Cytosolic access of Mycobacterium tuberculosis: critical impact of phagosomal acidification control and demonstration of occurrence in vivo

Abstract

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving--as recently confirmed--escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.

Fig 1. Detection of Mtb-mediated phagosome disruption by flow cytometry.

(A) Phagosomal rupture detected by CCF-4 FRET-based flow cytometry. Differentiated THP-1 cells were infected with Mtb, WT or ΔESX-1 strain (MOI = 1); NI = not infected. At 4 dpi, cells were successively stained with CCF-4 and anti-CD11b mAb, fixed, and their green (520 nm) vs. blue (447 nm) fluorescent signals were analyzed after gating on CD11b⁺ cells. Results are depicted as signal overlays of different groups as dot or contour blots. (B-C) Shown are ratios of MFI_{447 nm} / MFI_{520 nm} (B) and blue MFI_{447 nm} (C), calculated as described in Materials and Methods. (D) Differentiated THP-1 cells were infected with DsRed-expressing Mtb H37Rv strain (MOI = 0.3). At 4 dpi, cells were stained as in A. The cells having phagocytosed DsRed-Mtb (DsRed⁺) were first gated for their red signal and their green vs. blue CCF-4 signals were compared to the cells in the same culture that had not engulfed DsRed-Mtb (DsRed^-). The results are representative of at least 3 independent experiments.

Fig 2. Mtb-mediated phagosome disruption in different phagocyte types, relationship with infection dose and cell death.

(A) Comparative infectivity of WT and ΔESX-1 Mtb. BM-DC were infected with DsRed-WT or-ΔESX-1 strain and the red fluorescence was assessed by cytometry at 1 dpi. Percentages of cells having phagocytized DsRed-mycobacteria are indicated. (B) Detection of phagosomal rupture subsequent to infection with WT or ΔESX-1 strains, as determined by green vs. blue CCF-4 signals in BM-DC or BM-MΦ, infected with untagged Mtb, WT or ΔESX-1 (MOI = 1) at different time points, as detected after exclusion of cell debris and free bacteria by FSc/SSc gating and inclusion of CD11b⁺ cells. (C) MFI_{520 nm}, MFI_{447 nm} and MFI_{447 nm}/MFI_{520 nm} ratios in infected BM-DC or BM-MΦ at different time points. (D) Phagosomal rupture, monitored at 4 dpi by CCF-4 staining, in BM-DC infected with different MOI of WT or ΔESX-1 Mtb. (E) Percentages of dead cells, as determined by the use of Pacific Blue Dead/Live reagents, compared to those of cells displaying a CCF-4 blue shift. Due to the emission overlap of CCF-4-Coumarin and Pacific Blue fluorochromes, the two different assays were performed in separate tubes in parallel, in cells from the same BM-DC cultures. The results are representative of 2 independent experiments. Of note, the decrease of the dead cell percentage for WT Mtb at very high MOI is likely due to generation of cell debris, not anymore measurable by cytometry.

Fig 3. Early Mtb-mediated phagosomal rupture, relationship with secretion of type I IFNs.

(A) BM-DC were infected with Mtb WT at different MOI and the phagosomal rupture was assessed by CCF-4 staining at early time points of 3, 24 and 48 hpi. (B) MFI_{447 nm} of the infected cells at each time point and for different MOI of WT or ΔESX-1 strain. (C) IFN-α and-β concentrations, as quantified in the supernatants of the same infected cells by ELISA. *, ** = statistically significant, p<0.01 or p<0.001, respectively, as determined by the Student's t test. (D) Linear relationship between the amounts of IFN-β produced and Mtb-induced phagosomal rupture in BM-DC. Shown are representative data from 2 independent experiments.

Fig 4. Host Nramp-1 transporter counteracts the phagosomal rupture in Mtb-infected MΦ.

(A) Raw264.7 cells, parental or transfected with non-functional nramp-1S or functional nramp-1R allele, were infected with Mtb, WT or ΔESX-1 (MOI = 1). At 3 dpi, phagosomal rupture was monitored in CD11b⁺ cells. (B, C) The blue CCF-4 signal overlays (B) and MFI_{447 nm} (C) are plotted for different Raw264.7 cell lines infected with Mtb WT. ** = statistically significant, as determined by the Student's t test, p<0.001. (D) Mycobacterial loads in different Raw264.7 cell lines, infected with various MOI of WT Mtb, as determined at 3 dpi. (E-G) Raw264.7::nramp-1R were transfected with Nramp-1-specific or scramble siRNA and the effective gene silencing was checked 3 days later by qRT-PCR (E). The siRNA-treated Raw264.7::nramp-1R cells were then infected with WT Mtb (MOI = 1) and studied for phagosomal rupture at 3 dpi. The blue CCF-4 signal (F) and the MFI_{447 nm} (G) are plotted. The results are representative of at least 3 experiments.

Fig 5. Inhibition of phagosomal acidification intensifies phagosomal rupture in Mtb-infected phagocytes.

(A-B) Raw264.7::nramp-1R cells (A) or BM-DC from Sv129 nramp-1R mice (B) were treated with 20 nM of bafilomycin or DMSO 1h before infection with WT or ΔESX-1 Mtb (MOI = 1) and were assessed for phagosomal rupture at 4 dpi. (C) The intrinsic β-lactamase activity of WT Mtb, grown in Dubos broth with various pH, as measured by nitocephin, a chromogenic β-lactamase substrate. The results are representative of 2 experiments.

Fig 6. Mtb-mediated phagosomal rupture in different cell subsets ex vivo and in vivo.

(A) Low-density cells were isolated from C57BL/6 mouse lung parenchyma and infected ex vivo with WT or ΔESX-1 Mtb (MOI = 1). Monocytes/MΦ (CD11b⁺ CD11c^-) and DC (CD11b^int CD11c⁺) were assessed for CCF-4 signals at 4 dpi. (B) Phagosomal rupture detected in vivo in different Mtb-infected phagocyte subsets. C57BL/6 mice (n = 3) were injected i.v. with 1 x 10⁶ CFU of DsRed WT Mtb and at 3 weeks post infection. Alive low-density cells were isolated on Optiprep gradient from the spleen and were sequentially stained with CCF-4 and a cocktail of mAbs to distinguish neutrophils (CD11b^hi CD11c^- Ly6G⁺), MΦ/monocytes (CD11b^int CD11c^- Ly6G^-) or DC (CD11b^lo CD11c⁺ Ly6G^-). (C) Inside each innate cell subsets, the blue CCF-4 signals of the DsRed⁺ and DsRed^- cells were compared together. The results are representative of 2 independent experiments.

Fig 7. Detection of phagosomal rupture in vivo in Mtb-infected phagocytes.

(A-B) BM-DC from CD45.1 donors were left non-infected or were infected with Mtb WT or ΔESX-1 (MOI = 1) for 16h. Of note, cultures of CD45.1 BM-DC, infected with DsRed-mycobacteria in the same conditions, showed that >70% of cells had uptaken mycobacteria like shown in Fig. 2A. Cells were recovered and transferred i.n. (2 x 10⁶ cells/mouse) into CD45.2 congenic recipients. (B) Four days post-transfer, alive lung low-density cells from the recipients (n = 3/group) were isolated on Optiprep gradient and incubated with mAbs specific to CD11b and CD45.1, subsequent to incubation with CCF-4. The blue signals from the three experimental groups are overlaid. (C) Histograms show the comparative blue CCF-4 signals in the CD11b⁺ CD45.1⁺ cells from different groups at days 4 or 6 post transfer. * = statistically significant, as determined by the Student's t test, p<0.01. (D) Lung granuloma from C57BL/6 mice, infected via aerosol route with ≈200 CFU/mouse of WT Mtb, were removed at 6 weeks p.i. and were treated with collagenase and DNAse-I and enriched in low-density cells. In parallel to these cells, lung low-density cells from uninfected controls were assessed for CCF-4 blue switch. (E) CD11b vs. CD11c surface expression of the cells showing the increased CCF-4 shift (pink), compared to unstained negative control incubated with control Ig isotypes (gray). The results are representative of 2 independent experiments.