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. 2017 Apr 24;7:46666. doi: 10.1038/srep46666

Figure 4. Gene complementation of eccCa1 (Rv3870) restores EsxA secretion, EsxA specific T cell responses and increases virulence.

Figure 4

(a) Western blot comparing secretion of EsxA and Ag85B in culture filtrate and bacterial pellet from in vitro grown cultures of M.tb DK9897 and M.tb DK9897 complemented with the Rv3870-72 gene cassette (DK9897::pKG18). For clarity the blots are cropped, full-length blots can be seen in Supplementary Figure S2. Lung cells isolated from mice infected for six weeks with H37Rv or the complemented DK9897 strain (DK9897::pKG18) were stimulated with either Ag85B, EsxH (TB10.4) or EsxA protein. The following day the percentage of CD4+ CD3+ double-positive cells producing at least one of the cytokines TNF-α, IFN-γ, IL-2 or IL-17 in response to antigen stimulation was determined for individual mice (n = 4) by flow cytometry. (c) Individual lungs from mice infected with H37Rv, DK9897 or DK9897::pKG18 for six weeks were homogenized and plated in serial dilutions. After two weeks incubation, the number of mycobacteria was determined. Mean and SEMs are indicated, p = 0.03, two-tailed t-test.