Fig. 6
From: Structural basis for arginine glycosylation of host substrates by bacterial effector proteins
HEN motif plays a key role in NleB/SseK enzyme activity. a SseK1 mutants were generated and the cellular function in HEK293T cells was investigated. A non-reducing gel (right panel) was used to confirm the presence of the TRADD oligomer. Mutants in red represent mutations of residues proposed to be catalytically important. Data represent at least three repetitions. b The NF-κB level in A549-NF-κB luc cells was measured to check the enzymatic functions. Data represent the mean and standard deviation in triplicate. Multiple comparisons perform by one-way ANOVA followed by Turkey’s Multiple Comparison Test (**P < 0.01, ***P < 0.001 compare to WT). c Enzyme kinetic assays of SseK1 and SseK2, respectively. d In vitro glycosylation of FADD by NleB1, NleB2 (top panel) and SseK1 and SseK2 HEN mutants (bottom panel). e In vitro glycosylation of TRADD by NleB1, NleB2 (top panel) and SseK1 and SseK2 HEN mutants (bottom panel). f In vitro glycosylation of GAPDH by NleB1, NleB2 (top panel) and SseK1 and SseK2 HEN mutants (bottom panel). g Glycosylation of TRADD after co-transfection with either NleB1 or SseK1 (WT and HEN mutants) in HEK293T cells. FLAG-TRADD was immunoprecipitated and then immunoblotted using an anti-Arg-GlcNAc antibody. h Glycosylation of GAPDH after co-transfection with either NleB1 or SseK1 (WT and HEN mutants) in HEK293T cells. FLAG-TRADD was immunoprecipitated and then immunoblotted using an anti-Arg-GlcNAc antibody. i Colonization (log10 CFUs/g colon) of indicated C. rodentium strains (14 days post-gavage) in C57BL/6 J mice (n = 6). Asterisks indicate significantly different colonization magnitude as compared to WT; Kruskal-Wallis test. Uncropped blots are shown in Supplementary Figs. 21 and 22
