Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins
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.