Secreted acid phosphatase (SapM) of Mycobacterium tuberculosis is indispensable for arresting phagosomal maturation and growth of the pathogen in guinea pig tissues

Abstract

Tuberculosis (TB) is responsible for nearly 1.4 million deaths globally every year and continues to remain a serious threat to human health. The problem is further complicated by the growing incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizing the need for the development of new drugs against this disease. Phagosomal maturation arrest is an important strategy employed by Mycobacterium tuberculosis to evade the host immune system. Secretory acid phosphatase (SapM) of M.tuberculosis is known to dephosphorylate phosphotidylinositol 3-phosphate (PI3P) present on phagosomes. However, there have been divergent reports on the involvement of SapM in phagosomal maturation arrest in mycobacteria. This study was aimed at reascertaining the involvement of SapM in phagosomal maturation arrest in M.tuberculosis. Further, for the first time, we have also studied whether SapM is essential for the pathogenesis of M.tuberculosis. By deleting the sapM gene of M.tuberculosis, we demonstrate that MtbΔsapM is defective in the arrest of phagosomal maturation as well as for growth in human THP-1 macrophages. We further show that MtbΔsapM is severely attenuated for growth in the lungs and spleen of guinea pigs and has a significantly reduced ability to cause pathological damage in the host when compared with the parental strain. Also, the guinea pigs infected with MtbΔsapM exhibited a significantly enhanced survival when compared with M.tuberculosis infected animals. The importance of SapM in phagosomal maturation arrest as well as in the pathogenesis of M.tuberculosis establishes it as an attractive target for the development of new therapeutic molecules against tuberculosis.

Figure 1. Characterization of MtbΔsapM mutant.

(A) Confirmation of sapM gene deletion in M.tuberculosis by PCR. PCR was carried out by employing primers SapM-F and SapM-R to obtain 0.8 kb amplification in M.tuberculosis (lane 2) and 2.2 kb amplification in MtbΔsapM (lane 3). 100 bp and λHindIII markers were loaded in lanes 1 and 4, respectively. (B) Confirmation of sapM deletion in M.tuberculosis and complementation of sapM gene in the mutant by immunoblot analysis. 10 µg of culture filtrate of M.tuberculosis (lane 1), MtbΔsapM (lane 2) and MtbΔsapMComp (lane 3) were loaded on a 12% polyacrylamide gel and subjected to electrophoresis. Anti-SapM polyclonal antibody was employed to detect SapM that migrated as a protein band corresponding to a molecular mass of 28 kDa in the culture filtrate of M.tuberculosis (lane 1) and MtbΔsapMComp (lane 3) while the disruption of sapM in MtbΔsapM (lane 2) was confirmed by the absence of protein expression. (C) Disruption of sapM results in the loss of phosphatase activity. 120 µg of culture filtrate from M.tuberculosis, MtbΔsapM and MtbΔsapMComp were incubated with 20 mM pNPP and absorbance was measured at 405 nm as described in the materials and methods. Phosphatase activity was detected in M.tuberculosis and MtbΔsapMComp. However, no activity was obtained in MtbΔsapM. Data is the mean (±SE) of 3 independent experiments carried out in triplicates. ***, P<0.001 (One-way ANOVA). (D) Growth kinetics of M.tuberculosis, MtbΔsapM and MtbΔsapMComp in MB7H9 medium. Cultures were inoculated in duplicates with a starting absorbance (A_{600 nm}) of 0.05 and the growth was monitored for 12 days. There was no significant difference in the growth of any strain. The values of absorbance are represented as the mean (±SE) of two independent experiments carried out in duplicates.

Figure 2. Disruption of sapM impairs growth of MtbΔsapM in THP-1 macrophages and enhances phagosomal maturation.

(A) Influence of sapM deletion on the growth of M.tuberculosis in THP-1 cells. THP-1 cells were infected with M.tuberculosis, MtbΔsapM and MtbΔsapMComp 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 MtbΔsapM in comparison to M.tuberculosis and MtbΔsapMComp was observed from second day post-infection. The growth of MtbΔsapMComp was comparable to M.tuberculosis. The values are represented as the mean (±SE) of three independent infections and the experiment was repeated three times. ***, P<0.001 (Two way ANOVA). (B) Increased localization of MtbΔsapM in LysoTracker labeled compartments in THP-1 macrophages. Macrophages were infected with FITC labelled M.tuberculosis, MtbΔsapM and MtbΔsapMComp (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 sapM lead to accumulation of MtbΔsapM in acidified organelles (overlap of green and red images appears yellow) while the M.tuberculosis and MtbΔsapMComp were found in non-acidified organelles. The scale bars depict 5 µm. (C) Values indicate the percentage of phagosomes containing M.tuberculosis, MtbΔsapM or MtbΔsapMComp that colocalized with Lysotracker red. On infecting the macrophages with MtbΔsapM, 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 M.tuberculosis (23.1%) or MtbΔsapMComp (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. ***, P<0.001 (One way ANOVA).

Figure 3. Disruption of sapM in M.tuberculosis leads to the attenuation of the pathogen in guinea pigs.

The figure depicts bacillary load in the lungs and spleen of guinea pigs (n = 5) infected with M.tuberculosis, MtbΔsapM and MtbΔsapMComp at (A) 4 weeks, (B) 10 weeks and (C) 16 weeks post-infection. Guinea pigs infected with MtbΔsapM exhibited a significantly reduced bacillary load in the lungs as well as spleen when compared with the animals infected with either M.tuberculosis or MtbΔsapMComp. Each data point represents the Log₁₀ CFU value for an individual animal and the bar depicts mean (±SE) for each group. Missing data points represent the animals that succumbed to disease before the time of euthanasia. *, P<0.05; **, P<0.01; ***, P<0.001 (One way ANOVA).

Figure 4. Gross pathology of guinea pig organs infected with various M.tuberculosis strains at 10 weeks.

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 M.tuberculosis, MtbΔsapM and MtbΔsapMComp 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 MtbΔsapM infected animals exhibited fewer and smaller pulmonary, hepatic and splenic lesions when compared with the organs of guinea pigs infected with either M.tuberculosis or the MtbΔsapMComp. *, P<0.05; **, P<0.01 (Mann-Whitney U test).

Figure 5. Gross pathology of guinea pig organs infected with various M.tuberculosis strains at 16 weeks.

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 M.tuberculosis, MtbΔsapM and MtbΔsapMComp 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 MtbΔsapM 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 M.tuberculosis or the MtbΔsapMComp that exhibited a heavy involvement with numerous large sized tubercles and necrosis. *, P<0.05; **, P<0.01 (Mann-Whitney U test). Missing data points represent the animals that succumbed to disease before the time of euthanasia.

Figure 6. Histopathology of guinea pig organs infected with various M.tuberculosis strains at 10 weeks.

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 M.tuberculosis, MtbΔsapM and MtbΔsapMComp euthanized at 10 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). MtbΔsapM infected animals displayed a significant diminution in granulomatous infiltration in the lungs and liver when compared with numerous coalescing granulomas observed in the organs of animals infected with M.tuberculosis or MtbΔsapMComp. The scale bars depict 500 μm. *, P<0.05; **, P<0.01 (Mann-Whitney U test).

Figure 7. Histopathology of guinea pig organs infected with various M.tuberculosis strains at 16 weeks.

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 M.tuberculosis, MtbΔsapM and MtbΔsapMComp 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 M.tuberculosis or MtbΔsapMComp infected guinea pigs exhibited large areas of granulomatous inflammation, the animals infected with MtbΔsapM exhibited normal pulmonary and hepatic parenchyma. The scale bars depict 500 μm. *, P<0.05; ***, P<0.001 (Mann-Whitney U test). Missing data points represent the animals that succumbed to disease before the time of euthanasia.

Figure 8. Influence of disruption of sapM gene of M.tuberculosis on the survival of guinea pigs post-infection.

Guinea pigs aerogenically infected with 10–30 bacilli of either M.tuberculosis, MtbΔsapM or MtbΔsapMComp were monitored for survival (n = 10) up to 210 days post-infection. In case, the animals were infected with MtbΔsapM, 100% of the animals survived. While no animal survived in the M.tuberculosis infected group, 40% survivors were observed in case the animals were infected with MtbΔsapMComp. The median survival time for each infected group is mentioned in brackets. * represents the significant difference in comparison to the MtbΔsapM. **, P<0.01; ***, P<0.001 [Log-rank (Mantel-Cox) Test].