Reengineering Redox Sensitive GFP to Measure Mycothiol Redox Potential of Mycobacterium tuberculosis during Infection
Figure 5
Dynamic changes in intrabacterial EMSH during infection.
THP-1 cells were infected with Mrx1-roGFP2 expressing Mtb strains; (A) H37Rv, (B) BND 320, (C) Jal 2287, (D) 1934, (E) Jal 2261 (F) MYC 431 and (G) BCG at an moi of 10. (H) Naïve and (I) IFN-γ/LPS treated (activated) RAW 264.7 macrophages were infected with H37Rv (moi: 10). At indicated time points, cells were treated with NEM-PFA and ∼30,000 infected macrophages were analyzed by flow cytometry and intramycobacterial EMSH was measured as described in figure 4. The “0” h time point refers to time immediately after initial infection with H37Rv for 4 h. The percentage of bacilli in each subpopulation was calculated and plotted as a bar graph. Data shown is the result of at least six independent experiments performed in quadruplicate and data from biologically independent experiments were combined and represented as percentage of bacilli in each subpopulation ± standard deviation. p-values shown in the panel I were calculated by comparing EMSH-oxidized populations of resting and activated macrophages (* p<0.01).