Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis.

Figure 1

MT103 induces apoptosis on MEF by activation of the mitochondrial apoptotic route. Wild-type MEF (WT) and MEF knockouts for Bax, Bak, caspases 3 and 7(C3/7 DKO), caspase 9 (C9), Bak and Bax (Bax/Bak DKO), Bim, Bid were infected with MT103 (MOI 30 : 1) during seven days. (a and b) Cells were stained with annexinV and 7-AAD, and analysed by flow cytometry. Representative dot plots are shown in the right panels. Data in the graphs (left panels) are represented as mean±S.E.M. of four independent experiments. (c) Cells were stained with an anti-active caspase 3 antibody and analysed by flow cytometry. Representative histograms are shown in the upper panel. Black and red lines represent infected-C3/7 DKO MEF and the indicated MEF types, respectively. Data in the graph (lower panel) correspond with the ratio of mean fluorescent intensities (MFI) of the different infected MEF types versus the C3/7 DKO MEF control and are shown as mean±S.E.M. of three independent experiments. (d) Seven days post infection, 150 cells per well were seeded and incubated in fresh medium during eight additional days. Survival was calculated as percentage of colonies relative to the number of colonies in the non-infected controls. Data in the graphs are represented as mean±S.E.M. of two independent experiments. Statistical analysis was done using one-way ANOVA and Bonferroni's post hoc test comparing with MT103-infected wild-type cells. *P<0.05; **P<0.01; ***P<0.001

Figure 2

MT103 upregulates Bim in J774 cells. J774 cells were infected with MT103 (MOI 10 : 1) up to 72 h. Bim expression was analysed by RT-qPCR (a), or by western blot (b) at 24 h, 48 h and 72 h post infection. (a) Data in the graph are represented as mean±S.E.M. of three independent experiments. (b) Quantification of the western blot is represented as the ratio of Bim/Actin densities. A representative blot of three independent is shown in the figure

Figure 3

Bim expression is induced by virulent M. tuberculosis. (a and b) J774 cells were infected with MT103 or the live-attenuated vaccine candidate MTBVAC (MOI 10 : 1) for 72 h. (a) Cells were stained with annexinV and 7-AAD, and analysed by flow cytometry. Data in the graph are represented as mean±S.E.M. of three independent experiments. (b) A representative blot of two independent experiments is shown. Quantification of the western blot is represented as the ratio of Bim/Actin densities. (c and d) J774 cells were infected with live or heat-killed (HK) MT103 or MTBVAC bacteria. (c) Cells were stained with annexinV and 7-AAD, and analysed by flow cytometry. Data represent the mean±S.E.M. of two independent experiments. (d) A representative blot of two independent experiments is shown. Quantification of the western blot is represented as the ratio of Bim/Actin densities

Figure 4

siRNAs targeting Bim impair MT103-induced apoptosis. (a) Bim knockdown induced by specific siRNAs was confirmed by western blot. (b and c) Non-transfected and Bim-specific siRNAs-transfected J774 cells were infected with MT103 (MOI 5 : 1 and 10 : 1) during 72 h. (b) Cells were stained with annexinV and 7-AAD, and analysed by flow cytometry. Data in the graphs from four independent experiments are represented as mean±S.E.M. of the percentage of dead cells. (c) Percentage of GFP-positive cells at 0 and 72 h post infection was determined by flow cytometry. Data are shown as mean±S.E.M. of three independent experiments. Statistical analysis was done using one-way ANOVA and Bonferroni's post hoc test. *P<0.05; **P<0.01; ***P<0.001

Figure 5

Bim induction triggered by MT103 is mediated by p38MAPK. (a) J774 cells were infected with MT103 or the live-attenuated vaccine candidate MTBVAC (MOI 10 : 1) for up to 72 h. A time course of p38MAPK phosphorylation (P-p38) was analysed by western blot at 1, 4 and 72 h post infection. A representative blot of two independent experiments is shown. Quantification of the western blot is represented as the ratio of phospho-p38MAPK/actin densities. (b and c) Cells were infected with MT103 for 72 h in the presence or absence of SB203580 10 μM. (b) Cells were stained with annexinV and 7-AAD, and analysed by flow cytometry. Data are represented as mean±S.E.M. of two independent experiments. (c) A representative blot of two independent experiments is shown. Quantification of the western blot is represented as the ratio of Bim/actin densities