J Bacteriol Virol.  2020 Dec;50(4):246-256. 10.4167/jbv.2020.50.4.246.

Recombinant Rv0753c Protein of Mycobacterium tuberculosis Induces Apoptosis Through Reactive Oxygen Species-JNK Pathway in Macrophages

• Lee KI
• Choi S
• Choi HG
• Kebede SG
• Dang TB
• Back YW
• Park HS
• Kim HJ

• KMID: 2512144
• DOI: http://doi.org/10.4167/jbv.2020.50.4.246

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most important infectious diseases worldwide. Mtb and its culture filtrates or sonic extracts induce apoptosis in macrophages. However, there is a little known about Mtb components that modulate apoptosis and their regulating mechanism. We identified Rv0753c protein with apoptotic potential through searching the biologic active proteins from the multidimensional fractions of Mtb culture filtrate. Here, we investigated the apoptotic effects of Rv0753c on RAW264.7 cells. The recombinant Rv0753c induced RAW264.7 cells apoptosis in a caspase-9-dependent manner. Dissipation of the mitochondrial transmembrane potential (ΔΨ m ), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with Rv0753c. Enhanced reactive oxygen species (ROS) production was required for Rv0753c-mediated apoptosis. Furthermore, ROS-mediated JNK activation was major signaling pathway for Rv0753c-induced apoptosis. Moreover, Rv0753c-mediated apoptosis is dependent on TLR4. Altogether, these results suggest that Rv0753c induce apoptosis through ROS-JNK signaling pathway in RAW264.7 cells.

Keyword

Mycobacterium tuberculosis; RAW264.7 cells; Apoptosis; ROS; Rv0753c

Figure

• 

  Fig. 1 Rv0753c induces caspase-dependent apoptosis of RAW264.7 cells. (a) The purified 10 µg/mL of Rv0753c protein was subjected to SDS-PAGE and western blot analysis using a mouse anti-His antibody. (b) The apoptosis of RAW264.7 cells treated with Ag85 (10 µg/mL), STS (50 nM), and Rv0753c (1, 5, and 10 µg/mL), and the cells were harvested after 24 h. The macrophage cells were then stained with annexin V/PI. The percentage of cells in each quadrant is indicated. Data are expressed as the mean ± SD of at least three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 cells treated with media (MC, medium control) compared to cells treated with antigens. n.s., no significant difference. (c) The DNA fragmentation of RAW264.7 cells incubated with Ag85 (10 µg/mL), STS (50 nM), and Rv0753c (10 µg/mL) for 24 h were put to ELISA assay using kits for determining the levels of DNA fragmentation. Data are expressed as the mean ± SD of at least three experiments. **p < 0.01, ***p < 0.001 cells treated with media (MC, medium control) compared to cells treated with antigens. (d) Immunoblotting analysis of caspase-3 and -9, and PARP in RAW264.7 cells treated with Ag85 (10 µg/mL), LPS (100 ng/mL), Rv0753c (10 µg/mL), and STS (50 nM) for 24 h. The cell lysates were separated by SDS-PAGE, followed by immunoblotting analysis using antibodies against caspase-3, caspase-9, PARP, and β-actin. (e) RAW264.7 cells were incubated with Rv0753c (10 µg/mL) in the presence or absence of z-VAD-fmk (50 µM) for 24 h. The amounts of DNA fragmentation were then measured by ELISA. The results are expressed as the mean ± SD of three independent experiments. ***p < 0.001 cells incubated with Rv0753c only compared to those incubated with Rv0753c + z-VAD-fmk.

• 

  Fig. 2 Rv0753c induces ΔΨm collapse of RAW264.7 cells. (a) RAW264.7 cells were incubated with Ag85 (10 µg/mL), STS (50 nM), and Rv0753c (10 µg/mL) for 24 h. The macrophage cells were then stained with DiOC6 (10 nM). A loss of ΔΨm was assessed by flow cytometry and the percentage of positive cells is shown in each panel. The data are shown as the mean ± SD of three independent experiments. *p < 0.05, and **p < 0.01 media control (MC) cells compared to cells treated with antigens. n.s., no significant difference. (b) RAW264.7 cells were treated with or without Rv0753c (10 µg/mL) for 24 h. The levels of Bax and cytochrome c (Cyt c) in the cytosolic (Cyto) and mitochondrial (Mito) fractions were measured by immunoblotting. The expression of VDAC and α-tubulin were used as the loading controls of the mitochondrial and cytosolic fractions. The results are representative of three independent experiments.

• 

  Fig. 3 Rv0753c induces apoptosis through ROS production. (a) RAW264.7 cells were treated with Rv0753c (10 µg/mL), Ag85 (10 µg/mL), and H2O2 (500 nM) in the presence or absence of NAC (10 mM) for 24 h. The ROS levels were measured by flow cytometry after DCFDA (10 µm) treatment. The percentage of proliferating cells is shown in each panel. The data are shown as the mean ± SD of three independent experiments. **p < 0.01 MC compared to cells treated with antigens, ##p < 0.01 and ###p < 0.001 compared each antigen to the NAC pre-treated cells. n.s., no significant difference. (b) The apoptosis of RAW264.7 cells treated with the antigens in the presence or absence of NAC (10 mM) was measured by flow cytometry using Annexin V/PI staining. The macrophage cells were then stained with annexin V/PI. The percentage of cells in each quadrant is indicated. (c) RAW264.7 cells were pre-treated with NAC (5, 10, and 20 mM) for 1 h prior to incubation with Rv0753c (10 µg/mL) for 24 h. The quantity of DNA fragmentation were then measured by ELISA. Data are presented as the mean ± SD of three experiments. ***p < 0.001 compared to Rv0753c alone with each NAC pre-treated cells.

• 

  Fig. 4 Rv0753c-mediated JNK activation is associated with ROS production and apoptosis. (a) For MAPKs activation analysis, RAW264.7 cells were treated with 10 µg/ml Rv0753c for the indicated times. The cell lysates were analysed using an immunoblot analysis was performed using specific antibodies. The images show a representative example of three independent experiments showing similar results. (b) RAW264.7 cells were pre-incubated with NAC (5, 10, and 20 mM) for 1 h and then treated with Rv0753c (10 μg/ml) for 15 min. The cell lysates were analysed using an immunoblot analysis was performed using specific antibodies. The images show a representative example of three independent experiments showing similar results. (c) RAW264.7 cells were incubated with ERK inhibitor (U0126), p38 inhibitor (SB203580), and JNK inhibitor (SP600125) 10 µM for 1 h prior to the treatment with Rv0753c (10 µg/mL), or with UT (DMSO; vehicle control with Rv0753c). After 24 h, screening for the induction of apoptosis was performed using Annexin V/PI staining. Flow cytometric histograms are represented as the mean ± SD of three independent experiments. ***p < 0.001 Rv0753c alone compared to cells incubated with Rv0753c co-treated with MAPK inhibitors. n.s., no significant difference.

• 

  Fig. 5 Rv0753c induces apoptosis through TLR4 in RAW264.7 cells. RAW264.7 cells were transfected with control siRNA (200 nM), TLR2 siRNA (200 nM), and TLR4 siRNA (200 nM) and incubated for 6 h. The transfected RAW264.7 cells stimulated with Rv0753c (10 µg/mL) for 24 h. Apoptotic cells were assessed by flow cytometry and the percentage of annexin V/PI positive cells is shown in each panel. Data are the mean ± SD from three independent experiments. *p < 0.05 Rv0753c alone compared to cells incubated with Rv0753c, transfected siRNA TLR4 cells, and Rv0753c with transfected siRNA TLR2 cells n.s., no significant difference.

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