Fig. 4: EhaF is a secreted autotransporter translocating into the host cell cytosol during infection.
a The domain organization of EhaF with an N-terminal signal peptide (residues: 1–31), a passenger domain (residues: 32–309), and a C-terminal beta domain (residues: 310–369). b Immunoblot for EhaF-FLAG in the bacterial supernatant and pellet of H2O- or IPTG-treated BL21/pEmpty-FLAG, BL21/pΔSP-EhaF-FLAG (lacking signal peptide), and BL21/pEhaF-FLAG (full-length EhaF). c, d IFNβ and IL-6 secretion by BMDMs treated with supernatants (25 μl) from H2O or IPTG-treated BL21/pEmpty, BL21/pEhaF, or BL21/pΔSP-EhaF 30 min prior to treatment with 1 μg/ml LPS or wild-type EHEC or ΔEhaF (MOI = 50) at 6 h post-treatment. e, f Agar plating for bacterial counts (e) and immunoblots for EhaF-FLAG, EEA1, Rab7, LAMP1, Na + /K + ATPase, and GAPDH (f) in the cytosolic and residual fractions of uninfected BMDM or BMDM infected with the indicated strains for 1.5 h at an MOI of 50 obtained by 0.005% digitonin fractionation. g Transmission electron microscopy of BMDMs infected with IPTG-treated BL21/pEmpty-FLAG or BL21/pEhaF-FLAG at an MOI of 50 for 1.5 h and stained with gold-conjugated anti-FLAG antibody (red arrows show FLAG labeling). Scale bar=500 nm. h Immunoblot for EhaF-FLAG, IRF3, and p65 in the elute from FLAG immunoprecipitation (FLAG IP) or in the lysates (Input) from BMDMs at 1.5 h following infection with IPTG-treated BL21/pEmpty-FLAG or BL21/pEhaF-FLAG. c, d Data (mean ± SEM) were from three independent experiments and each dot is a mean of each experiment’s technical replicates. Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-test. p < 0.05 indicated statistical significance. Multiplicity adjusted p values are presented. b, e, f, g, h Data from one experiment representative of three independent experiments is shown. Source data are provided as a Source Data file.
