MPT64 is one of the secreted proteins from Mycobacterium tuberculosis. Little is known about its role in infection by Mycobacterium tuberculosis. In this study, we demonstrated that MPT64 could dose-dependently inhibit the apoptosis of RAW264.7 macrophages induced by PPD-BCG. Quantitative real-time PCR results showed that the expression of bcl-2 increased in macrophages treated with MPT64 compared with PPD-treated cells. Furthermore, the results provided strong evidence that bcl-2 up-regulation was positively controlled by miRNA-21. Finally, NF-κB was identified as the transcription factor for miRNA-21 using a ChIP assay. It can be concluded from our study that MPT64 could inhibit the apoptosis of RAW264.7 macrophages through the NF-κB-miRNA21-Bcl-2 pathway.

Citation: Wang Q, Liu S, Tang Y, Liu Q, Yao Y (2014) MPT64 Protein from Mycobacterium tuberculosis Inhibits Apoptosis of Macrophages through NF-kB-miRNA21-Bcl-2 Pathway. PLoS ONE 9(7): e100949. https://doi.org/10.1371/journal.pone.0100949

Editor: Riccardo Manganelli, University of Padova, Medical School, Italy

Received: February 26, 2014; Accepted: June 2, 2014; Published: July 7, 2014

Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This article is supported by the grants of National Natural Science Foundation (Nos. 31070121). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Infection with Mycobacterium tuberculosis (M.TB) remains a major cause of morbidity and mortality throughout the world, resulting in 3 million deaths and over 9 million new cases of tuberculosis each year [1]. The increased emergence of multidrug-resistant (MDR) strains of M.TB and co-infection with HIV have complicated the threat [2]. Hunting for new strategies to control TB requires a better understanding of the complex interactions between the host macrophages and Mycobacterium tuberculosis. To escape the host's immune response, M. tuberculosis utilizes many strategies to manipulate infected host macrophages. After phagocytosis by macrophages, Mycobacterium tuberculosis tries to survive and replicate in macrophages using different strategies that can resist fusion of the phagosome with the lysosome to avoid killing [3]. Recent research has focused on the interactions between host macrophages and Mycobacterium tuberculosis.

On one hand, macrophages may undergo apoptosis after the phagocytosis of Mycobacterium tuberculosis, which will result in the death of bacilli. Some reports demonstrate that apoptosis is an important way to kill Mycobacterium tuberculosis [4]–[6]. Apoptotic macrophages are abundant in human granulomas, and virulent strains of mycobacteria are less apoptogenic than attenuated strains are [7]–[8]. On the other hand, some components of Mycobacterium tuberculosis can inhibit the apoptosis of macrophages, which is helpful for the persistence of bacilli. Hence, apoptosis inhibition seems to be a virulence factor that is associated with the up-regulation of anti-apoptotic molecules and down-regulation of pro-apoptotic molecules.

In mycobacteria, several anti-apoptotic components have been identified, including SecA2, Rv3654v, Rv3655c and protein kinaseE (PknE) [9]–[11]. MPT64 is one of the proteins secreted from Mycobacterium tuberculosis, but little is known about its role during Mycobacterium tuberculosis infection. Here, we report that MPT64 protein from Mycobacterium tuberculosis can inhibit the apoptosis of RAW264.7 macrophages in vitro. The mechanism includes the up-regulation of bcl-2, the involvement of increased miRNA21 and the control of the transcription factor NF-κB.

RAW264.7 cells were cultured at 37°C and 5% CO2 in RPMI1640 supplemented with 10% FBS, streptomycin and penicillin. RAW264.7 cells were induced with phorbol myristate acetate (PMA) (Sigma) at 10 ng/ml final concentration for approximately 24 hours.

The mature coding region of mpt64 from M. tuberculosis was cloned into the BamH1 and Xhol1 sites of plasmid vector pGEX5T. MPT64 protein was produced as a GST-tagged recombinant protein. The recombinant protein was purified from E. coli k802 by affinity chromatography with Glutathione Sephrose-4B.

To remove the nonspecific effect of LPS, the purified MPT64 protein was treated by polymyxin B (Sigma) according to the manufacturer's instruction. Then, LPS was tested using a Limulus Assay Kit [GenScript (Nanjing) Co].

Purification of MPT64 was confirmed by Coomassie Brilliant Blue R-250 and by immunoblotting using a mAb specific for MPT64 (MyBiosource, San Diego, CA). The recombinant MPT64 protein was transferred onto a nitrocellulose membrane, and the membrane was probed with a 1∶1000 dilution of MPT64-specific antibody and with horseradish peroxide-conjugated secondary antibodies at a dilution of 1∶2000. Secondary antibody binding was detected using DAB substrate.

PMA-differentiated RAW264.7 cells were put into 24-well flat bottom tissue culture plates at a density of 1×105 cells per well. The cells were incubated at 37°C. Cells were washed, and medium was replaced 4 hours before treatment. Cells were incubated with BCG-PPD (10 µg/ml), or mixtures of PPD (10 µg/ml) and MPT64 at different concentrations from 10 to 20 µg/ml, respectively. To control the nonspecific effects of the GST tag itself, control cells were incubated with GST protein (20 µg/ml). Furthermore, the groups included treatment with heat-inactivated MPT64 (20 µg/ml) because the MPT64 was produced in E. coli. The different groups were incubated at 37°C and 5% CO2 for up to 16 hours before the apoptosis assay.

The apoptosis assay was performed with an Annexin V-FITC apoptosis detection kit (BD Bioscience). The treated macrophages were collected after cold PBS washing and digestion with trypsin at a concentration of 0.25%. After washing twice, the cells were resuspended in binding buffer. An aliquot of 500 µl was removed and mixed with 5 µl of Annexin V-FITC and 5 µl of Propidium iodide (PI). The mixture was incubated for 10 min at room temperature in the dark. Finally, the cells were analyzed by flow cytometry. For each condition, 10,000 events were collected, and the percentage of AnnexinV-positive and PI-negative cells was determined. The experiments were repeated three times, and the average values and standard deviations were calculated.

Cell lysates were centrifuged for 10 min at 4°C, and the supernatant was obtained. The protein concentration was determined using the Lowry method, and 30 µg protein was used for 15% SDS-PAGE. The separated proteins were electrophoretically transferred onto polyvinyldene difluoride (PVDF) membranes. After blocking with 5% non-fat milk in PBS, the membranes were probed overnight at 4°C with mAb against Bcl-2 at a dilution of 1∶1000. The bound antibodies were detected by incubation for 1 h at 37°C with horseradish peroxide-conjugated secondary antibodies (dilution 1/1000) for 1 h. Reactive bands were detected using an ECL chemiluminescence system (Santa Cruz).

Quantitative real-time polymerase chain reactions (QRT-PCR) were performed to confirm the differential expression of selected genes among the different groups. Primer sequences were designed based on alignments of candidate gene sequences. RNA samples were treated with DNA-free (Takara), following the manufacturer's instructions, to remove contaminating genomic DNA. Total RNA was reverse-transcribed using SuperScript II (Takara). Five microliters of the reverse transcription reaction was added to 45 µl of SYBR green PCR master mix (Takara). Forty cycles of amplification, data acquisition and data analysis were performed.

The sequences of the specific primers were as follows: 5′gtg aga agt gag gga cct tta tg 3′(forward) and 5′cac tca tta gcc ata tcc aac ttg 3′ (reverse) for bcl-2; 5′atg gag ctg cag agg atg 3′ (forward) and 5′tgt cca gcc cat gat ggt tc3′ (reverse) for bax; 5′cgg ttc cga tgc cct gag gct ctt 3′ (forward) and5′ cgt cac act tca tga tgg aat tga 3′ (reverse) for β-actin; 5′ tag ctt atc aga ctg atg ttg a 3′ for miRNA21.The PCR reaction program for bcl-2 was as follows: 1 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 15 sec at 57°C and 15 sec at 72°C. The PCR reaction program for bax amplification was as follows: 1 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 15 sec at 57°C and 15 sec at 72°C. The PCR reaction program for miR21 amplification was as follows: 1 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 15 sec at 57°C and 15 sec at 72°C.

The primers for bcl-2 3′UTR were designed as follows: (1) bcl2 3′UTR wild type primers (bcl2-utr-wt), forward primer, 5′cca ctg aga ctt ccc tgc tga 3′, and reverse primer, 5′ tgg gca cta cct gcg ttc 3′; (2) bcl2 3′UTR mutant primers (bcl2-utr-mut), forward primer, 5′ttc acg tac caa ttg tgc cga g 3′, and reverse primer, 5′ tgg gca cta cct gcg ttc 3′. The amplified sequences were inserted into the SpeI and HindIII sites of the pMIR-report™ luciferase vector, respectively.

RAW264.7 macrophages were seeded at 4×104 cells/well in flat-bottom tissue culture plates and co-transfected with 800 ng of pMIR-report-bcl2-utr-wt, 80 ng of pRL—TK (Promega) and 20 pmol miRNA21 by lipofectamine™ 2000 (Promega). The recombinant mutant plasmid pMIR-report-bcl2-utr-mut was also transfected using this method. miRNA-335 was used as control miRNA. The luciferase assay was performed using a Luciferase detection kit (Promega), as previously described [12]. Three wells were used for each Luciferase Assay sample.

ChIP experiments were performed in RAW264.7 cells using the ChIP assay kit (Millipore), as previously described [9], and NF-κB rabbit Ab (C-20, Santa Cruz Biotech, Inc.). Primers specific to the mouse miR-21 promoter are listed in Table 1.

https://doi.org/10.1371/journal.pone.0100949.t001

siRNA specific for bcl-2 or NF-κB was synthesized by GenePharma Co. The siRNA sequences for bcl-2 [13] were as follows: sense strand, 5′-AAGUACAUACAUUAUAAGCUG-3′; antisense strand, 5′CAGCUUAUAAUGUAUGUACUU-3′.The siRNA sequences for NF-κB p65 [14] were as follows: sense strand, 5′-AAGAAGCACAGAUACCACCAA-3′; antisense strand,5′ –UUGGUGGUAUCUGUGCUUCUU-3′.The siRNAs were dissolved in RNase-free water to a final concentration of 20 µM.

One day before transfection, RAW264.7 cells were seeded in 2 ml of complete medium without antibiotics in a 6-well plate, so the macrophages could reach 30–50% confluency for transfection. The transfection was performed according to the manufacturer's instruction. A total 5 µl of siRNA was added to 250 µl of Opti-MEM (Invitrogen), and 2.5 µl of Lipofectamine 2000 (invitrogen) was diluted in the same amount of medium. After incubation for 5 min at room temperature, the diluted siRNA was mixed gently with diluted Lipofectamine 2000 and incubated for 20 min at room temperature. The mixture was then added to the plates with 1.5 ml of serum-free, antibiotic-free medium.

After 16 h of transfection, apoptosis of RAW264.7 cells was detected. The cells were harvested, and total RNA was isolated with Trizol reagent (Takara), according to the manufacturer's instructions. Total RNA was reverse-transcribed using SuperScript II (Takara), followed by dilution of the reverse transcription products to 1∶20. Five microliters of the reverse transcription reaction was added to 45 µl of SYBR green PCR master mix (Takara). The sequences of the specific primers were: 5′gtg aga agt gag gga cct tta tg 3′(forward) and 5′cac tca tta gcc ata tcc aac ttg 3′ (reverse) for bcl-2; 5′cgg ttc cga tgc cct gag gct ctt 3′ (forward) and5′ cgt cac act tca tga tgg aat tga 3′ (reverse) for β-actin. The PCR reaction program was as follows: 1 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 15 sec at 57°C and 15 sec at 72°C. The levels of individual mRNA transcripts relative to the control were calculated using 2−△△ct. The experiment was repeated three times.

All data are represented as the means±SEM from three separate experiments.The difference between two groups was analyzed using Student's t-test.Differences were considered significant at P