Bacterial strains, plasmids and cell line employed in this study.

<p>* After generation of the mutants, all the strains were grown in the absence of kanamycin resulting in the loss of plasmid pJV53. The loss of plasmid pJV53 was confirmed by PCR analysis.</p

Histopathology of guinea pig organs infected with various <i>M.tuberculosis</i> strains at 16 weeks.

<p>The figure depicts representative lower magnification (20x) photomicrographs of H&E stained 5 µm sections of lung and liver of guinea pigs (n = 5) infected with <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> and MtbΔ<i>sapM</i>Comp euthanized at 16 weeks post-infection. Pulmonary and hepatic granuloma consolidations were graphically represented as % granuloma by box plot (median values are denoted by horizontal line, the mean is represented by ‘+’, inter quartile range by boxes, and the maximum and minimum values by whiskers). While the lungs and liver of <i>M.tuberculosis</i> or MtbΔ<i>sapM</i>Comp infected guinea pigs exhibited large areas of granulomatous inflammation, the animals infected with MtbΔ<i>sapM</i> exhibited normal pulmonary and hepatic parenchyma. The scale bars depict 500 μm. *, <i>P</i><0.05; ***, <i>P</i><0.001 (Mann-Whitney U test). Missing data points represent the animals that succumbed to disease before the time of euthanasia.</p

Influence of disruption of AP endonuclease(s) on the growth of <i>M.tuberculosis</i> in guinea pigs.

<p>The figure depicts bacillary load in the lungs and spleens of guinea pigs (n = 5) infected with wild-type <i>M.tuberculosis</i>, MtbΔ<i>end</i>, MtbΔ<i>end</i>Comp, MtbΔ<i>xthA</i> and MtbΔ<i>xthA</i>Comp and MtbΔ<i>end</i>Δ<i>xthA</i> at (A) 4 weeks and (B) 10 weeks post-infection. Guinea pigs belonging to all groups exhibited a comparable bacillary load in the lungs as well as in the spleen. Each data point represents the Log₁₀ CFU value for an individual animal and the bar depicts mean (±SE) for each group.</p

Influence of disruption of AP endonuclease(s) on the susceptibility of <i>M.tuberculosis</i> to DNA damaging agents.

<p>The figure represents the fraction of surviving bacteria after a 24(A) methyl methane sulfonate (MMS), (B) hydrogen peroxide (H₂O₂) and (C) mitomycin C (MMC). The results indicate that while both the AP endonucleases are important in protecting the pathogen against alkylation damage, End plays a more important role than XthA in protecting this pathogen against oxidative assault. End and XthA do not contribute to the repair of intrastrand and interstrand cross-linking of DNA. The percentage of the bacteria surviving after addition of the stress agent in comparison to the bacteria without the addition of stress agent is represented as the mean (±SE) of two independent experiments carried out in duplicates. NS, not significant; *, <i>P</i><0.05; **, <i>P</i><0.001 and ***, <i>P</i><0.001 (One way ANOVA).</p

Oligonucleotides employed in this study.

<p>Oligonucleotides employed in this study.</p

Gross pathology of guinea pig organs infected with variou<i>s M.tuberculosis</i> strains at 10 weeks.

<p>The figure depicts representative photographs of gross pathological lesions and graphical depiction of gross scores of lung, liver and spleen of guinea pigs (n = 5) infected with <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> and MtbΔ<i>sapM</i>Comp euthanized at 10 weeks post-infection. Each data point represents the score of an individual animal, and the bars depict medians (±interquartile range) for each group. Organs of the MtbΔ<i>sapM</i> infected animals exhibited fewer and smaller pulmonary, hepatic and splenic lesions when compared with the organs of guinea pigs infected with either <i>M.tuberculosis</i> or the MtbΔ<i>sapM</i>Comp. *, <i>P</i><0.05; **, <i>P</i><0.01 (Mann-Whitney U test).</p

Gross pathological and histopathological damage in the organs of infected guinea pigs.

<p>(A) The figure depicts representative photographs of gross pathological lesions and graphical depiction of gross scores of lungs, liver and spleen of guinea pigs (n = 5) infected with wild-type <i>M.tuberculosis</i> or MtbΔ<i>end</i>Δ<i>xthA</i> euthanized at 10 weeks post-infection. The organs of guinea pigs infected with either wild-type <i>M.tuberculosis</i> or MtbΔ<i>end</i>Δ<i>xthA</i> exhibited heavy involvement with numerous coalescing tubercles and necrosis. No significant differences were observed in the gross pathological scores for the lungs, liver and spleen of guinea pigs infected with either strain. Each data point represents the score of an individual animal and the bars depict medians (± interquartile ranges) for each group. (B) The figure depicts representative photomicrographs (20×) of hematoxylin and eosin (H&E) stained 5 µm sections of lung and liver of guinea pigs infected with either wild-type <i>M.tuberculosis</i> or MtbΔ<i>end</i>Δ<i>xthA</i> euthanized at 10 weeks post-infection. Similar histopathological damage was observed in the lung and liver of guinea pigs infected with MtbΔ<i>end</i>Δ<i>xthA</i> or wild-type <i>M.tuberculosis</i>. Infected animals exhibited numerous foci of granulomatous infiltration. The scale bars depict 500 µm.</p

Disruption of both the AP endonucleases impairs the growth of <i>M.tuberculosis</i> in THP-1 macrophages.

<p>THP-1 cells were infected with wild-type <i>M.tuberculosis</i>, MtbΔ<i>end</i>, MtbΔ<i>xthA</i> or MtbΔ<i>end</i>Δ<i>xthA</i> at an MOI of 1∶5 (bacteria∶macrophage). The number of intracellular viable bacteria was determined on each alternative day for 6 days. A significant attenuation in the growth of MtbΔ<i>end</i>Δ<i>xthA</i> in comparison to wild-type <i>M.tuberculosis</i> was observed on the 4^th and 6^th day of infection. The growth of single mutants MtbΔ<i>end</i> and MtbΔ<i>xthA</i> was comparable with wild-type <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).</p

Gross pathology of guinea pig organs infected with various <i>M.tuberculosis</i> strains at 16 weeks.

<p>The figure depicts representative photographs of gross pathological lesions and graphical depiction of gross scores of lung, liver and spleen of guinea pigs (n = 5) infected with <i>M.tuberculosis</i>, MtbΔ<i>sapM</i> and MtbΔ<i>sapM</i>Comp euthanized at 16 weeks post-infection. Each data point represents the score of an individual animal, and the bars depict medians (±interquartile range) for each group. Organs of the MtbΔ<i>sapM</i> infected animals exhibited minimal involvement with the presence of only a few visible tubercles when compared with the organs of guinea pigs infected with either <i>M.tuberculosis</i> or the MtbΔ<i>sapM</i>Comp that exhibited a heavy involvement with numerous large sized tubercles and necrosis. *, <i>P</i><0.05; **, <i>P</i><0.01 (Mann-Whitney U test). Missing data points represent the animals that succumbed to disease before the time of euthanasia.</p

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).</p