Fig 1.
SseI deamidates an essential glutamine residue in the switch II region of Gαi2.
(A) Western blot analysis of Gαi2. GST-Gαi2 was coexpressed without (control) and with SseI in E. coli. Purified GST-Gαi2 was immunoblotted and detected by the Gαi2-specific antibody (Gαi2) and by the deamidation-specific antibody (GαQE). (B) Gαi2 coexpressed with SseI was analyzed by HPLC-MS/MS spectrometry. Combined extracted ion chromatograms for m/z 455.2(2+) and 455.7(2+), corresponding to the tryptic peptides MFDVGGQR and MFDVGGER (amino acids 199–206) of Gαi2, are shown (see also S1B and S1C Fig). (Upper panel) Gαi2 coexpressed with inactive SseI-C178A. (Lower panel) Gαi2 coexpressed with active wt SseI. (C) Immunoblot analysis of recombinantly expressed Gαi2 incubated with wild type C-terminal part of SseIC (wt) or with 3 different mutant SseIC (C178A, H216A and D231A).
Fig 2.
Cell permeable SseI activates Gαi-dependent signal transduction pathways.
(A) Schematic representation of the cell permeable PMT-SseIC chimera. The C-terminal domain of SseI, encompassing amino acids 137–322 (SseIC), was fused to the receptor binding and translocation domain of PMT (PMT, amino acids 1–505). (B) Immunoblot analyses of HEK-293 cells incubated with indicated concentrations of PMT-SseIC or PMT-SseIC-C178A for 16 h. RIPA buffer lysates were prepared and immunoblots were performed to detect Gα deamidation, using the GαQE antibody. Equal loading was verified by detection of tubulin. (C) Comparison of PMT- and PMT-SseIC-induced Gα deamidation. PMT treatment of cells (1 nM, 16 h) led to two signals of deamidated Gα proteins in immunoblot analysis, migrating at the same size of Gαi and Gαq. PMT-SseIC (100 nM, 16 h) induced one deamidation signal at the size of Gαi. (D) PMT-SseIC blocks stimulation of the adenylyl cyclase (AC) activity. HeLa cells were transfected overnight (16 h) with the FRET sensor construct EPAC2-camps. Cells were left untreated (con) or were incubated with PMT (1 nM) or PMT-SseIC (100 nM) for 4 h. AC was stimulated with forskolin (40 μM, added at time point 0) and FRET measurement was performed. cAMP increase is depicted as normalized ratio of YFP/CFP of the sensor. (E) PMT-SseIC stimulates the PI3Kγ. HEK-293 cells were transfected with the PIP3 sensor GFP-Grp1PH and the PI3-kinase subunit p110γ. In addition, HEK-293 cells were transfected with the non-catalytic PI3-kinase γ subunit p101 as indicated. Cells were stimulated with PMT-SseIC or the inactive C178A mutant of PMT-SseIC (each 100 nM). After baseline measurement for 1 min fMLP (1 μM) was added and the measurement was continued for 5 min. Histogram shows the quantification of the membrane translocation of GFP-Grp1PH. Data depicted represent the mean ±SEM from 3 independent sets of experiments analyzing 17 or 18 cells in total. (F) Immunoblot analysis of BMDMs transiently transfected with GFP-SseI or GFP-SseI-C178A. Cells were incubated for 24 h, followed by serum starvation for 4 h. Cells were lysed and subjected to Western blot analysis with indicated antibodies. Representative immunoblots from one experiment are shown. See also S2 Fig.
Fig 3.
Cellular effects of SseI during infection.
(A) Fluorescence microscopy of fixed cells. RAW264.7 cells were infected with wild type (wt) S. Typhimurium or a ΔsseI-strain at a MOI of 1 for 5 h. Deamidation of Gα was detected utilizing the GαQE antibody and an Alexa 568-conjugated secondary antibody. Salmonella were identified by Salmonella O antiserum and an Alexa 488-conjugated secondary antibody. Deamidated Gα is depicted in red and Salmonella in green. Z planes showing Salmonella were maximum projected into one image. Scale bars = 5 μm. Quantification of images (right panel). Gα protein deamidation of Salmonella-infected or uninfected cells was calculated by determining the average intensity of Alexa 568 fluorescence of the whole cell area in the Z plane with internalized Salmonella. Significance was determined by two-tailed Student’s t-test. Data are means ±SEM. (n = 10 cells). (B-D) Serum-starved RAW264.7 cells were infected with a MOI of 30 for 30 min. (B) Time-resolved immunoblot analysis. At indicated times p.i., cells were lysed and processed for immunoblotting. Blotting membranes were treated with indicated antibodies (GαQE, p-Akt and Akt). Representative blots of 3 independent experiments are shown. Equal loading was verified by detection of Akt, presence of Salmonella was verified by Salmonella-O antigen staining. Right panel shows the quantification of p-Akt (Ser473) labeling over time after infection with wt- and ΔsseI-Salmonella strains from 3 independent experiments. Chemiluminescence intensity, given as area units [AU], was determined for each band. Statistical significance was assessed by Mann Whitney test. (C) Only macrophages infected with wild type (wt) Salmonella show strong phosphorylation of Akt. Cells were infected with pEGFP expressing wt- or ΔsseI-Salmonella Typhimurium for 30 min. 5 h p.i. cells were fixed and processed for immunofluorescence microscopy. Cells were stained for phospho-Akt Ser473 (red) and nuclei were stained with DAPI (blue). Scale bars = 20 μm. (D) Addition of the PI3K inhibitor LY29 leads to concentration dependent inhibition of Akt phosphorylation in wild type (wt) Salmonella-infected macrophages. 1.5 h p.i. cells were treated with the indicated concentrations of LY29 for 3.5 h. Thereafter, cells were lysed. Lysates were processed for immunoblotting with indicated antibodies (Akt, p-Akt(Ser473)). (E) Quantification of p-Akt (Ser473) immunoblots of infected cells treated with increased concentrations of LY29. Experiments were performed as depicted in (D). Intensity of labeled bands was normalized to untreated (con) cells. Values are means ±SEM from at least 3 independent experiments.
Fig 4.
Effects of SseI on host cell survival.
(A and B) RAW264.7 macrophages were infected with wild type (wt) or ΔsseI-Salmonella (MOI = 30; 30 min). 1.5 h p.i., cells were starved for 3.5 h in Earle’s balanced salt solution. The PI3K inhibitor LY29 (30 μM) was added 1.5 h p.i. where indicated. (A) The amount of LDH released into the medium was measured fluorometrically 5 h p.i.. ΔsseI-infected macrophages showed higher amounts of released LDH compared to wt-Salmonella infected cells. Data show means ±SEM from 3 independent experiments. Maximum LDH-release was obtained in the presence of 1% Triton X-100. Statistical significance was assessed using ANOVA (Bonferroni post-test). (B) Viability of RAW264.7 macrophages was fluorometrically measured 5 h p.i. by the ability of cells to reduce Resorufin. Experiments were performed in triplicates. Values are means ±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA (Bonferroni post-test).
Fig 5.
Influence of SseI on directed migration of dendritic cells.
(A-C) DCs were infected with GFP-expressing wild type (wt)- or ΔsseI-Salmonella (MOI = 30; 30 min). Then, cells were transferred into a collagen matrix. (A) A CCL19 gradient was applied and directed migration for 4 h was determined via time-lapse microscopy. Red dots mark the end of the migration tracks. Every track starts at x = 0 / y = 0. (B) Tracks of n = 30 randomly selected DCs for each condition from 3 independent experiments were quantified and the chemotactic index was determined with Chemotaxis and Migration tool V2.0 (Ibidi). (C) Similarly, the speed of migration was quantified. Statistical significance was assessed using ANOVA with Bonferroni post-test. (D) DCs infected with wt- or ΔsseI-Salmonella (MOI = 30; 30 min) were lysed at the indicated times and immunoblot analysis was performed with indicated antibodies (GαQE, p-Akt, Akt and actin). (E-G) Migration of DCs after ectopical expression of wild type (wt) SseI or SseI-C178A (C178A). Cells were transferred into a collagen matrix, a CCL19 gradient was applied, and directed migration (E) was determined by time-lapse microscopy (wt SseI, n = 43; SseI-C178A, n = 49). Cells were randomly selected from 3 independent experiments. (F, G) Quantification of the chemotactic index (F) and speed (G) of DCs. Statistical significance was assessed using unpaired, two-tailed Students t-test.
Fig 6.
Effects of G protein deamidation on chemotaxis.
(A) Wild type (wt)-, Gnai2-/-—and Gnai3-/-—DCs were infected with wt-Salmonella (wt S. Tm) (MOI = 30; 30 min) and the chemotactic indices were determined from at least n = 34 cells for each condition from 3 independent experiments as in Fig 5. Statistical significance was assessed using ANOVA and Bonferroni post-test. (B) Time-resolved immunoblot analysis of DCs derived from Gnai2-/—(upper panel), Gnai3-/—(lower panel) mice or wild type littermates (Gnai2+/+, upper panel; Gnai3+/+, lower panel) after infection with wild type Salmonella (S. Tm infection) (MOI = 30, 30 min) for indicated times. Proteins were analyzed by specific antibodies (GαQE, Gαi1+2 and actin). (C-E) Gnai2-/- DCs were transfected with Gαi2, Gαi2Q205E or empty vector. Chemotaxis in a collagen matrix (C) with CCL19 gradient was measured by cell tracking using time-lapse microscopy. Chemotactic index (D) and migration speed (E) were calculated as described in Fig 5. Statistical significance was assessed using ANOVA (Bonferroni post-test).