Bacterial strains and cell line
B. abortus 2308 strain (S2308) was obtained from the Center of Chinese Disease Prevention and Control (Beijing, China). The 2308ΔgntR mutant and 2308ΔgntR-C complementary strain were constructed and kept in our research laboratory . All Brucella strains were cultured in tryptic soy agar (TSA) or broth (TSB) (Difco, MI, USA) at 37°C in 5% CO2 (v/v). The murine macrophage RAW 264.7 line was obtained from the Cell Resource Center (Beijing, China). RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco Life Technologies, Rockville, MD, USA) at 37°C with 5% CO2 (v/v). All experimental procedures were performed in Biosafety Level 3 Laboratory (BSL-3).
The apoptosis was detected by flow cytometry, as previously described but with several modifications . Briefly, monolayers of RAW 264.7 macrophages of 1×106 cells/well were cultured in six-well plates for 24 h at 37°C under 5% CO2 and then infected with Brucella strains at a multiplicity of infection (MOI) of 100. Culture plates were centrifuged at 350 × g for 5 min at room temperature, and incubated at 37°C for 45 min. After washing twice with the medium without antibiotics, the infected cells were incubated for 60 min in the presence of 50 μg/mL gentamicin to kill the extracellular bacteria. Then, the cultures were placed in fresh DMEM containing 25 μg/mL gentamicin (defined as time zero) and incubated at 37°C. The control group comprised uninfected cells. At 24 h post-infection, the cells were washed with PBS, centrifuged at 900 × g for 5 min, and then resuspended with 500 μL of binding buffer mixed with 5 μL of fluorescein isothiocyanate (FITC)-labeled Annexin V (Annexin V-FITC) and 5 μL of propidium iodide (PI). After incubation in the dark at room temperature for 15 min, the cell samples were detected by flow cytometry (Life Technology, USA). All assays were performed thrice.
The expression of P62 protein in the cell was observed by confocal microscopy, as previously described with several modifications . Briefly, RAW 264.7 cells were infected with S2308 or 2308ΔGntR as described above. The control group comprised uninfected cells. At 24 h post-infection, the cells were fixed in 4% paraformaldehyde (PFA) for 10 min. Then, the cells were washed for 5 min with 1 mL of PBS containing 2 mg glycine. The cells were blocked with 1 mL of PBS containing 1% (v/v) bovine serum albumin (BSA) and 0.1% (v/v) Triton X-100 for 1 h. The cells were washed thrice with PBS and added with primary (rabbit anti-mouse P62 polyclonal antibody) antibody (Bioworld, Minneapolis, USA). Then, the cells were placed overnight at 4°C. Thereafter, the cells were washed thrice with PBS and added with secondary (green fluorescently tagged goat anti-rabbit IgG antibody) antibody (Bioworld, Minneapolis, USA) for 1 h at 37°C. Subsequently, the cells were washed thrice with PBS, and fluorescence was observed by confocal microscopy (ZEISS, Germany).
Macrophage infection and RNA extraction
Murine macrophage RAW 264.7 cells were used to detect the apoptosis and autophagy of S2308, 2308ΔgntR and 2308ΔgntR-C. The RAW 264.7 cells were infected with Brucella as described above. At 4, 8, 12 and 24 h post-infection, 1 mL TRIzol (Invitrogen, Carlsbad, CA, USA) was added to the cells for each well. RNA was also isolated from the uninfected cells as a negative control. Residual DNA in the samples was removed using DNase I (Promega, Madison, WI, USA). RNA concentration and purity were determined spectrophotometrically using an ND 1000 spectrophotometer (Thermo Scientific, Wilmington, USA). The total RNA from the Brucella-infected cells was extracted as previously described [32, 33]. All assays were performed in triplicate and repeated at least thrice.
Quantitative real time-PCR (qRT-PCR)
QRT-PCR was used to detect the expression levels of apoptosis-associated genes (caspase-3, caspase-8, Bax, and Bcl-2) and autophagy-associated genes (LC3-I, LC3-II, and p62), as previously published procedures with several modifications . The cDNA was generated from the total RNA by using a random hexamer primer and the SuperScript II reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The apoptosis- and autophagy-associated genes were selected. The primers used to amplify these genes are listed in Table 1. Samples were run in triplicate and amplified in a 20 μL reaction containing 2 × SYBR Premix Ex Taq II (Takara, Japan), 100 nM forward and reverse primers, and 1 μL of cDNA target. The mix was incubated for 5 min at 95°C, and then 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s were performed on a Roche LightCycler 480 II system (Roche, Basel, Switzerland). The relative transcriptional levels were determined by the methods of 2-ΔΔCt, as described previously . The expression of β-actin was used as a reference gene. All assays were performed in triplicate and repeated at least thrice.
Western blot (WB) analysis
To determine the expression levels of caspase-3 and P62 in the RAW 264.7 cells, we analyzed caspase-3 and P62 protein lysates by WB as previously described with several modifications [13, 36]. Briefly, the RAW 264.7 cells were infected with S2308 and 2308ΔGntR following the steps as described above. The control group comprised uninfected cells. At 24 h post-infection, the cells were lysed in ice-cold RIPA lysis buffer (Solarbio Science and Technology, Beijing, China) for 20 min and centrifuged at 13,400 × g for 20 min at 4°C. Protein lysates (50 μg total protein/lane) were separated by 12% SDS-PAGE and electrotransferred to a nitrocellulose (NC) membrane by using a semi-dry trans-blot cell (Bio-Rad, Hercules, CA, USA) at 200 Ma for 1 h in transfer buffer (100 Mm Tris-HCl, 150 Mm NaCl, 0.05 % Tween 20, Ph 7.2). Membranes were incubated in blocking solution (5% nonfat milk in Tris-buffered saline Tween-20 [TBST]) for 1 h at room temperature. Membranes were washed thrice with the TBST buffer. Subsequently, the membranes were incubated with primary (rabbit anti-mouse caspase-3 and P62 polyclonal antibody) antibodies (Bioworld, Minneapolis, USA) at 37°C for 1 h. The membrane was washed with TBST thrice and incubated with secondary (goat anti-rabbit horseradish peroxidase-labeled IgG antibody) antibodies (SBA, Birmingham, Al, USA) for 1 h in 5% milk/TBST at 37°C. After three washes, bound conjugate was visualized using an enhanced HRP-DAB substrate color kit (Tiangen Biotech Co. Ltd., Beijing, China). Western blott analysis was repeated thrice.
To determine the expression levels of LC3-II/I in the RAW 264.7 cells, we analyzed the LC3 protein lysates by WB, as previously described with several modifications [8, 37]. The RAW 264.7 cells were infected with S2308, 2308ΔGntR of 2308ΔGntR-C as described previously. At 24h post-infection, the samples (50 μg total protein/lane) were loaded onto SDS-PAGE gels in this experiment. The primary (rabbit anti-mouse LC3 antibody) antibody (Bioworld, Minneapolis, USA) and secondary (goat anti-rabbit horseradish peroxidase-labeled IgG antibody) antibody (SBA, Birmingham, Al, USA) were purchased from Cell Signaling Technology and used at dilutions of 1:1,000 and 1:2,000, respectively. The bound conjugate was visualized using an enhanced HRP-DAB substrate color kit (Tiangen Biotech Co. Ltd., Beijing, China). Western blot analysis was repeated thrice.
QRT-PCR data were analyzed using the Roche LightCycler480 1.5 software (Roche, Basel, Switzerland), and the relative quantification was obtained using the 2-ΔΔCt method. The expression levels of the apoptosis- and autophagy-related genes were expressed as mean ± standard deviation (SD). Statistical analysis was performed with Student’s unpaired t-test. The differences between groups were analyzed by analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post-test by comparing all the groups. The results were analyzed by the Fisher test and expressed in percentages. SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA) was used to analyze the differences between groups. P values of < 0.05 were considered statistically significant.