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First published online 10 February 2009
doi: 10.1242/jcs.040345

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Yersinia enterocolitica differentially modulates RhoG activity in host cells

Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Hamburg-Eppendorf, Martinistraβe 52, 20246 Hamburg, Germany

View larger version (21K): [in this window] [in a new window]   Fig. 1. GST-Elmo2NT specifically binds to active RhoG. Cos-7 cells were transfected with indicated myc-tagged Rho GTPase constructs, lysed and subjected to pull-down using GST-Elmo2NT. Proteins from pull-down and in cell lysates were detected by western blotting using anti-Myc antibody.

View larger version (96K): [in this window] [in a new window]   Fig. 2. Specific intracellular redistribution of GFP-Elmo2NT by active RhoG. Confocal fluorescence images of HUVEC co-transfected with GFP-Elmo2NT and myc-RhoGV12, myc-RhoGN17 or myc-TrioD1. GFP-RhoG was co-transfected with myc-TrioD1. Merge represents overlays of green and red fluorescence channel resulting in yellow colour. Myc-tagged constructs were stained with anti-myc antibody. GFP-Elmo2NT colocalises with constitutively active RhoGV12 but not with dominant-negative RhoGN17. GFP-Elmo2NT and GFP-RhoG translocate to TrioD1-induced dorsal ruffles. Bar, 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Differential recruitment and activation of RhoG by Y. enterocolitica. (A) HUVEC were transfected with GFP-Elmo2NT and then infected with Yersinia WA-C or Yersinia WA-314 for 10 minutes. Images show representative cells that display recruitment and essentially no recruitment of GFP-Elmo2NT, respectively. The WA-314-infected cell shows severe morphological alteration owing to Yop action. Merged images represent overlays of green fluorescence and phase-contrast channel. White boxes indicate areas that were enlarged about fourfold. Broken lines outline the cell borders. The graph shows quantification of GFP-Elmo2NT recruitment by indicated strains 10 and 45 minutes after infection. Percentage of total cell-associated bacteria showing GFP-Elmo2NT enriched at phagocytic cups was determined microscopically. Each bar represents mean±s.d. (error bars) of three different experiments with at least 33 cells analysed per experiment. Bars, 10 µm (upper panel) and 3 µm (lower panel). (B) HUVEC were transfected with GFP-RhoG (upper panel) or GFP-Elmo2NT (lower panel), and then infected with Yersinia WA-314 or WA-C. GFP-RhoG or GFP-Elmo2NT recruitment was visualised using live-cell imaging. Depicted still images were taken from representative movies at indicated time points (see also supplementary material Movie 1). Cells infected with WA-C show rapid closure of GFP-RhoG- and GFP-Elmo2NT phagocytic cups. By contrast, cells infected with WA-314 show severely impaired GFP-RhoG and GFP-Elmo2NT recruitment to phagocytic cups with alternating back and forth movements of the respective fluorescent proteins. Bars, 2 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 4. RhoG is activated by Yersinia Invasin. (A) Cos-7 cells were transfected with GFP-RhoG and infected with Yersinia WA-C or WA-314 (left panel), reacted with beads coated with GST-Invasin397 or GST (middle panel) or infected with Invasin-E. coli or YadA-E. coli for 30 minutes (right panel). Active GFP-RhoG was precipitated with GST-Elmo2NT and detected by western blotting using anti-GFP antibody. (B) HeLa cells were infected with Yersinia WA-C or WA-314 (left panel) or Invasin-E. coli (right panel) for 30 minutes, and active endogenous RhoG was precipitated with GST-Elmo2NT and detected by western blotting.

View larger version (31K): [in this window] [in a new window]   Fig. 5. RhoG is inactivated by YopE. (A) Cos-7 cells were co-transfected with myc-RhoG and either control plasmid (left lane), myc-TrioD1 (middle lane) or HA-Tiam1 (right lane). Active myc-RhoG was precipitated with GST-Elmo2NT and analysed by western blotting. Myc-RhoG and myc-TrioD1 were detected with anti-myc antibodies and HA-Tiam1 with anti-HA antibody. TrioD1 but not Tiam1 produced a strong RhoG activation. (B) Cos-7 cells were co-transfected with myc-RhoG and myc-TrioD1. After 24 hours, cells were infected with indicated Yersinia strains for 2 hours, lysed and subjected to GST-Elmo2NT pull-down. RhoG is downregulated by all strains except by YopE strain. (C) Experiments were performed as in B but cells were infected with indicated bacterial strains. (D) In vitro GAP assay. GTP-hydrolysis was measured at 650 nm absorbance after co-incubation of indicated proteins for 20 minutes. YopE and YopEMLD stimulated the GTPase activity of RhoG and Rac1 but not of Ras. p50RhoGAP was used as a positive control. Values are mean±s.d. (error bars) of three independent experiments.

View larger version (50K): [in this window] [in a new window]   Fig. 6. YopE reduces RhoG recruitment to Yersinia. HUVEC were transfected with GFP-RhoG and then infected with Yersinia WA-C(pTTSS+pYopER144A) or Yersinia WA-C(pTTSS+pYopE). Images show GFP-RhoG distribution (green fluorescence) after 15 minutes of infection. Merged images represent overlays of green fluorescence channel and phase contrast. White boxes indicate areas that were enlarged about fourfold. Broken lines outline the cell borders. For the bar chart, GFP-RhoG recruitment by indicated strains was quantified after 15 and 45 minutes of infection. Each bar represents mean±s.d. (error bars) of three different experiments with at least 33 cells analysed per experiment. Bars, 10 µm (upper panels) or 3 µm (lower panels).

View larger version (53K): [in this window] [in a new window]   Fig. 7. YopE and RhoG colocalise in ER and Golgi. (A) Confocal fluorescence images of HUVEC transfected with GFP-YopER144A. Golgi was immunostained with anti-GS27 antibody and ER with anti-calreticulin antibody. Merged images represent overlays of green and red channels, resulting in yellow colour. Boxes indicate areas that were enlarged two- to threefold and shown in insets or bottom panels (ZOOM). In the bottom-left panel, the putative Golgi- and ER localisation of YopER144A is outlined. Bars, 10 µm. (B) Confocal fluorescence images of HUVEC co-transfected with mCherry-YopER144A and GFP-RhoG, GFP-Rac1, GFP-Cdc42 and GFP-TC10. Merges represent overlays of green and red channel. Bars, 10 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 8. YopEMLD but not native YopE inactivates RhoA. Active RhoA was precipitated by GST-Rhotekin from Cos-7 cells infected with Yersinia strains WA-C(pTTSS+pYopE) or WA-C(pTTSS+pYopEMLD) for 2 hours. RhoA was detected by western blotting using anti-RhoA antibody.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Invasin stimulates and YopE inhibits Rac1 activity via RhoG. (A) Cos-7 cells were transfected with siRNA for RhoG and at indicated time points equal amounts of protein were analysed by western blotting using anti-RhoG and anti-Rac1 antibody. RhoG siRNA effectively reduced the level of endogenous RhoG protein at 48 hours but had no effect on Rac1 protein level (left panel). Cos-7 cells were transfected with non-targeting (nt) siRNA or with RhoG siRNA for 48 hours and then subjected to GST-PAK-CRIB pull-down assay. Rac1 was detected by western blotting using anti-Rac1 antibody (right panel). (B) HUVEC were left untransfected or transfected with RhoG siRNA (indicated by stars) for 48 hours and then infected or not with WA-C or Invasin-E. coli for 15 minutes. The level of active Rac1 was determined by GST-PAK-CRIB pull-down and western blotting using anti-Rac1 antibody. (C) HUVEC were transfected with RhoG siRNA or non-targeting (nt) siRNA for 48 hours and then infected with Yersinia WA-C for 60 minutes. Invasion of bacteria was determined using a double fluorescence method (see Materials and Methods). Bars represent mean±s.d. (error bars) of three independent experiments with at least 60 cells analysed per experiment. (D) Cos-7 cells were transfected with myc-Rac1 (lane 1 and 2) or co-transfected with myc-Rac1 and HA-Elmo-T618 (lane 3 and 4) and then infected with WA-C(pTTSS+pYopE) for 2 hours. The level of active myc-Rac1 was determined by GST-PAK-CRIB pull-down and western blotting using anti-myc antibody. HA-Elmo-T618 was detected with anti-HA antibody. (E) HUVEC were transfected with plasmids expressing myc-Rac1 (lane 1 and 2) or myc-Rac1 and HA-Tiam1 (lane 3 and 4), and infected with Yersinia strain WA-C(pTTSS+pYopE) for 2 hours. The level of active Rac1 was determined by GST-PAK-CRIB pull-down as described in D.

View larger version (20K): [in this window] [in a new window]   Fig. 10. Model for modulation of RhoG activity by Yersinia. During the invasive phase of infection, Yersinia binds to integrins via Invasin, which triggers activation of RhoG and stimulation of Rac1 through the Elmo/Dock180 module. Activation of RhoG and Rac1 leads to cellular responses, such as invasion, phagocytosis, caspase 1 activation and production of interleukins. During the subsequent immunosuppressive phase, Yersinia injects effector Yops through its TTSS. The effector YopE acting as a GAP inactivates RhoG and Rac1, and reverses the specific cellular responses.

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