The strains used in these experiments were B. melitensis strain 16M (Library for Brucellosis of Chinese Centre for Disease Control and Prevention) and Bacillus subtilis strain BAA12545 (gifted by Professor Wenge Hu; College of Life Sciences, Shihezi University, Shihezi, Xinjiang, China), which was engineered for its sacB gene and used for the screening of mutant strains by counter-selection. B. melitensis was grown on Brucella agar (BD Biosciences, Franklin Lakes, NJ, USA). Sucrose medium was utilized for sacB counter-selection as previously described (18).

The RAW264.7 macrophage cell line was purchased from the China Academy Typical Culture Preservation Committee cell library (Shanghai, China). The cells were cultured at 37°C with 5% CO₂ in complete tissue culture medium (c-DMEM) consisting of Dulbecco's modified Eagle's medium (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Genomic DNA of B. melitensis strain 16M was used as the template for amplification of OMP31 upstream and downstream segments by PCR. The primer sequences used are shown in Table I. The flanking sequences were separately amplified, and overlap extension PCR (19) was then employed to combine the flanking sequences to generate contiguous DNA sequences of the OMP31 fragment. The fragment was cloned into a pMD18-T vector (D101A; Takara Biotechnology Co., Ltd., Dalian, China). Following SphI/XhoI digestion of the recombinants, the target fragments were introduced into a pGEM-7zf (+) suicide vector (P2251; Promega Corp., Beijing, China). The sacB gene of Bacillus subtilis was amplified by PCR and inserted into the recombinant pGEM-7zf (+) vector. Thus, the suicide plasmid pGB-OMP31 was generated. The pGB-OMP31 plasmid was introduced into B. melitensis strain 16M by electroporation as described previously (20). Ampicillin-resistant and sucrose-sensitive integrants were selected according to the study of Campos et al (20). The mutant, designated 16MΔOMP31, was identified by western blot assay and PCR tests (21) following subculture for 15 generations. Conventional microbiological tests, including assays of lipopolysaccharide antigens, phage typing, dye sensitivities, CO2 requirements, H2S production, and metabolic properties (21), were carried out to identify its phenotype.

RAW264.7 macrophages were plated in 12-well plates in DMEM without antibiotics at a concentration of 5×10⁵ cells per well and incubated overnight at 37°C with 5% (vol/vol) CO₂. The RAW264.7 cells were then infected with B. melitensis 16M or the mutant strain 16MΔOMP31 in triplicate wells of a 24-well plate at a multiplicity of infection (MOI) of 100:1. Following 4 h of incubation at 37°C in an atmosphere containing 5% CO₂, the cells were washed three times with phosphate-buffered saline (PBS, pH 7.0) and treated with 50 µg/ml gentamicin to kill extracellular Brucella. The infected cells were then lysed with 0.1% Triton X-100 in PBS at 2, 4, 8, 12 and 24 h post infection. The number of viable colony forming units (CFUs) was determined by plating a series of 1:10 dilutions on tryptic soy agar plates.

RAW264.7 cells cultured in 12-well plates were infected with B. melitensis 16M or mutant strain at a MOI of 100:1 in triplicate wells as described above. At 2, 4, 8, 12 and 24 h post infection, the RAW264.7 cells were washed with PBS and harvested. Apoptosis was then investigated. Briefly, B. melitensis 16M or mutant strain-infected RAW264.7 cells were incubated with Annexin V and propidium iodide (PI) using an Annexin V-FLUOS staining kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) at room temperature for 20 min. Immediately afterwards, apoptotic RAW264.7 cells were detected using flow cytometry.

Cells cultured in 12-well plates were infected with B. melitensis 16M or mutant strain in triplicate wells as described above. At 2, 4, 8, 12 and 24 h post infection, TNF-α production in the culture supernatants was tested using an ELISA kit (BD Biosciences).

Cells cultured in 12-well plates were infected with B. melitensis 16M or mutant strain in triplicate wells as described above. Total RNA of the RAW264.7 cells was isolated using TRIzol reagent (CW0580Sl; CWBio, Beijing, China) and was further purified using a Qiagen RNeasy mini kit (Qiagen, Inc., Valencia, CA, USA), used according to the manufacturers' protocols. A Nanodrop 2100 instrument (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) was used to assess the concentrations and quality of the RNA samples. Reverse transcription, cDNA quantification and cDNA amplification were performed exactly as described in the instructions of the Super RT One Step RT-PCR kit (CW0742S; CWBio). Specific primers used are listed in Table II. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. Relative mRNA levels of a particular gene in mutant strains were normalized using the 2^−ΔΔCq method with GAPDH as a reference gene (22).

RAW264.7 cells cultured in 12-well plates were infected with B. melitensis 16M or mutant strain at a MOI of 100:1 in triplicate wells as described above. At 12 h post infection, Cyt c from the cells was detected using a Cyt c ELISA kit (Elabscience, Wuhan, China) according to the manufacturer's protocol.

RAW264.7 cells cultured in 12-well plates were infected with B. melitensis 16M or mutant strain at a MOI of 100:1 in triplicate wells as described above. At 4 h post infection, the concentrations of caspase-3, −8 and −9 were measured using caspase-3, −8 and −9 ELISA kits (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's instructions.

To determine the statistical significance of the immune response to the parent and mutant, differences were analyzed using analysis of variance tests and least significant difference post-hoc tests, with SPSS statistical analysis software (version 13.0; SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The OMP31 gene deletion mutant based on B. melitensis 16M was successfully constructed. To assess the mutant strain, conventional microbiology tests were carried out to identify its phenotype. These results showed that the lipopolysaccharide of the mutant strain remained smooth and the mutant exhibited the same traits as Brucella melitensis 16M (data not shown), including lipopolysaccharide antigens, phage typing, dye sensitivities, CO₂ requirements, H₂S production, and metabolic properties.

Brucella has developed the ability to inhibit apoptosis to benefit intracellular replication. To assess the intracellular replication of the bacteria in RAW264.7 macrophages, the number of CFUs was counted following infection with Brucella melitensis 16M or the mutant strain. The results demonstrated that at 2–10 h post-infection, particularly at 4 h post-infection, parent stain impaired the ability of the bacteria to replicate in RAW264.7 cells. However, at 10–24 h post infection, the OMP31 mutant strain impaired the ability of the bacteria to replicate in RAW264.7 cells.

Whether the deletion of OMP31, in addition to impairing the intracellular colony formation of Brucella in macrophages, also affected the apoptosis level was investigated. To evaluate the apoptosis of RAW264.7 cells induced by B. melitensis 16M or the mutant, flow cytometric analysis was conducted. The results demonstrated that the deletion of OMP31 increased the proportion of apoptotic RAW264.7 cells from 2 h post infection (Fig. 1). At 2–24 h post-infection, the mutant strain triggered more apoptosis cells than the parent strain. However, at 2–4 h post-infection, the mutant strain promoted RAW264.7 apoptosis and the parent strain inhibited apoptosis slightly. At 4–24 h post-infection, both the parent and mutant strains promoted RAW264.7 apoptosis.

The cytokine TNF-α plays a key role in immunity and inflammation by inducing cellular responses such as apoptosis (23). Therefore, following the detection of apoptosis, the TNF-α levels (RNA and secretion) induced by B. melitensis 16M or the mutant strain were detected. The results showed that the deletion of OMP31 increased the levels of TNF-α for the mutant strain in comparison with those for B. melitensis 16M from 4 h after infection for RNA (Fig. 2A), and 8 h post infection for protein (Fig. 2B). At 2–12 h post-infection, the concentration and transcriptional level of TNF-α increased rapidly, triggered by both mutant stain and parent strain, although the mutant stain induced greater TNF-α expression.

Triggering of the TNF receptor-1 (TNFR-1) by TNF-α may induce classical apoptosis by activating the initiator protease caspase-8 in the death receptor pathway (24). TNF-α induced apoptosis is mediated via the cell surface receptor TNFR-1 and involves the formation of two signaling complexes that sequentially activate NF-κB and caspases (25). Other studies have suggested that TNF-α may trigger apoptosis via an additional mechanism, involving constituents of acidic vesicles that can generate ceramides as second messengers such as Bcl-2 and activation of caspase-3 (26,27). Therefore, caspase-3 and 8 were detected by ELISA. The levels of caspase-3 and −8 were increased after 4 h post infection with the mutant strain 16MΔOMP31 (Fig. 3), to a greater extent than with the parent strain.

Bax, a member of the Bcl-2 protein family, is involved in the control of apoptotic events, and may accelerate apoptosis (28). Bcl-2 prolongs cell survival following the Bax-induced release of Cyt c (29). High levels of Bcl-2 inhibit apoptosis by attenuating the release of Cyt c while high levels of Bax induce apoptosis by binding to mitochondrial membranes and increasing the permeability of the membranes, facilitating the release of Cyt c (30). Cyt c activates caspase cascades in the cytosol, ultimately leading to cell death (31). To identify the possible pathway underlying the effects of OMP31, following the increase of TNF-α expression and apoptosis percentage, the transcriptional levels of Bcl-2 and Bax were detected. The present results for Bcl-2 (Fig. 4A) showed that the deletion of OMP31 reduced the bcl-2 level from 8 to 24 h post infection, although not at 4 h post infection. The results for Bax (Fig. 4B) revealed that the deletion of OMP31 raised the Bax level from 2 to 24 h post infection, but not at 4 h post infection. At 4–24 h post-infection, the mutant strain and parent strain both impaired Bcl-2 transcription and induced Bax transcription. Similarly, the mutant strain induced a greater change than the parent strain.

Cyt c is an essential component of the mitochondrial respiratory chain. It is a soluble protein, localized in the intermembrane space, and is loosely attached to the surface of the inner mitochondrial membrane. Cyt c release from mitochondria is a key event that has an important role in initiating the process of apoptosis in mammalian cells (32). To further investigate the pathway involved in the apoptosis induced by the deletion of OMP31, Cyt c release from infected cells was detected in the present study. The results showed that the deletion of OMP31 raised the concentration of Cyt c released after infection for 12 h (Fig. 4C). At 2–12 h post-infection, the concentration of Cyt c rapidly increased, triggered by both the mutant and parent strains.

Caspases are a family of proteases with major roles in apoptosis. They are considered as downstream effectors of apoptosis, with caspase activation being an irreversible step in apoptotic signaling. Caspase-3, −6 and −7, known as executioner caspases, are downstream of the caspase-8 or −9 activation cascade (33,34). In a previous experiment of the present study, caspase-3 and −8 levels were found to be raised by the mutant strain. To further define the pathway induced by the deletion of OMP31, following the observation of a high level of Cyt c release, the levels of caspase-9 were detected (Fig. 4D). The results showed that caspase-9 levels were increased more than caspase-8 levels at 4 h post infection with the mutant strain. The mutant strain triggered more caspase-3 and −9 than the parent strain, and the concentration of caspase-9 was greater than that of caspase-3.