Figure 3. EsxA is expressed but not secreted in the M.tb DK9897 isolate and not recognized by T cells after infection.

(a) In vitro growth of mycobacterial strains H37Rv, Erdman, BCG, and DK9897. Exponentially growing bacteria cultures (OD₆₀₀~0.3) were diluted to a concentration of 2 × 10⁵ CFU/mL in 7H9 medium and growth was measured for up to 14 days. Data from one of two independent experiments. (b) Six weeks after infection with either M.tb strain Erdman or DK9897, cells were isolated from individual lungs and stimulated in vitro with either Ag85B (gray bars) or EsxA (black bars) protein. The percentage of CD4 T cells producing at least one of the cytokines, IFN-γ, TNF-α, IL-2 or IL-17 in response to stimulation was measured by flow cytometry for each group (n = 4). Mean values and SEM’s are shown. (c) The presence of EsxA and Ag85B proteins in bacteria pellet and culture supernatant was determined by Western blotting using in vitro cultures of M.tb Erdman and M.tb DK9897 and protein specific antibodies. For clarity the blots are cropped, full-length blots can be seen in Supplementary Figure S2.