Sub-vacuolar compartments are the source of redox heterogeneity within <i>Mtb</i> population during infection.

<p>THP-1 cells were infected with H37Rv expressing Mrx1-roGFP2 (moi: 10). At 24 h p.i. infected cells were treated by NEM-PFA. Cells were then stained for EEA1 and LC3 and analyzed by confocal microscopy for measuring ratiometric sensor response in <i>Mtb</i> co-localized within early endosomes and autophagosomes, respectively. In case of lysosomes, the cells were first pre-treated with Lysotracker followed by NEM-PFA fixation. (A) False color ratio confocal image of <i>Mtb</i> (∼80) inside THP-1 at 24 h p.i. <i>E_MSH</i> of bacilli was measured using <i>in vitro</i> calibration curve (SI <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003902#ppat.1003902.s005" target="_blank">Figure S5A</a>). (B) Representative bacilli for each subpopulation are shown. Co-localization of <i>Mtb</i> H37Rv in (C) endosomes, (D) lysosomes, and (E) autophagosomes. The co-localization is demonstrated in the merged images, where green indicates bacteria, red indicates phagosomal markers, and yellow indicates a positive correlation. False color ratio images were generated as described in SI <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003902#s4" target="_blank"><i>Materials and Methods</i></a>. Small dashed line boxes indicate co-localized bacilli and large solid line boxes represent the enlarged view of the one of the co-localized bacilli. Numbers represent <i>E_MSH</i> in millivolts. <i>E_MSH</i> of co-localized bacilli (≥50) is calculated and distribution is shown in scatter plot. In each panel, scatter plot depicts quantification of microscopy data. Each point on the plot represents a bacterium. Bar represents mean values. p-values were calculated by one way ANOVA followed by Tukey's HSD statistical test (* p<0.01). Percentage of bacilli in each subpopulation is represented as a stacked bar graph in every panel. Color bar corresponds to the 405/488 nm ratios ranging from 0 to 1. Data shown is the representative of at least three independent experiments.