Figure 3

T4SS-dependent translocation of potential Brucella effectors. A, B Western blot analysis of the expression of TEM1-tagged proteins. Bacterial lysates from the B. melitensis M5 strain and M5∆virB123 strain expressing TEM1-fusion proteins were analysed via SDS‒PAGE followed by western blot analysis, with an anti-β-lactamase antibody used as the primary antibody. Brucella GAPDH was used as a loading reference. A Expression of C-terminally TEM1-tagged Brucella proteins. Lane M: prestained protein marker VII (8–195 kDa). Lanes 1–5: B. melitensis M5 strains expressing TEM1-fusion proteins; Lanes 6–10: B. melitensis M5∆virB123 strains expressing TEM1-fusion proteins. Lanes 1 and 6: negative control protein GST-TEM1 (55.34 kDa); Lanes 2 and 7: positive control protein BPE123-TEM1 (45.86 kDa); Lanes 3 and 8: RS15060-TEM1 (64.99 kDa); Lanes 4 and 9: RS10635-TEM1 (64.83 kDa); Lanes 5 and 10: RS14565-TEM1 (61.92 kDa). B Expression of N-terminally TEM1-tagged Brucella proteins. Lane M: prestained protein marker VII (8–195 kDa). Lanes 1–4: B. melitensis M5 strains expressing the TEM1-fusion protein; Lanes 5–8: B. melitensis M5∆virB123 strains expressing the TEM1-fusion protein. Lanes 1 and 5: TEM1-GST, a negative control protein (55.47 kDa); Lanes 2 and 6: TEM1-VceC, a positive control protein expressing the full length of VceC (74.21 kDa); Lanes 3 and 7: TEM1-RS10585 (56.12 kDa); Lanes 4 and 8: TEM1-RS15060 (65.12 kDa). C, E Quantification of the translocation of C-terminally TEM1-tagged Brucella proteins; D, F quantification of the translocation of N-terminally TEM1-tagged Brucella proteins. The cytosolic translocation of β-lactamases by the M5 strain and M5∆virB123 strain expressing different TEM1 fusion proteins was assessed by fluorescence plate-reader detection and fluorescence microscopy at 5 hpi in RAW 264.7 cells. GST-TEM1 and TEM1-GST were used as negative controls (green fluorescence), and BPE123-TEM1 and TEM1-VceC served as positive controls (blue fluorescence). C, D The response ratios of three independent wells were calculated, and all the data were normalized so that the negative control wells had a mean value of 1.0 (N = 3, means ± SD, two-way ANOVA). E, F Representative fluorescence micrographs of control proteins (GST, VceC and BPE123) and fusion proteins with TEM1 tags located at the C-terminus (E) or N-terminus (F) in strains M5 and M5∆virB123, respectively, are shown. **p < 0.01; ***p < 0.001; ns: nonsignificant difference.