Mycobacterial escape from macrophage phagosomes to the cytoplasm represents an alternate adaptation mechanism

Abstract

Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.

Figure 1. Mtb strain-dependent differences in intracellular localization.

(A) Representative Transmission electron micrographs of infected cells showing Mtb existing in absence of a vesicle (i), (ii), (iii) in comparison to Mtb in vesicle structures (iv). Black arrows emphasize contiguity of the bacterial wall with the macrophage cytoplasm. Distinct vesicles (marked by red arrows), (iv) for certain vesicle associated strains of Mtb. Left side panels are low power images of the macrophage showing Mtb infection. Right side panels are blown up images of adjacent images (black box) showing Mtb in vesicle free and vesicle associated forms. Magnification: 2 um and 200 nm (i), 500 nm and 20 nm (ii), 2 um and 200 nm and (iii) and 200 nm and 500 nm (iv). More than 100 cells were investigated from multiple experiments. (B) Representative transmission electron micrographs showing Mtb particles (yellow arrows) in phagosomal preparations. The two upper figures show bacteria that are closely associated with vesicular membranes. The lower micrographs show free bacteria as identified by yellow arrows. More than 100 cells were viewed from 3 separate experiments (C) 3D Visualization of intracellular bacteria using electron tomography. Top panel shows a vesicle-enclosed H37Rv bacterium (top, left panel), which was reconstructed using isocontour volume rendering (top, right panel). The bottom panel shows a cytoplasmic JAL2287 bacterium (left panel), where a similar volume rendering shows clear intracellular electron dense bodies and continuity with the cytoplasmic mass (right panel). “M” refers to Mtb. (D) Relative proportion of intracellular bacteria that were cytosolic in cells infected for 24 hr with each of the strains. Values were determined by counting bacteria in >100 cell sections (mean ± S.D.). Bacillary load was enumerated after staining of the cells with Ziehl-Neelsen stain, and results are given in panel E as the mean (±S.E.) value for >200 cells.

Figure 2. Strain-distinctive intracellular niche preferences are also retained in vivo.

Groups of eight mice each were infected through the aerosol route with the individual Mtb strains (100–150 bacilli/lung13). At 15 days later these mice were sacrificed and the lungs harvested. Lungs from two mice in each group were taken for TEM imaging and panels (A,B) shows the representative micrographs obtained. Panel (A) shows two representative images of lung cells containing phagosome-enclosed bacteria for the H37Rv (marked by black box) where phagosomal membranes (marked by red arrows) surrounding the bacteria can be distinguished from the bacterial cell wall (marked by yellow arrows). Lower image shows the blown up Mtb structure, boxed (black) in the upper low power shot showing the lung field. Magnification–1 um. 70–100 cells were observed in lung sections for all lung investigations. Panel B confirms cytosolic residence of JAL2287 and MYC431 bacteria in the lung cell field (marked by black box). Upper panels represent low power images showing the lung macrophages followed by their respective high power shots in the lower panels. Continuity of the bacterial wall (black arrows) of the Mtb (Black box) from the low power is shown in the images. Magnification–500 nm and 1 um (upper) and lower–200 nm in both cases. Panel (C-(i)) and (C-(ii)) show the blown up images showing distinct absence and presence of vesicle structure in lung macrophages, for the indicated strains respectively. Yellow arrows mark the bacterial wall, whereas, the vesicle membrane is marked by red arrows. Field from left side images of both panels (marked by boxes-red and black) are blown up to show the Mtb structures in the adjacent panels Magnification: 500 nm. Lysates were generated from lungs of the remaining six mice per group, and plated for determination of the Cfu values (D). Values are the mean ± S.D.

Figure 3. Time-course study of THP-1 cells infected with H37Rv and JAL2287.

(A) Representative images of cells infected with either H37Rv (i–iii) or JAL2287 (iv–vi) and a TEM analysis was performed at various times thereafter. Results shown are for 2 (i, iv), 24 (ii, v), and 36 (iii, vi) hours p–i. Magnification, 500 nm for (i), (v) and (vi); 1 nm for (ii) and (iii), 100 nm for (iv). >200 cells were observed from multiple infections. (B) Comparison of time-dependent changes in bacterial load (green line) versus the corresponding changes in the proportion of cytoplasmic bacteria (purple line) in JAL2287-infected cells. The Y-axis gives either the bacterial load (cfu/well (x10³)), or the cytosolic bacteria as a percent of the total number of bacteria/cell. (C) A composite of images reflecting both cytoplasmic (white box) and vesicle-associated bacteria in cells after a 1 hr infection period with JAL2287 (top left panel, magnification–2 um). The boxed regions are also shown at a higher magnification (bottom and right panels, magnification 500 and 200 nm respectively) to confirm their cytoplasmic localization. The “bulls eye” appearance characteristic of Mtb is also evident here. >200 cells were observed from multiple experiments. (D) Another phagosome from this experiment (magnification, 100 nm) where one of the bacteria appears to be in the “entry mode” (white arrows), while another appears exiting the phagosome (white box), with its membrane fused to the phagosome (black arrows) membrane. Non-specific debris that may be of bacterial origin can be seen. (E) PED-6 fluorescence was monitored at 24 hr p–i. by confocal microscopy in infected THP1 cells. The mean fluorescence intensity (MFI)/cell of PED6 hydrolysis/cell is expressed in infected cells, values are expressed as a ratio over the corresponding value obtained for uninfected cells (>200 cell observed, ± S.D., n = 3). Panel F shows the results of a linear regression analysis performed between the values of PED-6 fluorescence intensities in panel E (PF6), and the percent cytosolic bacteria obtained in Fig. 1D. The positive correlation implies a link between host cytosolic phospholipase A2 activation and phagosomal escape of Mtb.

Figure 4. Cytosolic translocation of Mtb is facilitated through PLA₂ activation.

(A) THP-1 cells were infected with the Mtb strains either in the absence (−) or presence (+) of the PLA₂ inhibitor AACOCF3. At 24 hrs later, the cells were fixed and processed for TEM. The percent of intracellular bacteria distributed between the endosomal (blue) and cytosolic (red) compartments was determined by averaging the values from more than 100 cell sections in each case. In panel (B), Mtb-infected THP-1 cells were either left untreated (blue line) or treated (red line) with AACOCF3. At indicated times, cells were lysed and the intracellular bacillary load determined in terms of the cfu counts. (C) Infected THP-1 cells were either left untreated or treated with rapamycin for 6 hrs. Lysates were then generated at 60 hrs p–i, and the bacterial load determined in terms of the cfu counts. Results are expressed as a percent reduction in cfu values in rapamycin treated cells, compared to those in the untreated cells in each case (mean ± S.D., n = 3). (D) Results of a linear regression analysis performed between the data in panel (C) (rapamycin sensitivity), and that in Fig. 1D (% Cytosolic bacteria). In panel (E), cells infected with the indicated Mtb strains for 24 hr were either left untreated (blue bars), or treated with rapamycin for 6 hrs (red bars). Subsequently, LC3-II levels were measured by confocal microscopy and bars show mean (±S.E.) fluorescence intensity per cell (MFI/cell). Values were averaged over 80–100 cells in each case. In Panel (F), Mtb infected cells were either left untreated (blue bars) or treated with Interferon gamma for 48 hrs (red bars). LC3-II levels were measured by confocal microscopy and bars show mean fluorescence intensity per cell (±S.E) after averaging the values obtained for over 100 cells in each case.