 10 µM nocodazole. Means and standard deviations of 3 samples per strain, each analyzed in triplicate, from a single experiment is shown. Data are representative of 3 independent experiments.

https://doi.org/10.1371/journal.ppat.1003598.g003

The ΔlegG1 mutant strain was as cytotoxic for RAW264.7 macrophages as wild-type bacteria, since macrophages infected with either strain were vacuolized to the same extent (Figure 3C), and the percentage of cells permeable for propidium iodide was similar (Figure 3D). However, the overproduction of LegG1 reduced cytotoxicity significantly. The reduction of cytotoxicity was specific for LegG1, as overproduction of other effector proteins such as SidC or SidM significantly increased cytotoxicity (Figure S6A). Cytotoxicity reduction by LegG1 did not seem to be due to an impairment of type IV secretion, since translocation of the Icm/Dot substrate SidC was not affected (Figure S6B). Furthermore, L. pneumophila lacking or overexpressing legG1 was taken up with the same efficiency as wild-type bacteria by amoebae (Figure S7). The uptake of L. pneumophila is promoted by the Icm/Dot T4SS [40], and therefore, this finding also indicates that the overproduction of LegG1 does not simply obstruct the T4SS.

To test further effects of LegG1 on host cells, we assessed the fragmentation of the Golgi apparatus by L. pneumophila by using transmission electron microscopy (Fig. 3E). Upon infection of RAW264.7 macrophages with wild-type L. pneumophila the Golgi cisternae were almost completely disrupted, while in cells infected with a ΔicmT mutant strain the Golgi was preserved to an extent similar to uninfected cells. In macrophages infected with L. pneumophila ΔlegG1 the Golgi cisternae were conserved to an intermediate degree and appeared shorter than in uninfected cells.

Finally, in absence of legG1, the same number of LCVs accumulated the ER/LCV marker calnexin, suggesting that LegG1 does not affect the fusion of the pathogen vacuole with the ER (Figure S7C). Together, these results indicate that LegG1 is an Icm/Dot-translocated L. pneumophila virulence factor that localizes to the LCV membrane, is dispensable for bacterial uptake, and promotes intracellular replication in protozoan and mammalian phagocytes.

LegG1 stabilizes microtubules in L. pneumophila-infected phagocytes

Ran controls a number of cellular processes, some of which involve microtubule assembly and microtubule-dependent trafficking processes [11], [13]. To study a possible role of microtubule polymerization and LegG1 for intracellular replication of L. pneumophila, macrophages were treated with the microtubule-depolymerizing agent nocodazole, and the fate of GFP-producing wild-type, ΔicmT or ΔlegG1 mutant bacteria was monitored (Figure 3F). The growth rate of wild-type L. pneumophila in nocodazole-treated cells was somewhat lower compared to control cells treated with DMSO only. However, the difference in the growth rate of the ΔlegG1 strain in nocodazole-treated cells compared to control cells was much more pronounced, indicating that in the absence of the Ran activator LegG1 the depolymerization of microtubules is more deleterious for intracellular bacterial growth. ΔicmT mutant bacteria were unable to grow and were killed to the same extent in macrophages treated with nocodazole. These results suggest that the depolymerization of microtubules by nocodazole and the absence of the Ran activator LegG1 synergistically compromise intracellular growth of L. pneumophila.

Next, we used confocal laser scanning fluorescence microscopy to test whether Ran activation by LegG1 promotes microtubule polymerization in L. pneumophila-infected phagocytes. For this purpose, a D. discoideum strain producing GFP-α-tubulin was infected with red fluorescent L. pneumophila wild-type, ΔicmT, ΔlegG1 or ΔlegG1 expressing M45-legG1 (Figure 4A). In amoebae infected with wild-type L. pneumophila microtubules were polymerized to a greater extent compared with cells infected with the ΔlegG1 or ΔicmT mutant strains. More than 50% of the cells infected with ΔlegG1 showed a less dense microtubule network, and the phenotype was complemented by the legG1 gene. In D. discoideum infected with the complemented strain, the majority of microtubules emanated from the centrosome and reached the cell cortex, similar to amoebae infected with wild-type L. pneumophila.

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Figure 4. LegG1 stabilizes microtubules in L. pneumophila-infected phagocytes.

(A, B) Microtubules were analyzed by confocal laser scanning fluorescence microscopy in (A) D. discoideum producing tubulin-GFP or (B) RAW264.7 macrophages infected (MOI 10, 2 h (amoebae), 4 h (macrophages)) with DsRed-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR077 or with ΔlegG1/pER5 (M45-LegG1). The macrophages were immuno-labeled for α-tubulin (green) and SidC (blue) and, as controls, treated with taxol or nocodazole (30 µM). (C, D) Microtubules were analyzed by STED microscopy in RAW264.7 macrophages infected (MOI 10, 4 h) with GFP-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR076 or with ΔlegG1/pER4 (M45-LegG1) and immuno-labeled for (C) α-tubulin (grey), or (D) α-tubulin (green) and SidC (blue). Uninfected macrophages were treated with taxol or nocodazole (30 µM) as control. Bars, 1 µm (A), 5 µm (B, C), 0.5 µm (D). (E) Microtubule polymerization was analyzed by anti-tubulin Western blot in RAW264.7 macrophages infected (MOI 10, 4 h) with GFP-producing L. pneumophila wild-type, ΔicmT or ΔlegG1 harboring pCR033, or with ΔlegG1/pSU19 (M45-LegG1). As a control, uninfected macrophages were treated with taxol or nocodazole (30 µM). Homogenates of the macrophages were centrifuged (20'000×g, 30 min) to separate polymerized tubulin in the pellet (P) from soluble tubulin in the supernatant (S), and the amount of microtubule polymerization was assessed by α-tubulin Western blot (upper panel). Actin was used as a control. A representative experiment is shown, and the ratio of polymerized to soluble α-tubulin is indicated (R). The graph (lower panel) shows means and standard deviations of 3 independent experiments.

https://doi.org/10.1371/journal.ppat.1003598.g004

Similarly, in RAW264.7 macrophages infected with red fluorescent L. pneumophila wild-type, microtubules were polymerized to a greater extent, compared to cells infected with the ΔlegG1 mutant strain (Figure 4B). Approximately 50% of the cells infected with ΔlegG1 showed a less dense microtubule network, and again the phenotype was complemented upon providing the legG1 gene on a plasmid. In macrophages infected with ΔicmT, microtubules were polymerized to a similar extent as in cells infected with wild-type L. pneumophila. Uninfected macrophages treated with taxol or nocodazole served as controls for microtubule polymerization or depolymerization, respectively.

The effect of the above L. pneumophila strains on microtubule polymerization in macrophages was further analyzed by stimulated emission depletion (STED) microscopy. This super-resolution immuno-fluorescence microscopy analysis confirmed that microtubules were polymerized to a greater extent in macrophages infected with green-fluorescent wild-type L. pneumophila or ΔicmT, compared to cells infected with ΔlegG1, and the phenotype was complemented by providing the legG1 gene on a plasmid (Figure 4C). Moreover, a high magnification inspection of the microtubule network in the vicinity of LCVs revealed an α-tubulin accumulation on 11% or 49% of LCVs harboring either wild-type L. pneumophila or the complemented ΔlegG1 strain, but not on LCVs harboring ΔicmT or ΔlegG1 mutant bacteria (Figure 4C, inset; Figure 4D).

The amount of polymerized or non-polymerized microtubules in L. pneumophila-infected macrophages was also assessed by Western blot using an anti-tubulin antibody (Figure 4E). Lysates of infected macrophages were subjected to a low and a high speed centrifugation step, and the quantity of microtubules in the pellet and the supernatant was compared. This approach revealed that macrophages infected with L. pneumophila lacking legG1 contained smaller amounts of polymerized microtubules compared to cells infected with wild-type bacteria, and the phenotype was complemented by overexpression of legG1. In summary, different approaches revealed that L. pneumophila promotes microtubule polymerization in amoebae and macrophages in a LegG1-dependent manner to efficiently replicate intracellularly.

Microbial microinjection of LegG1 promotes microtubule polymerization

As an alternative approach to analyze the effect of a single effector protein, LegG1, on host cells, we delivered the effector into host cells by microbial microinjection using the Yersinia enterocolitica strain WA (pT3SS). This Yersinia “toolbox” strain produces the Ysc type III secretion system (T3SS), yet lacks all endogenous T3SS effectors [41], [42]. N-terminal fragments of the Y. enterocolitica RhoG/Rac1 GAP YopE (YopE_1–53, YopE_1–138) are not cytotoxic but mediate secretion and translocation of hybrid proteins through the T3SS. Fusions of YopE_1–53 or YopE_1–138 with LegG1 or SidM were produced and secreted into the bacterial supernatant via the T3SS upon calcium depletion with 5 mM EGTA (Figure 5A). Moreover, dependent on the T3SS and sensitive to the protonophore CCCP (carbonyl cyanide m-chlorophenyl-hydrazone) heterologously produced YopE_1–53-LegG1 was translocated into HeLa cells by Y. enterocolitica WA (pT3SS) (Figure 5B).

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Figure 5. Microbial microinjection of LegG1 promotes microtubule polymerization.

(A) Yersinia enterocolitica strain WA (pT3SS), encoding the Ysc T3SS but lacking type III-secreted effectors, produced and secreted fusion proteins of YopE_1–53 or YopE_1–138 with the Legionella Ran activator LegG1 or the Rab1 GEF SidM. The proteins in the pellet (P) or supernatant (S) were precipitated by chloroform/methanol treatment and visualized by Western blot using an antibody against YopE. (B) HeLa cells were infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE_1–53-LegG1, washed several times and lysed with 1% digitonin. After centrifugation, proteins in the pellet (intact bacteria, debris) and in the supernatant (translocated bacterial/soluble host proteins) were precipitated, and the fusion protein was visualized by Western blot using an anti-YopE antibody. Controls: HeLa cells alone, bacteria treated with 1% digitonin, 1% SDS or with the T3SS inhibitor CCCP (50 µM). (C) Fluorescence microscopy of YopE_1–138-LegG1 translocation into HeLa cells infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE_1–138-LegG1, YopE_1–138-SidM or YopE. Controls: uninfected cells or cells treated with 30 µM taxol or nocodazole. The cells were immuno-stained for α-tubulin and YopE, and nuclei were labeled with DAPI. Bars, 10 µm. (D) Fluorescence microscopy of YopE_1–53-LegG1 translocation into HeLa cells treated with nocodazole (1 µM, 1 h) and infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1. The cells were immuno-stained for α-tubulin (green) and YopE (red), nuclei were labeled with DAPI (grey). Bars, 40 µm or 10 µm (insets). (E) Western blot of YopE_1–53-LegG1 translocation into HeLa cells treated with nocodazole (1 µM, 1 h) and infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1. The soluble microtubule fraction in the cell supernatant (S) and the pellet (P) was analyzed with an anti-α-tubulin antibody; ratio of polymerized to soluble α-tubulin (R). (F) Fluorescence microscopy of A549 cells treated or not with siRNA against Ran and infected (MOI 10, 2 h) with Y. enterocolitica WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1. The cells were immuno-stained for α-tubulin (green) and YopE (red), and nuclei were labeled with DAPI (grey). Bars, 10 µm or 5 µm (insets). The data shown is representative of 3 independent experiments (A–F).

https://doi.org/10.1371/journal.ppat.1003598.g005

To analyze the translocation of heterologously produced effector fusion proteins, HeLa cells were infected for 2 h with Y. enterocolitica WA (pT3SS) producing YopE_1–138-LegG1, YopE_1–138-SidM or YopE, and the morphology and microtubules were analyzed by immuno-fluorescence microscopy (Figure 5C). Uninfected cells or cells infected with WA (pT3SS) producing YopE_1–138-LegG1 showed a similar morphology and microtubule network. In contrast, cells infected with WA (pT3SS) producing YopE_1–138-SidM or full length Y. enterocolitica YopE, a RhoG/Rac1 GAP [43], rounded up and the microtubules disintegrated. Uninfected HeLa cells treated with 30 µM taxol or nocodazole served as controls.

In order to visualize more subtle effects of LegG1 on the microtubule network, HeLa cells were pretreated for 1 h with 1 µM nocodazole prior to infection for 2 h with Y. enterocolitica WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1 (Figure 5D). Immuno-fluorescence microscopy indicated that under these conditions, cells infected with Y. enterocolitica producing YopE_1–53-LegG1 contained a denser microtubule network, compared with cells infected with bacteria producing YopE_1–53 or uninfected cells. Throughout the cell body, the injection of YopE_1–53-LegG1 caused the formation of a larger number of microtubule bundles, which emanated from the peri-nuclear region and radiated towards the cell cortex.

Microtubule polymerization triggered by Y. enterocolitica WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1 was also quantified by Western blot using an anti-α-tubulin antibody (Figure 5E). This approach confirmed that LegG1 injected into HeLa cells significantly increased the amount of insoluble tubulin in the pellet and thus caused microtubule polymerization. Taken together, these results indicated that heterologously produced LegG1 delivered into eukaryotic cells via microbial microinjection promotes polymerization of microtubules.

Finally, to assess whether LegG1 requires the Ran GTPase to exert its effect on microtubule polymerization, A549 cells (Fig. S1) were treated with siRNA oligonucleotides silencing Ran for 2 days, followed by 1 µM nocodazole for 1 h and infection with Y. enterocolitica WA (pT3SS) producing YopE_1–53 or YopE_1–53-LegG1 for 2 h (Figure 5F). Under these conditions, LegG1 triggered microtubule condensation only in cells treated with AllStars negative control siRNA, but not in cells depleted for Ran. While 83% of the control cells infected with Y. enterocolitica producing YopE_1–53-LegG1 contained a dense tubulin network, only 45% of the Ran-depleted cells did so. This experiment indicates that Ran is essential for LegG1 to promote microtubule polymerization.

LCVs harboring L. pneumophila ΔlegG1 are stalled

Shortly after formation, LCVs rapidly move within D. discoideum cells, and the pathogen vacuoles are transported along microtubules [6]. The dynamics of LCVs harboring either L. pneumophila wild-type (Movie S1) or ΔlegG1 mutant bacteria (Movie S2) was assessed by real-time confocal laser scanning fluorescence microscopy using calnexin-GFP-producing D. discoideum and DsRed-producing L. pneumophila. Two hours post infection the LCV motility was recorded for 5 min with images taken every 15 s (Figure 6A). While LCVs harboring wild-type L. pneumophila were very motile and rapidly moved along microtubules, LCVs harboring ΔlegG1 mutant bacteria were drastically slowed down and hardly moved. The velocity of LCVs was quantified by tracking the migration distance of LCVs over time. These experiments revealed that LCVs harboring wild-type L. pneumophila moved with a speed of about 40 nm/s within the D. discoideum cells, while LCVs harboring ΔlegG1 mutant bacteria moved with a 3–4 times lower speed and were almost stalled (Figure 6B).

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Figure 6. LCVs harboring L. pneumophila ΔlegG1 show impaired motility.

(A) Real-time fluorescence microscopy of LCV motility in D. discoideum producing calnexin-GFP and infected (MOI 10, 2 h) with DsRed-producing L. pneumophila wild-type or ΔlegG1 mutant bacteria harboring pSW001. Two hours post infection, trafficking of LCVs was recorded by laser confocal scanning microscopy for 5 min with images taken every 15 s. Bars, 1 µm. (B) The velocity of LCVs was quantified by tracking the migration distance of LCVs over time (n = 50/strain; ***, p<0.001).

https://doi.org/10.1371/journal.ppat.1003598.g006

Bacteria, cells, growth conditions and infection

Bacteria and cells are listed in Table S1. L. pneumophila strains were grown for 3 days on CYE agar plates containing charcoal yeast extract, buffered with N-(2-acetamido)-2-amino-ethanesulfonic acid (ACES). Liquid cultures were inoculated in AYE medium at an OD₆₀₀ of 0.1 and grown at 37°C to an OD₆₀₀ of 3.0 (21–22 h). Chloramphenicol (Cam; 5 µg/ml) and IPTG (1 mM) were added when needed.

Murine RAW264.7, as well as human HeLa and A549 lung epithelial carcinoma cells were cultivated in RPMI 1640 medium amended with 10% heat-inactivated fetal bovine serum and 1% glutamine (all from Life Technology). D. discoideum strains (Table S1) were grown and transfected by electroporation as described [47], [49], and A. castellanii (ATCC 30234) was propagated as described [50].

The infection of phagocytes by L. pneumophila wild-type, ΔicmT or ΔlegG1 mutant strains producing GFP was analyzed as described using A. castellanii, D. discoideum or murine RAW264.7 macrophages as host cells [32], [47], [48], [50], [51]. Briefly, the phagocytes were infected with L. pneumophila grown for 21–22 h in AYE broth (MOI 1–50), the infection was synchronized by centrifugation (450×g, 10 min, RT), and the infected phagocytes were incubated at 37°C, 30°C or 25°C (D. discoideum) for the time indicated.

Chromosomal deletion of legG1 and plasmid construction

All plasmids and oligonucleotides used are listed in Table S1 or Table S2, respectively. DNA manipulations were performed according to standard protocols, and plasmids were isolated using commercially available kits from Macherey-Nagel. All PCR fragments were sequenced.

The chromosomal deletion of legG1 was performed as described [50]: 800 bp upstream and downstream fragments of legG1 (lpg1976) were amplified by PCR using the primer pairs oSU94/oSU95 and oSU96/oSU97, respectively, and chromosomal L. pneumophila DNA as a template. Both fragments were inserted by a four way ligation into a pGEM-T easy vector with a Kan^R cassette in-between using BamHI sites and adenosine overhangs, yielding plasmid pSU1. Clones were analyzed by restriction digestion and sequencing. The Kan^R cassette flanked by upstream and downstream fragments was transferred into the pLAW344 suicide plasmid using NotI, yielding plasmid pSU2. L. pneumophila JR32 was transformed by electroporation with pSU2 and selected for Kan^R/Suc^R and Cam^S colonies. Positive clones were tested by PCR, using the primers oSU94, oSU97, oKan3′ and oKan5′, and by sequencing.

Translational M45-fusion proteins of legG1 were constructed by PCR amplification using the primer pairs: oCR158/oCR160 or oER3/oER7 and chromosomal DNA of L. pneumophila JR32 as a template. The fragments were cut with the appropriate restriction enzymes and inserted into pMMB207-RBS-C-M45 (pCR33), pMMB207-C-RBS-gfp-RBS (pCR76), pMMB207-C-RBS-DsRed-RBS (pCR77), yielding the plasmids pSU19, pER4 and pER5, respectively. The plasmids pSU17 and pSU26 (encoding RanA-GFP or RanBP1-GFP) were constructed by PCR-amplification of the corresponding genes using the oligonucleotides listed in Table S2. D. discoideum cDNA was used as a template, cut with NsiI and inserted into pSW102 cut with the same restriction enzyme.

Plasmids encoding LegG1-His₆ (pER2) or His₆-LegG1 (pER3) were constructed using the primers oER4/oER6 or oCR158/oER05, respectively, and chromosomal DNA from L. pneumophila strain JR32 as a template. The PCR fragment was cut with NcoI/SalI or with BamHI/SalI and cloned into the vector pET-28a. Plasmid pER35 encoding LegG1_N223A-His₆ was obtained with the QuickChange protocol using the primers oER22 and oER23, and plasmid pER2 as a template.

To construct plasmids encoding YopE_1–53 (5.5 kDa) or YopE_1–138 (14.8 kDa) fusion proteins produced in Y. enterocolitica, the legG1 gene was amplified by PCR using the oligonucleotides oER158/oER3 and chromosomal DNA of L. pneumophila JR32 as template. The PCR fragment was cut with BamHI/SalI and cloned into pCJYE53-G3 or pCJYE138-G3 cut with the same enzymes, yielding pGP3 and pGP4, respectively. The genes sidM, sidC or sidC_P4C were released from plasmid pEB189, pCR6 or pHP56, respectively, by digestion with BamHI/SalI and cloned into pCJYE53-G3 or pCJYE138-G3 cut with the same enzymes. To liberate gfp from pCJYE53-G3 or pCJYE138-G3, the plasmids were cut with BamHI/SalI, filled in with Klenow polymerase and re-ligated.

Translocation assay

To determine Icm/Dot-dependent translocation into host cells of LegG1, 5×10⁵/ml RAW264.7 macrophages were seeded onto 96-well plates in a final volume of 100 µl/well and incubated at 37°C overnight. The macrophages were infected with L. pneumophila (MOI 20, 1 h, 37°C) producing TEM β-lactamase fusion proteins (kindly provided by X. Charpentier), grown for 21 h in AYE supplemented with 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside). 20 µl of 6-fold CCF4/AM substrate (Invitrogen) was added to each well. After 90 min incubation, the fluorescence was measured with a fluorescence plate reader (FluoStar Optima, BMG Labtech) using an excitation wave length of 410 nm, and an emission of 450 nm or 520 nm, respectively. The Icm/Dot substrate LepA served as a positive control and the cytoplasmic protein FabI as a negative control.

Purification of His-tagged LegG1

The constructs pER2 or pER3 were transformed into E. coli BL21(DE3) grown aerobically in LB medium at 37°C and induced with 1 mM IPTG during exponential growth for 4 h at 30°C. Alternatively, pER2 was transformed into E. coli BL21-CodonPlus (DE3)-RIL and grown in 5 l LB medium at 37°C to an OD₆₀₀ of 0.5 to 0.7 before expression was induced with 0.5 mM IPTG at 20°C overnight. Cells were harvested by centrifugation (7'000×g, 30 min, 4°C), suspended and homogenized in lysis buffer (50 mM Tris/HCl, pH 7.5, 10% glycerol, 10 mM β-mercaptoethanol (βME), 0.5 mM PMSF, 30 ng ml⁻¹ DNase), disrupted at 10,000 psi by a French Press, and the suspension was cleared by centrifugation (11'000×g, 30 min, 4°C).

His₆-tagged LegG1 was purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (Qiagen) equilibrated with buffer E (50 mM Tris/HCl, pH 8.0, 10% glycerol, 10 mM βME, 10 mM imidazole), eluted with buffer E containing 250 mM imidazole and dialyzed overnight against elution buffer lacking imidazole (4°C). Some preparations of His₆-LegG1 were further purified by gel filtration (Superdex 75 16/600; GE Healthcare, Munich, Germany) using a buffer containing 20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl₂, and 2 mM DTE (dithioerythritol).

Guanine nucleotide exchange assay

For the GEF assay Ran GTPase was preparatively loaded with fluorescent mantGDP (2′/3′-O-(N-methyl-anthraniloyl)-guanosine-5′-diphosphate). To this end, purified human Ran was incubated for 2 h RT in the presence of EDTA in five times molar excess over MgCl₂ and mantGDP in five times molar excess over Ran protein. Unbound nucleotides were removed by buffer exchange using a NAP-5 column (GE Healthcare, Munich, Germany) eluting the protein with exchange buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 2 mM DTE, 1 mM MgCl₂, 1 µM mantGDP). Collected fractions containing the protein were pooled and concentrated using a Spin-X UF 500 (Corning, Munich, Germany) concentrator.

The GEF-assay was performed in 1 ml fluorescence buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 2 mM MgCl₂, 2 mM DTE, 1 µM GTP) at 25°C using a fluorescence spectrometer (Fluoromax-4, Horiba Jobin Yvon). The excitation and emission wavelength of mantGDP is 345 nm and 440 nm, respectively. The release of mantGDP was monitored via the change in mant-fluorescence after addition of recombinant LegG1 or RCC1, respectively, in the presence of 100 µM GTP. The fluorescence traces have been corrected for dilution.

Pulldown and Western blot

Ran(GTP) was identified in cell lysates by pulldown experiments using reagents from New East Biosciences. To this end, RAW264.7 macrophages in T75 tissue culture flasks were infected (MOI 20, 1 h), lysed with lysis buffer (50 mM Tris-HCl, pH 8.0, 130 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 1% Triton X-100) containing a protease inhibitor tablet (complete; Roche) and incubated with an anti-Ran(GTP) antibody (1∶2000, New East Biosciences) together with protein A/G-agarose beads for 2 h. The beads were then washed 4 times with lysis buffer, subjected to SDS-PAGE and analyzed by Western blot using an anti-Ran antibody (1∶1000, Abcam). As positive and negative controls, uninfected cells were treated with γ-S-GTP or GDP in presence of 20 mM EDTA before incubation with the anti-Ran(GTP) antibody. Alternatively, A549 cells were washed three times with cold PBS before lysis, incubated for 30 min at 30°C with γ-S-GTP (100 µM) or GDP (1 mM), together with purified His₆-LegG1, heat-inactivated His₆-LegG1, LegG-His₆, LegG1_N223A-His₆, or human RCC1, and Ran(GTP) was immuno-precipitated as described above.

To separate polymerized and soluble tubulin, RAW264.7 macrophages seeded in 6-well plates were infected with L. pneumophila strains (MOI 10, 4 h), lysed with microtubule stabilization buffer (0.1 M PIPES, pH 7.6, 2 M glycerol, 5 mM MgCl₂, 2 mM EGTA, 0.5% Triton X-100, 4 µM taxol, protease inhibitors) and centrifuged at low speed (240×g, 10 min, RT) and high speed (20'000×g, 10 min). The protein concentration of each fraction was determined with Bradford reagent, and identical concentrations of each sample were separated by SDS-PAGE. Anti-α-tubulin (1∶6000, Abcam) and anti-actin (1∶500, Abcam) antibodies were used for Western blots.

Uptake and cytotoxicity assays

For uptake experiments A. castellanii, D. discoideum or RAW264.7 macrophages infected in 24-well plates (MOI 50, 1 h) were washed, detached by scraping and analyzed by flow cytometry. To quantify uptake, an uptake index was defined as the product of the number of cells above the gate threshold and the fluorescence intensity of the cells. Equal fluorescence intensities of different L. pneumophila strains were checked by a plate reader.

To determine cytotoxicity of different L. pneumophila strains, 2.5×10⁵ RAW264.7 macrophages were seeded onto a 24-well plate. The cells were infected with L. pneumophila (MOI 10, 4 h), and a bacterial input control was plated on CYE plates. After the infection, the medium was collected and replaced with PBS. To detach the infected macrophages, the plate was shaken vigorously at 1400 rpm for one minute on an Eppendorf plate incubator. The supernatant containing detached cells was combined with the stored media and centrifuged (240×g, 10 min). The cells were suspended in 0.5 ml PBS containing 1 µg/µl propidium iodide (15 min, 25°C, in the dark) and quantified by flow cytometry.

Intracellular replication and competition

To analyze intracellular replication of L. pneumophila, exponentially growing D. discoideum were washed with Sörensen phosphate buffer (2 mM Na₂HPO₄, 15 mM KH₂PO₄, pH 6.0) containing 50 µM CaCl₂ (SorC), seeded into a 96-well plate (1×10⁵ cells/ml MB medium) and allowed to adhere for 1–2 h. RAW264.7 macrophages were seeded (1×10⁵ cells/ml RPMI medium) one day before infection. L. pneumophila grown for 21 h in AYE broth was diluted in MB or RPMI and used for infection (MOI 1). After centrifugation, the infected phagocytes were incubated at 30°C (A. castellanii) or 37°C (macrophages), a bacterial input control was plated, and samples were taken at the time points indicated (t₀ = 10 min post infection).

Single round intracellular growth of GFP-producing L. pneumophila was assayed in A. castellanii or RAW264.7 macrophages as described [52]. Fresh medium was added to the cultures two days before the experiment. One day before the experiment, the cells were suspended and seeded into a black 96-well clear bottom plate (Perkin-Elmer) at a density of 2×10⁴ (amoebae) or 8×10⁴ (macrophages) cells/well and allowed to adhere overnight. Overnight cultures of L. pneumophila harboring pNT28 (GFP) were grown in AYE/Cam to an OD₆₀₀ of 3.0 (∼2×10⁹ bacteria/ml) and diluted to 8×10⁶ bacteria/ml in LoFlo low fluorescence medium (Formedium). The cells were infected (MOI 20) with 100 µl of diluted L. pneumophila, centrifuged and incubated at 30°C (amoebae) or 37°C (macrophages) for 48 h or longer. Nocodazole was added directly to the infection at the concentrations indicated. The GFP fluorescence was quantified at multiple time points using a plate reader (FluoStar Optima, BMG Labtech). To correlate fluorescence readings with bacterial viability, the cells were lysed at set time points using 0.8% saponin, dilutions were plated on CYE plates, and CFU were counted.

For the competition assays, A. castellanii (2×10⁴ per well, 96-well plate) in Ac buffer was co-infected (MOI 0.01) each with wild-type L. pneumophila and the Kan-resistant mutant strain to be tested. The infected amoebae were grown for 21 days at 37°C. Every third day the supernatant was combined with amoebae lysed with 0.8% saponin, diluted 1∶1000, and fresh amoebae were infected (50 µl homogenate per 200 µl amoebae culture volume). Aliquots were plated on CYE agar plates containing Kan (10 µg/ml) or not to determine CFU.

RNA interference

For the RNA interference experiments, A549 cells were grown in 96-well plates and treated for 2 days with a final concentration of 10 nM of the siRNA oligonucleotides indicated (Table S3). To this end, the siRNA stock (10 µM) was diluted 1∶15 in RNAse-free water, and 3 µl of diluted siRNA was added per well. Allstars siRNA (Qiagen) was used as a negative control. Subsequently, 24.25 µl RPMI medium without FCS was mixed with 0.75 µl HiPerFect transfection reagent (Qiagen), added to the well, mixed and incubated for 5–10 min at RT. In the meantime, cells were diluted in RPMI medium with 10% FCS, 175 µl of the diluted cells (2×10⁴ cells) were added on top of each siRNA-HiPerFect transfection complex and incubated for 48 h. The cells were then infected (MOI 10) with GFP-expressing L. pneumophila wild-type grown for 21 h, diluted in RPMI, centrifuged and incubated for 1 h. The infected cells were washed 3 times with pre-warmed medium containing 10% FCS and incubated for 24 h (well plate was kept moist with water in extra wells). To determine intracellular growth of L. pneumophila, GFP-fluorescence was measured using a plate reader (FluoStar Optima, BMG Labtech).

Microbial microinjection

For microbial microinjection the Y. enterocolitica strain WA (pT3SS) was used [41]. This Yersinia “toolbox” strain harbors a mini pYV virulence plasmid encoding the Ysc T3SS and the YadA adhesin, yet lacks all endogenous type III-secreted effectors [42], [53]. The T3SS recognizes an N-terminal secretion/translocation signal of Yersinia effectors. In particular, N-terminal fragments of the RhoG/Rac1 GAP YopE (YopE_1–53, YopE_1–138) are not cytotoxic but sufficient to mediate secretion and translocation of hybrid proteins. Fusion proteins composed of YopE_1–53 or YopE_1–138 and L. pneumophila LegG1, SidM, SidC or its PtdIns(4)P-binding domain SidC_P4C were produced in Y. enterocolitica WA (pT3SS) and analyzed by Western blot using a polyclonal anti-YopE antibody (1∶5000; gift from J. Heesemann). The production of YopE served as a positive control.

T3SS-dependent protein secretion was triggered by calcium depletion with EGTA essentially as described [53]. Briefly, Y. enterocolitica cultures grown overnight in BHI broth at 27°C were diluted 1∶20 in 10 ml BHI and grown at 37°C for another 1.5 h, followed by the addition of 10 ml of a solution containing 15 mM MgCl₂, 5 mM EGTA and 0.2% glucose for 2 h (final OD₆₀₀ 0.6–0.7). Subsequently, the cells were centrifuged (5000×g, 10 min), and the pellet was washed with PBS and suspended in 500 µl SDS PAGE sample buffer. The supernatant was sterile filtered, precipitated with TCA (10% v/v, 1 h, on ice) and centrifuged (20'000×g, 30 min, 4°C). The resulting pellet was suspended in 3 ml cold acetone (15 min, −20°C), centrifuged (20'000×g, 5 min, 4°C), washed once with each 1 ml cold acetone, and suspended in 50 µl SDS PAGE sample buffer. YopE and YopE fusion proteins were visualized by Western blot using an anti-YopE antibody.

Protein translocation into HeLa cells was determined by subcellular fractionation essentially as described [53], [54]. Y. enterocolitica producing YopE_1–53 or YopE_1–53-LegG1 were grown over night in BHI at 27°C, diluted 1∶20 into fresh media, grown for another 2 h at 37°C and used to infect HeLa cells (MOI 10) seeded at a density of 2×10⁷ in T75 tissue culture flasks the day before the experiment. Alternatively, the cells were infected with L. pneumophila (MOI 100) as described above. Two hours post infection the HeLa cells were washed several times with PBS and lysed with 1% digitonin. This treatment selectively lysed the host cells, while the bacteria remained intact. After centrifugation, proteins in the pellet (intact bacteria, debris) and in the supernatant (translocated bacterial proteins, soluble host proteins) were precipitated with methanol/chloroform, and YopE_1–53, YopE_1–53-LegG1 or SidC was visualized by Western blot using anti-YopE (1∶5000) or anti-SidC (1∶1000) antibodies. As controls, HeLa cells alone were used or bacteria treated with 1% digitonin, 1% SDS or 50 µM of the protonophore CCCP, a potent inhibitor of bacterial T3SSs [55], [56].

To analyze protein translocation by fluorescence microscopy, HeLa cells in 24-well plates containing sterile cover slips were infected (MOI 10, 2 h) with Y. enterocolitica producing YopE_1–138-LegG1, YopE_1–138-SidM or YopE. Uninfected cells or cells treated with 30 µM taxol or nocodazole served as controls. Alternatively, HeLa or A549 cells were treated with 1 µM nocodazole (1 h) and left uninfected or were subsequently infected (MOI 10, 2 h) with Y. enterocolitica producing YopE_1–53 or YopE_1–53-LegG1. Where indicated A549 cells were treated with siRNA two days before infection as described above. The cells were immuno-stained for α-tubulin (1∶600; Abcam) and YopE (1∶5000), and nuclei were labeled with DAPI (1 µg/ml).

(Real-time) fluorescence microscopy

Fluorescence microscopy of D. discoideum producing GFP fusion proteins was performed as described [47], [48], [51]. Briefly, exponentially growing cells were seeded on sterile coverslips in 24-well plates at 2.5×10⁵ per well in 1 ml HL5 medium and let grow over night. L. pneumophila cultures grown for 21–22 h in AYE liquid medium were diluted in HL5 medium and used for infection (MOI 50). The infection was synchronized by centrifugation, and the infected cells were incubated at 25°C for the time indicated and fixed on cover slips.

To obtain homogenates, infected amoebae were washed with cold SorC one hour post infection, suspended in homogenization buffer (20 mM HEPES, 250 mM sucrose, 0.5 mM EGTA, pH 7.2) [57] and lysed by seven passages through a ball homogenizer (Isobiotech) using an exclusion size of 8 µm. The homogenate was centrifuged onto coverslips coated with poly-L-lysine, fixed with 4% paraformaldehyde (PFA) for 30 min at 4°C and blocked with 1% BSA in SorC for 30 min.

The coverslips containing homogenates were incubated for 1 h at RT on parafilm with 40 µl of primary antibody diluted in blocking buffer (affinity purified rabbit anti-SidC (1∶100) [48]; anti-M45 1∶300, Genovac AG) and washed 3 times. Appropriate secondary antibodies were diluted 1∶200 in blocking buffer and incubated for 1 h at RT. Finally, the coverslips were washed and mounted using Vectashield (Vector Laboratories) supplemented with 1 µg/ml DAPI to stain DNA. The samples were viewed with a Leica TCS SP5 confocal microscope (HCX PL APO CS, objective 63×/1.4-0.60 oil; Leica Microsystems, Mannheim, Germany).

Microtubule cytoskeleton analysis was performed with RAW264.7 macrophages or D. discoideum infected with DsRed- or GFP-producing L. pneumophila (MOI 10, 4 h, 37°C). Subsequently, the infected phagocytes were washed once with Brb80, and fixed (50% Brb80, 0.1% Triton X-100, 0.5% glutaraldehyde) for 5 min. After washing with SorC, samples were blocked with 1 mg/ml sodium borohydrate in SorC for 10 min. The samples were stained with the anti-α-tubulin antibody WA3 (gift from M. Schleicher) and anti-SidC (1∶100) on parafilm for 1 h. Appropriate secondary antibodies were used at a dilution of 1∶200 (confocal microscopy) or 1∶400 (STED microscopy). 10 cells per coded sample were assessed for the degree of tubulin polymerization.

For real-time fluorescence microscopy exponentially growing D. discoideum amoebae producing calnexin-GFP were seeded in life-cell imaging dishes (Ibidi) in LoFlo medium containing G418 (20 µg/ml) to a total cell number of 5×10⁵ cells one day prior to the experiment. Before infection with L. pneumophila strains producing DsRed (MOI 10), the cells were washed with LoFlo. Ascorbic acid was added to the cells (20 mg/ml). After two hours incubation at 25°C with no centrifugation, an agar overlay was prepared and the infected cells were observed (recorded) for 5 min. Images were taken every 15 s.

STED microscopy

Stimulated emission depletion (STED) microscopy was employed for sub-diffraction resolution fluorescence imaging on a custom-made in-house setup. The system's basic principle is described elsewhere [58]. The system used is capable of acquiring one channel with confocal and two channels with STED resolution quasi-simultaneously. For imaging GFP-producing Legionella and Atto 655-coupled anti-α-tubulin, we used excitation/emission wavelengths of 488±3 nm/520±14 nm and 637±5 nm/685±20 nm, respectively. The STED wavelength for Atto 655 was 750±10 nm. Beam powers for acquisition were 0.5–4.5 µW, and 7 µW for GFP and Atto 655, respectively, as measured in front of the objective. STED beam powers were 1.4 mW for Atto 655. To reduce crosstalk, pulses for various channels were separated in time by varying optical path lengths. A home-built electronic gating device transmitted detector signals occurring at the correct time to the acquisition hardware, and rejected crosstalk signals occurring at other times. Dichroics and filters were purchased from AHF, Tübingen. The supercontinuum laser source was a SC450-PP-HE system running at 1 MHz, manufactured by Fianium Ltd, Southampton. For beam-scanning, we used a YANUS IV scan head from Till Photonics, Munich. The objective was a Leica 100×/1.4.

STED image processing: For acquiring images, movies comprised of 5–20 frames were acquired at an exposure time per pixel of 20–50 µs. All frames were then added up, and a non-local means algorithm (Buades et al., 2005) was applied via a matlab script (Peyré et al., 2007) with algorithm parameters being radius of search window: 1, radius of similarity window: 10, degree of filtering: 10.

Transmission electron microscopy

For transmission electron microscopy cells were fixed by adding 2% glutaraldehyde (GA) in 100 mM PIPES, pH 7.0, to the culture medium at a 1∶1 volume ratio. After 5 min, the supernatant was discarded, replaced with fresh 1% GA in the same PIPES buffer and incubated overnight at RT. For the subsequent epoxy resin embedding, either (i) cells were scraped, spun down and re-suspended in non-supplemented PIPES buffer, or (ii) the fixative of adherent cells was exchanged with non-supplemented PIPES buffer.

For epoxy resin embedding, the suspended or adherent cells were washed two times with 100 mM cacodylate buffer, post-fixed with 2% OsO₄ solution containing 1.5% potassium ferricyanide for 1 hour, and stained en block with 1.5% aqueous uranyl acetate for 30 min. The suspended cells were then dehydrated using a graded ethanol series and propylene oxide, and embedded in epoxy resin (Sigma-Aldrich; St. Louis/MO, USA). The cell monolayer was dehydrated using a graded ethanol series and detached from the bottom of the culture dish by dissolving the plastic with propylene oxide followed by rapid, vigorous pipetting. The cells were then washed four times with propylene oxide to remove the remains of dissolved plastic and thereafter embedded in epoxy resin. Ultrathin sections of 60–70 nm were cut with an ultramicrotome (Leica Ultracut UCT), stained with 0.2% lead citrate (Taab; Berks, England) in 0.1 M NaOH for 20 s and examined in a Philips CM100 transmission electron microscope.

For the stereological analysis of Golgi cisternae, two whole sections from two blocks of each sample were systematically sampled at 900× magnification in order to estimate the cytoplasmic area (volume). Within these micrographs all areas containing identifiable Golgi cisternae were selected and imaged at 8.900× magnification. To analyze the micrographs, a stereological test grid with horizontal and vertical lines was used. The total volume of the cytoplasm, which represented a reference space, was estimated by counting the number of test points over the cytoplasm. In order to estimate the total length of the Golgi cisternal membrane, the number of intersections of all identifiable Golgi membrane with horizontal test lines was counted. In addition, the number of Golgi cisternae - defined as an elongated enclosed membrane profile with a length minimum twice its breadth - was counted in these images. From these values the ratios of the total number of intersections (length) of Golgi cisternal membranes to the total volume of cytoplasm (relative surface density of cisternae) was estimated, as was the relative number of cisternae per cytoplasmic volume for the different conditions. At least 80 cell profiles were analyzed for each sample, and two independent experiments were performed. Within each experiment indices of the estimated values were calculated as ratios compared to the wild-type sample.

Supporting information

Supporting information includes eight figures (Figure S1, S2, S3, S4, S5, S6, S7, S8), three tables (Table S1, S2, S3) and two movies (Movie S1, S2) and can be found with this article online.

Accession numbers

The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the proteins discussed in this paper are L. pneumophila Icm/Dot T4SS (Y15044), LegG1/Lpg1976 (YP_095992), SidC (AY504673), D. discoideum calnexin (AF073837), human Ran GTPase (CAG29343) and RanBP1 (CAG30442).