Figure 4.

Insertion of ST at the C-terminus of EsxA didn’t affect Mm intracellular survival and allowed post-secretional labeling of EsxA by SpyCatcher(SC)-GFP. (a) The total lysate of Mm(EsxA-ST) were incubated with the purified SpyCatcher(SC)-GFP. The purified EsxA was incubated with SC-GFP as a control. The samples were subjected to Western blots. EsxA, EsxA-ST and EsxA-ST-SC-GFP were detected with anti-EsxA antibody. (b) SC-GFP and EsxA-ST-SC-GFP were detected with anti-GFP antibody. (c) The mCherry-expressing Mm(EsxA-ST) and Mm(WT) were incubated with SC-GFP. The images were taken under a LSM700 confocal fluorescence microscopy with the same configuration. For each strain, 12 random sights were taken from two replicate wells. The scale bar represents 20 µm. (d) The Green/Red overlap ratio in the randomly selected fields was quantified. The SC-GFP labeling assay was replicated for three times and the data is presented as mean ± SD. The statistical analysis was performed with t-test. ****< 0.0001. (e) The culture filtrates (CF) and cell lysates (CL) of Mm(WT) and Mm(EsxA-ST) were applied to Western blots, and the expression and secretion of EsxA or EsxA-ST were detected with anti-EsxA antibody. GroEL and Ag85B were detected as loading controls for CF and CL, respectively. GroEL was also detected in CF to make sure the EsxA-ST could be successfully secreted. (f) A549 cells were infected with Mm(WT), Mm(EsxA-ST) and Mm(ΔEsxA:B) at MOI = 2, respectively. At 2, 24 and 48 hpi, the cells were collected and subjected to intracellular survival tests. For each strain, the CFU of 24 and 48 hpi was counted and calculated to the percentage of the CFU at 2 hpi. The experiment was replicated for three times and the data is presented as mean ± SD. The statistical analysis was performed with One-way ANOVA method, followed by Holm-Sidak multiple comparisons. ****< 0.0001