Figure 2
Translocation of Brucella putative effector proteins. A, B Western blot analysis of the expression of TEM1-tagged proteins. Bacterial lysates from the B. melitensis M5 strain expressing the TEM1-fusion protein were resolved by SDS‒PAGE, followed by western blot analysis with an anti-β-lactamase antibody 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); Lane 1: negative control protein GST-TEM1 (55.34 kDa); Lane 2: positive control protein BPE123-TEM1 (45.86 kDa); Lane 3: RS15060-TEM1 (64.99 kDa); Lane 4: RS10635-TEM1 (64.83 kDa); Lane 5: RS14565-TEM1 (61.92 kDa); Lane 6: RS00230-TEM1 (192.73 kDa); Lane 7: RS08185-TEM1 (70.97 kDa). B Expression of N-terminally TEM1-tagged Brucella proteins. Lane M: prestained protein marker VII (8–195 kDa); Lane 1: negative control protein TEM1-GST (55.47 kDa); Lane 2: positive control protein TEM1-VceC (74.21 kDa); Lane 3: TEM1-RS10585 (56.12 kDa); Lane 4: TEM1-RS15060 (65.12 kDa); Lane 5: TEM1-RS14565 (62.05 kDa); Lane 6: TEM1-RS00230 (192.84 kDa); Lane 7: TEM1-RS08185 (71.10 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-lactamase by the B. melitensis M5 strain expressing different TEM1 fusion proteins was assessed via fluorescence plate-reader detection and fluorescence microscopy in RAW 264.7 cells at 5 hpi. GST-TEM1 and TEM1-GST were used as negative controls (green fluorescence), and BPE123-TEM1 and TEM1-VceC were used 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, one-way ANOVA). (E–F) Representative fluorescence micrographs from individual assay wells of control proteins (GST, VceC and BPE123) and selected TEM1 fusion proteins tagged at either the C-terminus (E) or N-terminus (F). *p < 0.05; **p < 0.01; ***p < 0.001; ns: nonsignificant difference.