The human non-small-cell lung carcinoma A549 cell line was obtained from The Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in modified RPMI-1640 medium (HyClone, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, New Zealand) at 37°C in a humidified incubator with a 5% CO₂ atmosphere.

The wild-type strain of M. smegmatis MC₂155 strain was obtained from the Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University. Cells were grown in 7H9/7H10 Middlebrook (BD, USA) broth supplemented with 0.05% Tween 80, OADC (BD, USA), and 0.2% glycerol (v/v). Cells were grown at 37°C with continuous agitation (220 rpm).

To produce recombinant HBHA (rHBHA) in Escherichia coli, the corresponding genes were PCR amplified using M. tuberculosis H37Rv (ATCC27294) DNA as the template and the following oligonucleotide primers: F primer (5′-CACGGATCCATGGCTGAAAACTCGAACAT-3′) and R primer (5′-CTGAAGCTTACTACTTCTGGGTGACCTTC-3′). The forward and reverse primers contained the underlined BamHI and HindIII restriction sites, respectively. The PCR products were digested with BamHI and HindIII and cloned into the PQE80L vector (Laboratory Animal Center, The Fourth Military Medical University), and the resulting clones were sequenced (Sangon Biotech). E. coli strain BL21 (DE3), which expresses His-tagged protein, was grown in Luria–Bertani broth supplemented with 100 μg/ml ampicillin. After induction with 0.3 mM isopropy-β-D-thiogalactoside for 5 h, the cells were lysed by sonication for 30 min. The rEC-HBHA was purified by heparin–Sepharose chromatography and further purified using a His-GraviTrap purification kit (GE Healthcare, USA) in accordance with the manufacturer's instructions.

The fast-growing, nonpathogenic M. smegmatis MC₂155 strain (Snapper et al., 1990), which lacks the hbhA gene, was electroporated to obtain a mycobacteria recombinant strain that could be easily manipulated in most laboratories (Delogu et al., 2004). The shuttle plasmid vector pMV261 or pMN437, which contains GFP promoter and the full-length hbhA gene, was transformed through electroporation and grown in 7H9 broth containing kanamycin (15 μg/ml) and hygromycin B (20 μg/ml). Such vector was obtained from Dr. Babak Javid of the Medical College of Tsinghua University. E. coli strain BL21 (Takara, Japan) was used for transformation and protein expression and cultivated under standard conditions. The purified protein of HBHA was prepared by our laboratory. The hbhA gene was excised using the enzymes BamHI and HindIII and cloned at the same site of the mycobacterial shuttle vector pMV261. Alternatively, the hbhA gene was excised using the enzymes ClaI and HindIII, cloned at the same site of the pMN437 vector, and electroporated (2.5 kV, 25 μF, and 1000 Ω) for the preparation of M. smegmatis competent cells using standard techniques (Bardarov et al., 1997).

To analyze the formation of autophagosome, A549 cells were starved for 90 min and subsequently treated with HBHA (8 μg/ml) for 90 min or 3-MA (100 μg/ml), an inhibitor of autophagy, for 4 h. For the infection assay, A549 cells were infected with MS-GFP and rMS-HBHA-GFP at the multiplicity of infection (MOI) of 10:1 for 18 h. Afterward, the A549 cells were washed three times with PBS and treated with 50 μM MDC in an incubator for 15 min. The cells were again washed three times with PBS and then immediately observed under a laser confocal microscope (FV10i, Olympus, Tokyo, Japan).

The A549 cells were cultured in glass bottom cell culture dish (NEST, Hong Kong, China) and starved for 90 min. Afterward, A549 cells were treated with HBHA (8 μg/ml) for 90 min or 3-MA (100 μg/ml) for 4 h. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were treated with 0.3% Triton-100 20 min at room temperature to increase permeability. Subsequently, they were blocked with heat-inactivated fetal bovine serum (Gibco, New Zealand) for 30 min at room temperature. The cells were incubated with polyclonal antiLC3 antibody at 4°C overnight. After washing with PBS, cells were incubated with antirabbit Cy3 fluorescent secondary antibody (1:500 final dilution; BBI Life Sciences, China), and they were observed by laser confocal microscopy (FV10i, Olympus, Tokyo, Japan).

To detect the expression of the key proteins of autophagy, A549 cells were starved for 90 min and treated with HBHA of different concentrations for 90 min or with rapamycin or rapamycin plus 3-MA (100 μg/ml) for 4 h. To confirm the effect of HBHA on the autophagic flux of A549 cells, cells were treated with 350 nM bafilomycin A1 (BAF A1; Abcam, USA) to prevent lysosomal degradation. For caspase-3 detection, A549 cells were treated with HBHA for 18 h in the presence or absence of caspase inhibitor Z-VAD-FMK (20 μM). The cells were then lysed in cell lysis buffer with phenylmethylsulfonyl fluoride, phosphatase inhibitor, and protease inhibitor according to the instructions of the manufacturer of the protein extraction kit. The protein concentration in the lysate was quantified using a BCA protein assay kit. Equal amounts of protein from each sample were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequent to incubation in blocking buffer (LICOR, Odyssey, USA) for 1 h, the membranes were incubated with monoclonal primary antibodies against ATG5/LC3B/Beclin-1 (Abcam, USA)/Caspase-3 (GeneTex, USA) HBHA (antiHBHA was prepared by our laboratory) and β-actin (Abcam, USA) overnight at 4°C. The membranes were then incubated with a horseradish peroxidase-conjugated goat antirabbit IgG secondary antibody (LICOR, Odyssey, USA) or antimouse IgG secondary antibody (LICOR, Odyssey, USA) for 2 h. The bands were detected using a dual-color infrared laser (LICOR, Odyssey, USA), and the protein levels were quantitated by densitometry using Gel-Pro Analyzer software (Media Cybernetics, Inc., Rockville, MD, USA).

The A549 cells were seeded at a density of 1 × 10⁶ cells/well in six-well plates and incubated with HBHA of different concentrations for 24 h in the presence or absence of rapamycin or 3-MA. Alternatively, A549 cells were infected with MS or rMS-HBHA at the MOI of 10:1 for 24 h. Afterward, the culture supernatant was collected and added to a 96-well plate. The caspase inhibitor Z-VAD-FKM (20 μM) was added into cells 1 h prior to HBHA treatment or infection. Specific RIPK1 inhibitor Necrostatin-1 (Nec-1) (Sigma) was used at 30 μM. A LDH cytotoxicity kit was then used according to the manufacturer's instructions. Specifically, dinitrophenylhydrazine and sodium hydroxide solution were detected at 450 nm using a microplate reader.

For the assay, A549 cells were seeded onto 24-well plates at a density of 4 × 10⁵ cells/well. The cells were infected with MS or rMS-HBHA at the MOI of 10:1 for 3 h in the presence or absence of rapamycin or 3-MA at 37°C in 5% CO₂. For caspase inhibition, Z-VAD-FKM was added into cells 1 h prior to infection. Amikacin (200 mg/ml) was then added for 3 h to kill extracellular bacteria. At indicated time points, the cells were washed three times with basal RPMI 1640 medium, and viable intracellular bacteria were released by incubation with 0.5 ml of 0.1% Triton X-100 in sterile water for 10 min. The samples were mixed vigorously with 0.5 ml of 7H9 broth. Serial 10-fold dilutions of lysates were prepared in 7H9 broth and plated on 7H10 agar for determination of CFU numbers.

All experiments were repeated at least three times. Statistical analyses were performed using SPSS 17.0. Data are expressed as the mean ± standard deviation and were analyzed using one-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of HBHA on the autophagy of A549 cells, Western blot assay was performed to detect the expression of LC3 and Beclin-1. LC3 and Beclin-1, which were starved for 1 h, were significantly induced in A549. However, when HBHA was added after 90 min, the expression of LC3 and Beclin-1 was suppressed in a dose-dependent manner (Figures 1A–D). In particular, 8 μg/ml HBHA could remarkably inhibit the expression of starved LC3 and Beclin-1, and the ATG5 expression was not affected by HBHA (Figure 1C). Subsequently, to measure the effect of HBHA on the autophagic flux of A549 cells, the cells were treated with BAF A1 to prevent lysosomal degradation. Compared with BAF A1 alone, HBHA treatment with BAF A1 could decrease LC3II levels. This result confirmed that HBHA inhibited the synthesis of autophagy-related membranes (Figures 1E,F). When rapamycin or 3-MA was used to induce or inhibit autophagy, the LC3 expression could be suppressed by 3-MA. Similar to 3-MA, the LC3 expression was inhibited in a dose-dependent manner when HBHA was added (Figures 1G,H).

To further explore the effect of HBHA on autophagy, we observed the formation of autophagosomes using MDC staining and immunofluorescence staining of LC3 in A549 cells. The number of MDC foci increased after starvation, but it could be reversed by 3-MA. Similarly, HBHA decreased the number of MDC foci (Figures 2A,B), and the number of LC3 puncta decreased significantly after HBHA addition (Figures 2C,D). Taken together, these results demonstrated that autophagy maturation could be considerably suppressed by HBHA.

We transferred HBHA into MS MC₂155 to construct recombinant rMS-HBHA. To confirm that HBHA was successfully expressed in rMS-HBHA, the total proteins of MS and rMS-HBHA were extracted. Western blot analysis was performed to detect the expression of HBHA protein using anti-HBHA antibody. The results showed that HBHA was not expressed in the parent strain MS MC₂155, but expressed in the rMS-HBHA (Figure 3A). To further confirm the inhibition role of HBHA on autophagy, A549 cells were infected with MS or rMS-HBHA. Western blot analysis results showed that rMS-HBHA inhibited the expression of starvation-induced LC3, but the wild-type strain did not (Figures 3B,C). We constructed GFP-expressing MS and rMS-HBHA, infected A549, and performed MDC staining to examine autophagosome formation. The number of MDC foci increased after starvation, and rMS-HBHA significantly decreased the number of MDC foci (Figures 3D,E). These results suggested that rMS-HBHA inhibited the maturation of autophagy and consistent with those observed with pure HBHA protein.

To determine whether autophagy inhibition could promote survival of rMS-HBHA within A549 cells, A549 cells were infected with WT MS or rMS-HBHA at the MOI of 10:1. Cells were lysed at 1, 10, or 18 h post-infection, and lysates were diluted and plated on agar plates to determine the number of viable intracellular bacteria. MS and rMS-HBHA showed no significant difference in growth curve (Figure 4A). However, rMS-HBHA showed higher survival and replication capacity within A549 cells than those of wild-type MS (Figure 4B). Autophagy activation in MS-infected A549 cells by rapamycin could decrease the number of intracellular MS, but showed no significant effect on rMS-HBHA (Figure 4C). Moreover, autophagy inhibition in MS-infected A549 cells by 3-MA could increase the number of intracellular MS (Figure 4D). These findings suggested that HBHA may enhance the capacity of MS to infect and survive within A549 cells through inhibition of autophagy.

To determine the fate of infected A549 cells, A549 cells were treated with HBHA of different concentrations or infected with MS or rMS-HBHA at the MOI of 10:1 for 24 h. The culture supernatant was collected to measure the released LDH. We found that HBHA protein treatment could induce large amount of cell death (Figure 5A). Similarly, rMS-HBHA infection could induce great number of cell death but not wild-type MS (Figure 5B). Activation of autophagy in rMS-HBHA-infected A549 cells by rapamycin showed no effect on cell death (Figure 5C). Furthermore, inhibition of autophagy in MS-infected A549 cells by 3-MA could slightly increase cell death but showed no effect on rMS-HBHA-infected A549 cells (Figure 5D). These results suggested that HBHA could promote cell death in infected A549 cells.

To investigate the form of HBHA-induced cell death, the RIPK1 inhibitor Nec-1 and caspase inhibitor Z-VAD-FMK was used. HBHA- or rMS-HBHA infection-induced cell death was not affected after Nec-1 treatment (Figures 6A,B). These results suggested necroptosis was not involved in this process. However, HBHA- or rMS-HBHA infection-induced cell death was significantly attenuated after Z-VAD-FMK treatment (Figures 6C,D). This result suggested that apoptosis may participate in this process. To further confirm the results, the cleaved-caspase-3 activity was detected by Western blot. HBHA treatment could increase the expression of cleaved-caspase-3 in a dose-dependent manner, and treatment with Z-VAD-FMK could inhibit the activation of caspase-3 (Figures 6E,F). These results indicated that HBHA protein treatment or rMS-HBHA infection could induce apoptosis of A549 cells through activation of caspase-3.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.