Deletion of the transcriptional regulator GntR affects apoptosis and autophagy in Brucella abortus-infected RAW 264.7 cells

Background: Brucellosis is an important zoonotic disease caused by the pathogen Brucella. Regulating apoptosis and autophagy is the prerequisite for the intracellular survival of Brucella. GntR is an important transcriptional regulator of Brucella that can regulate the expression of many target genes, and then play a regulatory role in many cell processes, including regulation apoptosis and autophagy. Therefore, understanding the relationship between GntR and apoptosis and autophagy is crucial to comprehending the pathogenic mechanism of Brucella. Methods: In the present study, we described the inuence of GntR on apoptosis and autophagy after the infection of RAW 264.7 cells with Brucella. We constructed the GntR mutant strain (2308ΔGntR) of Brucella abortus 2308 (S2308). Following the infection of the RAW 264.7 cells with S2308 and 2308ΔGntR, apoptosis and autophagy were detected. Results: Western blot analysis and ow cytometry analysis indicated that the apoptosis rate of the 2308ΔGntR-infected group was remarkably higher than that of the S2308-infected group. Confocal laser microscopy experiments indicated the presence of the P62 protein as punctate aggregates in the 2308ΔGntR group. Conclusion: These results showed that 2308ΔGntR promoted apoptosis and inhibited autophagy in the RAW 264.7 cells during Brucella infection.


Introduction
Brucella spp. are intracellular parasites that cause the a worldwide zoonotic disease called brucellosis in animals and humans [1]. Brucellosis brings enormous economic burdens for developing countries [2].
Apoptosis is a programmed cell death process, which is an essential physiological process that regulates tissue mass and architecture during normal tissue development [10]. Apoptosis is an active form of cell death characterized by various morphological and biochemical features [11]. Francisella tularensis could induce apoptosis in murine macrophages [12]. Brucella could also regulate apoptosis in macrophages [13]. Autophagy is a major protein degradation system. Autophagy is ubiquitous in eukaryotic cells, and it can preserve the homeostasis of the intracellular environment [8]. Autophagy is also a vital intracellular mechanism that prevents microbial infection [8,14]. Autophagy participates in cellular functions as well [15]. Therefore, apoptosis and autophagy play a crucial role in regulating a series of important cell life activities.
Bcl-2 is an inhibitor of apoptosis. Increased levels of Bcl-2 expression in cells could prevent apoptosis [16]. Stimulating factors could inhibit Bcl-2 expression and induce apoptosis [17]. Bax can join to Bak at the outer membrane of the mitochondria, collapse the mitochondrial membrane potential, and consequently trigger the cell apoptosis [18]. The outbalance ratio of Bcl-2 and proapoptotic Bax plays a pivotal role in apoptosis [19]. The imbalance of Bcl-2 and proapoptotic Bax leads to apoptosis [19]. Caspase-3 and − 8 are the key regulators of the apoptotic response, and they interact with the calpain family, a group of cysteine proteases, in the course of diseases [20]. Caspase-3 and − 8 are activated in the death receptor pathway of apoptosis. Caspase-3 and − 8 overexpression could enhance apoptotic activity [21]. Autophagy is an intracellular bulk degradation system. P62 is an autophagic substrate [22].
When autophagy is enhanced, the P62 substrate is cleared [23]. However, when the inhibitor 3methyladenineis used to inhibit autophagy, the P62 substrate accumulates [24]. P62 can be used as a marker of autophagy [25]. Microtubule-associated protein light chain 3 (LC3) is a mammalian homolog of yeast Atg8. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine [26]. Therefore, LC3 has been used as a speci c marker to monitor autophagy.
GntR is a transcriptional regulator. It is involved in the regulation of biological functions in many pathogens, including stress response and nutrient uptake [27,28]. GntR is an important virulence factor in Brucella [29]. In addition, GntR affects the expression of the virulence genes of Brucella and the secretion of in ammatory cytokines during infection [29,30]. However, the intracellular functions of GntR, apoptosis, and autophagy remain unknown.
In the present study, the roles of GntR in apoptosis and autophagy were detected. This study aimed to determine the function of GntR in apoptosis and autophagy during Brucella infection and to provide new ideas and clues for further elucidating the mechanisms of the intracellular survival and pathogenesis of Brucella.

Materials And Methods
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 [30]. All Brucella strains were cultured in tryptic soy agar (TSA) or broth (TSB) (Difco, MI, USA) at 37°C in 5% CO 2 (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 modi ed 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% CO 2 (v/v). All experimental procedures were performed in Biosafety Level 3 Laboratory (BSL-3).

Flow cytometry
The apoptosis was detected by ow cytometry, as previously described but with several modi cations [31]. Brie y, monolayers of RAW 264.7 macrophages of 1×10 6 cells/well were cultured in six-well plates for 24 h at 37°C under 5% CO 2 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 (de ned 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 uorescein 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 ow cytometry (Life Technology, USA). All assays were performed thrice.

Confocal microscopy
The expression of P62 protein in the cell was observed by confocal microscopy, as previously described with several modi cations [13]. Brie y, 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 xed 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 uorescently 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 uorescence 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 Scienti c, 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 modi cations [34]. 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 ampli ed 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 [35]. 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 modi cations [13,36]. Brie y, the RAW 264.7 cells were infected with S2308 and 2308ΔGntR following the steps as described above. The 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 modi cations [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.

Statistical analysis
QRT-PCR data were analyzed using the Roche LightCycler480 1.5 software (Roche, Basel, Switzerland), and the relative quanti cation 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 signi cant 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 signi cant.

Apoptosis in Brucella-infected cells
At 24 h after the S2308 and 2308ΔGntR infection of the RAW 264.7 cells, the percentages of apoptotic cells were detected by ow cytometry. The percentages of apoptotic cells of the S2308-infected group and 2308ΔGntR-infected group were 3.30% ± 0.50% and 16.80% ± 1.30%, respectively (Fig. 1). The results indicated that the percentages of apoptotic cells of the S2308-infected group were signi cantly lower than those of the 2308ΔGntR-infected group (P < 0.05). Hence, S2308 can inhibit apoptosis in RAW 264.7 cells.

GntR affects the expression levels of apoptosis-associated genes in Brucella-infected cells
To test the effect of GntR on apoptosis-associated genes, we analyzed the expression levels of caspase-3, caspase-8, Bax, and Bcl-2 in Brucella-infected cells. Following 2308ΔGntR infection, the relative expression levels of caspase-3, caspase-8, and Bax in the RAW 264.7 cells were signi cantly higher than those in the S2308-infected cells (P < 0.01, Fig. 2A, 2B, and 2C). The relative expression level of Bcl-2 in the 2308ΔGntR-infected cells was signi cantly lower than that in the S2308-infected cells (P < 0.05, Fig.  2D). The ratio of Bax/Bcl-2 in the 2308ΔGntR-infected cells was signi cantly higher than that in the S2308-infected cells (Fig. 2E). These results indicated that the 2308ΔGntR mutant affected the expression levels of the apoptosis-associated genes in the RAW 264.7 cells.

GntR affects the expression level of caspase-3 protein in Brucella-infected cells
We used WB to analyze the expression levels of caspase-3 protein. The results showed that the expression levels of caspase-3 protein in the 2308ΔGntR-infected cells were signi cantly higher than those in the S2308-infected cells (P < 0.01, Fig. 2F). Hence, the 2308ΔGntR mutant affected the expression levels of caspase-3 protein in the RAW 264.7 cells.

Localization expression levels of P62 protein in Brucella-infected cells
To further observe the expression of P62 protein in the cell, we conducted an experiment using a confocal laser scanning microscope. The focal points of P62 protein at 24 h post infection were observed by confocal microscopy. P62 showed punctate aggregates (Fig. 3A). P62 protein was dispersed in the control cells while the experimental groups showed punctate aggregates (Fig 3A). After S2308 and 2308ΔGntR infection, the P62 punctate aggregates became increasingly signi cant (Fig 3A), indicating that cell autophagy was inhibited after the infection of Brucella. Especially under 2308ΔGntR infection, the intracellular P62 punctate aggregates (Fig 3A) increased, indicating that autophagy was inhibited. Hence, 2308ΔGntR inhibited autophagy in the RAW 264.7 cells.
2308ΔGntR affects the expression levels of autophagy-associated genes The relative expression levels of p62, LC3-II, and LC3-I were computed using the 2 -ΔΔCt method. Following the infection of the RAW 264.7 cells with S2308 and 2308ΔGntR, the relative expression levels of p62 and LC3-I in the 2308ΔGntR-infected cells were slightly higher than those in the S2308-infected cells (Fig. 4A  and 4B). No signi cant difference was observed between the 2308ΔGntR-and S2308-infected groups (P > 0.05). The relative expression levels of LC3-II in the 2308ΔGntR-infected cells were signi cantly lower than those in S2308-infected cells (P < 0.01, Fig. 4C). The ratio of LC3-II/LC3-I in the 2308ΔGntR-infected cells was signi cantly lower than that in the S2308-infected cells (P < 0.01, Fig. 4D). Hence, the 2308ΔGntR mutant affected the expression levels of the autophagy-associated genes in the RAW 264.7 cells.
In the present study, we evaluated whether 2308ΔGntR could inhibit the expression of LC3-II. The ratio of LC3-II/I was decreased in the 2308ΔGntR-infected cells (Fig. 4E and 4F). Furthermore, the complemented strain 2308ΔGntR-C promoted autophagy ( Fig. 4E and 4F). Collectively, these results indicated that 2308ΔGntR affected autophagy in the RAW 264.7 cells.

GntR affects expression levels of P62 protein in Brucella-infected cells
We used WB to analyze the expression levels of P62 protein. The results showed that the P62 protein expression levels in the 2308ΔGntR-infected cells were signi cantly higher than those in the S2308infected cells (P < 0.05, Fig. 3B and 3C). Hence, 2308ΔGntR affected the expression levels of P62 protein in the RAW 264.7 cells.

Discussion
Brucellosis is a worldwide zoonosis, which remains prominent in many countries. The transcriptional regulatory system is one of the main virulence systems of Brucella. The transcriptional regulator GntR of Brucella plays a key role in the pathogenic process of Brucella [29]. GntR modulates various biological processes, including cytotoxicity [30], secretion of in ammatory cytokines [30], antibiotic production [38], antibiotic resistance [39], and plasmid transfer [40]. Transcriptional regulator GntR regulates the expression levels of many genes in Brucella [41]. However, the cellular functions of GntR are still unknown. In the present study, we demonstrated that the transcriptional regulator GntR participated in apoptosis and autophagy during Brucella infection.
Apoptosis plays an important role in the pathogenicity of pathogens. Brucella may inhibit the apoptosis of host cells to reproduce in cells. However, the speci c mechanism remains unclear. In this study, we used 2308ΔGntR mutant and parental strain S2308 to infect RAW 264.7 cells. Our results were the same as those of previous studies [13,34]. The percentages of apoptotic cells of the 2308ΔGntR-infected cells were signi cantly higher than those of the S2308-infected ones. Wild-type B. abortus 2308 is a smooth Brucella, which inhibits apoptosis for its long-term survival in host cells. We con rmed that the virulence of 2308ΔGntR was lower than that of the parental strain [29]. The survival time of the 2308ΔGntR mutant strain in the host cells was short [29]. These results may be related to apoptosis.
In this study, the ratio of Bax/Bcl-2 in the 2308ΔGntR-infected group was signi cantly higher than that in the S2308-infected group. In addition, the mRNA expression levels of the apoptosis-related genes caspase-3 and caspase-8 were up-regulated in 2308ΔGntR. Hence, 2308ΔGntR promoted apoptosis in the RAW 264.7 cells. The activation of caspase-3 indicated apoptosis [42]. Our results suggested that 2308ΔGntR enhanced caspase-3 protein expression. Moreover, the results indicated that Brucella transcriptional regulator GntR was involved in apoptosis and that the 2308ΔGntR mutant promoted apoptosis in the RAW 264.7 cells.
Autophagy is a cellular pathway that plays an important in development and survival. Autophagy has an important signi cance in Brucella intracellular tra cking. In epithelial cells, B. abortus is transited through autophagosomes [43]. Thus, the regulation of autophagy may be a crucial target of B. abortus. We detected the localization expression levels of P62 protein by confocal laser scanning microscopy. We found that the P62 punctate aggregates in the 2308ΔGntR-infected cells became more signi cant than those in the S2308-infected cells. The lack of P62 degradation indicates that 2308ΔGntR inhibits autophagy in RAW 264.7 cells.
Autophagy is related to microbial infection [44]. LC3 is a major marker of autophagy and a Brucellainduced autophagic response is related to LC3 [45]. After 2308ΔGntR infected the cells in this work, the ratio of LC3-II/LC3-I was decreased. These results indicated that 2308ΔGntR inhibited autophagy in the RAW 264.7 cells. Furthermore, P62 protein expression levels increased after 2308ΔGntR infected the cells. In sum, these results indicated that the Brucella transcriptional regulator GntR was involved in autophagy. They also con rmed that the 2308ΔGntR mutant inhibited autophagy in the RAW 264.7 cells.
In sum, we found that the mRNA relative expression levels of caspase-3, caspase-8, Bax, p62, and LC3-I and the ratio of Bax/Bcl-2 increased more signi cantly in 2308ΔGntR than in S2308. Moreover, the mRNA relative expression levels of Bcl-2 and LC3-II and the ratio of LC3-II/LC3-I decreased more signi cantly in 2308ΔGntR than in S2308. WB, ow cytometry, and confocal laser microscopy indicated that 2308ΔGntR promoted apoptosis and inhibited autophagy in the RAW 264.7 cells during Brucella infection. These results indicated that GntR was involved in the apoptosis and autophagy in the Brucella-infected cells. These results provide new ideas and clues for further elucidating the molecular mechanism of Brucella pathogenicity.