Disruption of <i>sapM</i> impairs growth of MtbΔ<i>sapM</i> in THP-1 macrophages and enhances phagosomal maturation.

<p>(A) Influence of <i>sapM</i> deletion on the growth of <i>M.tuberculosis</i> in THP-1 cells. THP-1 cells were infected with <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> and MtbΔ<i>sapM</i>Comp separately at an MOI of 1∶3 (bacteria:macrophage). The number of intracellular viable bacteria were determined on each alternative day for 6 days. A significant attenuation in the growth of MtbΔ<i>sapM</i> in comparison to <i>M.tuberculosis</i> and MtbΔ<i>sapM</i>Comp was observed from second day post-infection. The growth of MtbΔ<i>sapM</i>Comp was comparable to <i>M.tuberculosis</i>. The values are represented as the mean (±SE) of three independent infections and the experiment was repeated three times. ***, <i>P</i><0.001 (Two way ANOVA). (B) Increased localization of MtbΔ<i>sapM</i> in LysoTracker labeled compartments in THP-1 macrophages. Macrophages were infected with FITC labelled <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> and MtbΔ<i>sapM</i>Comp (green) separately. After a 4 h incubation, the THP-1 cells were washed twice with fresh RPMI media and treated with 200 µg/ml amikacin for 2 h at 37°C to remove extracellular bacteria. Subsequently, the THP-1 cells were incubated with 50 nM Lysotracker red in RPMI (supplemented with 10% FBS) for 1 h. After this 7 h post infection period, the THP-1 cells were once washed with fresh RPMI media and fixed with 4% paraformaldehyde in PBS and observed under confocal microscope as described in the materials and methods. Representative fluorescent images depict that disruption of <i>sapM</i> lead to accumulation of MtbΔ<i>sapM</i> in acidified organelles (overlap of green and red images appears yellow) while the <i>M.tuberculosis</i> and MtbΔ<i>sapM</i>Comp were found in non-acidified organelles. The scale bars depict 5 µm. (C) Values indicate the percentage of phagosomes containing <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> or MtbΔ<i>sapM</i>Comp that colocalized with Lysotracker red. On infecting the macrophages with MtbΔ<i>sapM</i>, it was observed that a significantly higher percentage of phagosomes containing mutant bacteria (∼86.57%) colocalized with LysoTracker red when compared with the phagosomes containing either <i>M.tuberculosis</i> (23.1%) or MtbΔ<i>sapM</i>Comp (22.72%). Data is the mean (±SE) of 3 independent experiments carried out in triplicates, with a minimum of 100 phagosomes counted per experiment for each sample. ***, <i>P</i><0.001 (One way ANOVA).