SIRT2 Inhibition Enhances Mitochondrial Apoptosis in Brucella-Infected Bovine Placental Trophoblast Cells

doi:10.21203/rs.3.rs-4940464/v1

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SIRT2 Inhibition Enhances Mitochondrial Apoptosis in Brucella-Infected Bovine Placental Trophoblast Cells

https://doi.org/10.21203/rs.3.rs-4940464/v1

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Brucella being a successful pathogen, employs a plethora of immune evasion mechanisms. This contributes to pathogenesis, persistence and also limits the efficacy of available treatment. Increasing understanding of host-pathogen interactions suggests that integrating host-directed strategies with existing anti-Brucella treatments could lead to more effective bacterial clearance and a reduction in drug-resistant strains. SIRT2 is a nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase found in mammals. It can deacetylate various transcription factors and regulatory proteins, playing a crucial role in host-pathogen interactions and pathogen infection-induced apoptosis. In this study, we investigate the role of SIRT2 in Brucella-induced cell apoptosis using bovine placental trophoblast cells. Our results indicate that B. abortus A19 infection upregulates SIRT2 protein expression and significantly induces mitochondrial apoptosis in these cells. Furthermore, Inhibition of SIRT2 exacerbates B. abortus A19-induced mitochondrial apoptosis and markedly inhibits intracellular bacterial survival. These results prove the role of SIRT2 in Brucella pathogenesis and the mechanism of action.

Brucella

Bovine placental trophoblast cells

SIRT2

Mitochondrial apoptosis

Brucella spp. are Gram-negative, facultative intracellular bacteria belonging to the class Alphaproteobacteria. Brucellosis, caused by Brucella spp., is a major zoonotic disease globally. In animals, it can lead to abortion and male infertility, while in humans, it presents with diverse symptoms including Malta fever, night sweats, anorexia, polyarthritis, meningitis, and pneumonia [1]. Currently, there is no internationally recognized safe and effective brucellosis vaccine in human [2]. Traditional treatment of brucellosis involves the use of multiple antibiotics, leading to an increase in drug-resistant cases [3]. This indicates a significant limitation of relying solely on microbial-targeted therapy.

Brucella have the ability to invade various host cells, including macrophages and trophoblasts [4]. To survive and proliferate in the harsh intracellular environment, Brucella has evolved various virulence factors or systems to evade immune response mechanisms, such as lipopolysaccharides (LPS), the Type IV secretion system (T4SS), and outer membrane proteins (OMPs) [5–7]. The immune system consists of innate immunity and adaptive immunity [8, 9]. Innate immunity serves as the first line of defense against pathogen invasion, relying on the host's pattern recognition receptors (PRRs) to identify pathogen-associated molecular patterns (PAMPs). This recognition triggers the production of interferons (IFNs) and inflammatory cytokines to eliminate the pathogens [10–13]. As the second line of defense, adaptive immunity enhances antibody production by activating antigen-presenting cells such as dendritic cells, phagocytes, and cytotoxic lymphocytes. During long-term interactions with the host, Brucella has evolved to use stealth strategies to evade, disrupt, or suppress immune responses, ultimately leading to chronic infections.

SIRT2 is a mammalian nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase primarily localized in the cytoplasm, functioning as the only major cytoplasmic Sirtuin protein [14]. Its activities are largely attributed to deacetylation targets within the cytoplasm. However, during mitosis and pathogen infection, SIRT2 shuttles to the nucleus and regulates chromatin condensation [15]. A small fraction of SIRT2 is also found in the endoplasmic reticulum-golgi intermediate compartment (ERGIC), where it modulates epigenetics and membrane transport [16]. Recent research has revealed a small amount of SIRT2 localized to mitochondria, participating in the regulation of mitochondrial fission and fusion [17]. SIRT2 interacts with numerous substrates, including histones and non-histone proteins, to regulate their biological activities. Its biological functions mainly involve the regulation of the host-pathogen interactions, cell cycle, metabolism, cancer and immunity [18–23]. The antibacterial effects of the SIRT2 inhibitor AGK2 have been confirmed in Mycobacterium tuberculosis, Listeria, Staphylococcus aureus, and Salmonella [18, 24–27]. In infections caused by Mycobacterium tuberculosis and Listeria, SIRT2 activity promotes bacterial survival and infection [18, 24]. In Staphylococcus aureus infection, SIRT2 may exacerbate the severity of the infection [27]. For Salmonella infection, SIRT2 demonstrates a dual role, aiding in bacterial clearance while potentially weakening host resistance [28]. However, the role of SIRT2 in Brucella infection remains unclear and requires further research to elucidate its role in the process of Brucella infection.

In this study, we used multiple methods to demonstrate that B. abortus A19 infection leads to an upregulation of SIRT2 protein expression in bovine placental trophoblast cells, significantly inducing apoptosis and mitochondrial damage. Inhibition of SIRT2 exacerbated B. abortus A19-induced mitochondrial apoptosis, significantly inhibiting bacterial intracellular survival. These findings elucidate the biological function of SIRT2 in Brucella infection and provide a theoretical basis for exploring effective host-directed therapies for brucellosis.

2.1 Biosafety Statement

All experiments were conducted in accordance with the "Regulations on Biosafety of Pathogenic Microorganism Laboratory" (2004) No. 424 issued by the State Council of the People's Republic of China and were approved by the Northwest A&F University Biosafety Committee.

2.2 Bacterial Strains and Mammalian Cells

The B. abortus vaccine strain A19 was provided by the Shaanxi Veterinary Drug Supervision Institute and cultured on tryptic soy agar (TSA) for 72 h or in tryptic soy broth (TSB) with shaking for 48 h. All procedures involving live B. abortus A19 strains were conducted in a biosafety level 3 (BSL-3) facility. The bovine placental trophoblast cells (BTCs) were provided by Beijing Agricultural College and cultured in DMEM/F-12 medium containing 10% fetal bovine serum (Thermo Scientific, Waltham, MA, USA) at 37℃ with 5% CO₂.

2.3 BTCs Infection Assay and AGK2 Treatment

BTCs in the logarithmic growth phase were seeded into 6-well plates at a density of 4×10⁵ cells per well and cultured for 12 h. Three bacterial strains were cultured, centrifuged at 5,500×g for 15 min, washed with PBS, and resuspended for colony-forming unit (CFU) counting on TSA plates. The cells were infected with B. abortus A19 at an MOI of 200. After 4 h, the cells were washed three times with PBS and cultured in DMEM/F-12 containing 50 µg/mL kanamycin for 1 hour to eliminate extracellular bacteria. Then, the medium was replaced with DMEM containing 10% FBS and 25 µg/mL kanamycin. At this point, AGK2 (10 µM, TargetMol, USA) was added to the cultures as needed.

2.4 Flow Cytometry Analysis

The proportion of apoptotic cells was measured using flow cytometry (Keygen, KGA105, Nanjing, China). Briefly, after infection with B. abortus A19, the cells were centrifuged, washed twice with PBS, and incubated with 5 µL of FITC-labeled Annexin V and 5 µL of PI for 20 min at room temperature in the dark. Analysis was performed using a flow cytometer (BD FACSAria™ III, Franklin Lakes, NJ, USA) and data were processed with Flowjo. At least 3×10⁴ cells were analyzed for each measurement.

2.5 Western Blotting

Infected BTCs were centrifuged, washed with PBS, and lysed in an ice-bath using lysis buffer (Keygen, KGP10100, Nanjing, China) for 30 min. The lysates were then centrifuged at 12,000×g at 4℃ for 15 min, and the supernatants were collected. Protein concentrations were measured using a BCA kit (Keygen, KGP903, Nanjing, China). SDS loading buffer was added, and samples were boiled at 100℃ for 10 min. Proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 10% nonfat milk for 2 h and incubated with the following primary antibodies for 24 h at 4℃: Bax (1:2000; Abmart, Shanghai, China), Caspase-3 (1:1000; Proteintech, Wuhan, China), SIRT2 (1:1000; Proteintech), P53 (1:2500; Proteintech), P53 (acetyl K370) (1:1000; MCE, America), Cyt-C (1:1000; Proteintech), DRP1 (1:1500; MCE), and β-actin (1:5000; Proteintech). After washing, the blots were incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. The signal was detected using an ECL chemiluminescence kit (Beyotime, P0018FS, Shanghai, China) and imaged with a gel imaging system (GE Amersham Imager 800, Boston, NY, USA).

2.6 Assessment of Mitochondrial Membrane Potential Change

BTCs in the logarithmic growth phase were seeded into 24-well plates at a density of 3×10⁴ cells per well. After 48 h of B. abortus A19 infection, the medium was aspirated, and the cells were washed twice with PBS, each wash lasting 5 min. JC-1 staining solution (Beyotime, Shanghai, China) was prepared according to the manufacturer's instructions and added to the wells. The cells were then incubated at 37℃ with 5% CO₂ for 30 min. After incubation, the cells were washed twice with 1×Buffer to remove excess staining solution and 2 mL of fresh RPMI-1640 (Hyclone, Logan, UT, USA) was added. The changes in mitochondrial membrane potential were observed using a confocal laser scanning microscope, and images were captured for analysis.

2.7 Cellular ATP Detection

Cellular ATP levels were measured using the ATP Assay Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. BTCs in the logarithmic growth phase were seeded into 6-well plates at a density of 4×10⁵ cells per well. After 48 h of B. abortus A19 infection, the culture medium was removed, and 200 µL of lysis buffer was added. The cells were lysed thoroughly by repeated pipetting. The lysates were centrifuged at 12,000×g for 5 min at 4℃, and the supernatant was collected for analysis. One hundred microliters of ATP detection solution were added to each sample and incubated at room temperature for 5 min to eliminate background ATP. An additional 20 µL of solution was then added to further reduce background. RLU values were measured using a multifunctional enzyme label reader (BioTek, Winooski, VE, USA). ATP concentration was calculated based on a standard curve (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM ATP) and converted to nmol/mg protein based on the protein concentration.

2.8 Measurement of Reactive Oxygen Species Formation

Intracellular reactive oxygen species (ROS) levels were measured using a 2’,7’-dichlorofluorescein diacetate (DCFH-DA) probe (Beyotime, Shanghai, China), which is oxidation-sensitive and non-fluorescent until oxidized to DCF. BTCs in the logarithmic growth phase were seeded into 24-well plates at a density of 3×10⁴ cells per well. After 48 h of B. abortus A19 infection, cells were collected and incubated with DCFH-DA (diluted to 10 mM in serum-free medium at 1:1000) for 20 min at 37℃. The cells were then washed three times, observed for green fluorescence using a fluorescence inverted microscope, and photographed for analysis.

2.9 RNA Isolation and Quantitative Real-Time PCR

Total cellular RNA was extracted using TRIzol (TaKaRa, Tokyo, Japan) and reverse-transcribed into cDNA using the Evo M-MLV RT reagent kit (AG Bio, Changsha, China). A 20 ng sample of cDNA was used as a template for analyzing mRNA levels by quantitative real-time PCR (qPCR) with the SYBR qPCR Master Mix kit (Vazyme Bio, Nanjing, China) on the Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Primer sequences are listed in Table S1. Expression levels were evaluated using the 2^−ΔΔCt method. mRNA expression was normalized to the GAPDH gene, which served as the control in all samples.

2.10 Extraction of Nuclear and Cytoplasmic Proteins

Nuclear and cytoplasmic proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (WANLEIBIO, Shenyang, China) according to the manufacturer's instructions. BTCs in the logarithmic growth phase were seeded into 6-well plates at a density of 4×10⁵ cells per well. After 48 h of B. abortus A19 infection, cell samples were collected. An appropriate amount of Cytoplasmic Protein Reagent A containing PMSF was added, mixed thoroughly, and incubated on ice for 15 min. Next, Cytoplasmic Protein Reagent B was added, mixed well, and incubated on ice for 1 minute, followed by centrifugation. The supernatant containing the cytoplasmic proteins was collected and transferred to an EP tube. The remaining pellet was resuspended in 50 µL of Nuclear Protein Reagent, mixed thoroughly, and incubated on ice for 30 min with vortexing every 2 min. The mixture was then centrifuged at 12,000 rpm for 10 min at 4℃. The supernatant, containing the nuclear proteins, was collected.

2.11 Immunofluorescence

BTCs were seeded on glass coverslips in 24-well plates at a density of 3×10⁴ cells per well. After 24 h of incubation with B. abortus A19, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 30 min at room temperature. The slides were then washed three times with PBS. For immunostaining, the slides were incubated overnight at 4℃ with primary antibodies Caspase-3 (1:500; Wuhan, China, Proteintech), SIRT2 (1:500; Proteintech). The following day, the slides were stained for 2 h at room temperature with Alexa-Fluor 488-conjugated donkey anti-rabbit IgG (for SIRT2, 1:500, A32790, ThermoFisher). Nuclei were stained with DAPI for 15 min. After each incubation step, the slides were washed four times with PBS. Images were captured using a confocal microscope (A1R; Nikon, Tokyo, Japan).

2.12 CFU Counting of Infected BTCs

BTCs were inoculated with B. abortus A19 at a multiplicity of infection (MOI) of 200. At the indicated time points, cells were washed three times with PBS and lysed with PBS containing 0.5% Triton X-100 for 10 min. The lysates were serially diluted with PBS, plated on TSA, and incubated at 37℃ for 72 h.

2.13 Statistical Analysis

All experiments were independently repeated at least three times, and the data are expressed as the mean ± standard deviation (SD) of the three replicate experiments. Statistical analysis was performed using SPSS software (SPSS, Inc., Cary, NC, USA). Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p-value of < 0.05 was considered statistically significant.

3.1 Brucella infection induces apoptosis in BTCs

The modulation of host cell apoptosis is a typical survival strategy used by infectious pathogens. The impact of B. abortus A19 infection on cell apoptosis in BTCs was examined using flow cytometry with Annexin V and propidium iodide staining. The apoptotic rate after 48 h was 15.23% for the infected group, compared with 10.57% for the control group (Fig. 1A, B). Subsequent immunofluorescence showed that infection with B. abortus A19 for 48 h resulted in increased expression of total caspase-3 (Fig. 1C, D). These results indicate that B. abortus A19 induces apoptosis in BTCs after 48 hours of infection.

3.2 Brucella infection induces mitochondrial damage in BTCs

Mitochondria are the sites of energy synthesis and storage, as well as material metabolism and energy conversion in cells. As executors of apoptosis, mitochondria induce cell apoptosis when damaged. Therefore, we investigated whether B. abortus A19 affects mitochondrial damage in BTCs after 48 h of infection. JC-1 staining was used to evaluate the extent of mitochondrial damage, and we observed a significant reduction in the mitochondrial membrane potential in the B. abortus A19 infection group (Fig. 2A). Additionally, using fluorescence probes DCFH-DA to detect ROS levels, we found that the ROS levels in the BTCs of the infection group were significantly higher than those in the control group (Fig. 2B), while ATP levels were significantly lower (Fig. 2C). These results indicate that mitochondrial function in BTCs is impaired by B. abortus A19 infection. Furthermore, we used RT-qPCR to detect changes in the expression of mitochondrial DNA (mtDNA1-6) and nuclear genome genes that control mitochondrial DNA replication (POLG, SSBP1, TOP1). The results showed that 48 h after infection with B. abortus A19, the mRNA expression of mtDNA2-6, POLG, SSBP1, and TOP1 in BTCs was significantly higher compared to the control group (Fig. 2D-F). These findings suggest that B. abortus A19 infection induces mitochondrial damage in BTCs.

3.3 Brucella infection upregulates the expression of SIRT2 in BTCs

To investigate the potential role of SIRT2 in BTCs infected with B. abortus A19, we examined the expression changes of SIRT2 after 48 hours of infection. We found that SIRT2 protein expression in BTCs was up-regulated by B. abortus A19 infection at 48 hours (Fig. 3A, B). It is known that SIRT2 is the only major Sirtuin primarily localized in the cytoplasm; however, during mitosis and pathogen infection, SIRT2 shuttles to the nucleus and regulates chromatin condensation [23]. To explore whether this phenomenon occurs during B. abortus A19 infection in BTCs, we analyzed the expression changes of SIRT2 in the cytoplasm and nucleus by Western blot. The results indicated that SIRT2 protein expression was down-regulated in the cytoplasm and upregulated in the nucleus by B. abortus A19 infection at 48 h (Fig. 3C, D). To further confirm the protein expression levels of SIRT2, we used immunofluorescence to detect the expression of SIRT2 in BTCs at 48 h post-infection. The results showed that SIRT2 expression was significantly higher in the infected group compared to the control group (Fig. 3E, F), which were consistent with the protein expression levels.

3.4 SIRT2 inhibits Brucella-induced mitochondrial apoptosis in BTCs.

To assess the role of SIRT2 in regulating Brucella-induced apoptosis, BTCs were infected with B. abortus A19, followed by the addition of 10 µM AGK2, a SIRT2 inhibitor, to the fresh culture medium. We employed flow cytometry to evaluate the impact of SIRT2 inhibition on apoptosis in BTCs. The results showed that SIRT2 inhibition significantly increased the apoptosis rate in the infected group (Fig. 4A, B), which suggested that SIRT2 inhibits Brucella-induced apoptosis.

It is known that a small amount of SIRT2 is localized in mitochondria and participates in regulating mitochondrial function [26]. Therefore, we hypothesized that SIRT2 might influence Brucella-induced apoptosis by regulating the mitochondrial apoptosis pathway. To verify this hypothesis, we measured changes in mitochondrial membrane potential and ROS levels. The results showed that inhibition of SIRT2 by B. abortus A19 infection led to a decrease in mitochondrial membrane potential (Fig. 4C) and an increase in ROS levels (Fig. 4D). We then used Western blot to examine the effects of SIRT2 inhibition on the expression of Caspase-3, Bax, P53, acetylated P53 (K370), Cyt-C, and DRP1 in BTCs. We observed that SIRT2 inhibition increased Caspase-3 and Bax expression (Fig. 4E-G), which induced apoptosis. It also decreased P53 protein levels (Fig. 4E, H) and increased acetylated P53 (K370) levels (Fig. 4E, I), indicating that SIRT2 may regulate apoptosis by deacetylating P53 (K370). Additionally, Cyt-C expression increased (Fig. 4J, K), affecting the mitochondrial electron transport chain, and DRP1 expression increased (Fig. 4J, L), implying disrupted mitochondrial fission in BTCs infected with B. abortus A19.

3.5 SIRT2 Promotes Brucella Replication

To determine the effect of SIRT2 on the replication of Brucella, we infected BTCs with B. abortus A19 and then added 10 µM of the SIRT2 inhibitor AGK2. Bacterial colony counts were measured at 0 h, 6 h, 12 h, 24 h, and 48 h. The results showed that there were no significant differences in colony counts at 0 h, 6 h, and 12 h. However, at 24 h and 48 h, inhibition of SIRT2 significantly reduced the number of intracellular Brucella in BTCs (Fig. 5). These findings indicate that SIRT2 promotes Brucella replication.

The primary pathogenic mechanism of Brucella involves its complex interaction with host cells, where regulating host cell apoptosis is a necessary step for Brucella to achieve its intracellular lifecycle [29, 30]. Brucella can either promote or delay apoptosis under different conditions. Caspase3, one of the most crucial executors in the apoptosis process, plays a major role in apoptosis. Previous research indicates that B. abortus infection in macrophages activates Nedd4, leading to the degradation of Calpain2, which in turn inhibits the activation of Caspase3, thus suppressing apoptosis [31]. ROS serves as the second messenger in apoptosis [32]. When cells receive apoptotic signals, ROS levels increase, which may lead to increased Ca²⁺ influx, upregulation of Bax, opening of the mitochondrial permeability transition pore (MPTP), activation of trypsin, and ultimately cell death [33]. Different levels of ROS determine whether cells undergo apoptosis, necrosis, or transition from apoptosis to necrosis. B. melitensis 16M can regulate the effect of the AIR domain on apoptosis in mouse macrophages through the ROS signaling pathway. As infection time increases, B. melitensis 16M enhances its ability to promote apoptosis. AIR can also affect B. melitensis 16M-induced apoptosis via the ROS pathway [34]. In this study, B. abortus A19 induced apoptosis in bovine placental trophoblast cells 48 h post-infection, with an apoptosis rate reaching 15.23%. This infection also elevated the expression of key apoptotic pathway proteins Caspase3.

Mitochondria play a critical role in the apoptosis process. When the intrinsic apoptotic pathway, characterized by the opening of the mitochondrial permeability transition pore and the loss of mitochondrial membrane potential, is activated, Cytochrome C (Cyt-C) is released from mitochondria via Bax and Bak from the Bcl-2-like family. In the cytoplasm, Cyt-C activates caspases, leading to programmed cell death [35, 36]. Typically, when a host is infected by pathogenic microorganisms, mitochondria can induce an immune response to eliminate the infected cells. Consequently, some pathogens may evolve various escape mechanisms to avoid being cleared by the host, while mitochondria may evolve corresponding mechanisms to resist pathogen infection. Studies have shown that Pseudomonas aeruginosa can induce mitochondrial damage, leading to lung injury and multiple organ failure [37]. Salmonella Typhimurium has also been found to cause cytosolic leakage of mtDNA and promote cGAS-STING signaling [38]. Both Mycobacterium abscessus and Mycobacterium tuberculosis can induce mitochondrial damage and mtDNA-mediated inflammatory signaling through inflammasome or cGAS-STING pathways [39, 40]. In this study, B. abortus A19 infection in bovine placental trophoblast cells led to decreased mitochondrial membrane potential, increased ROS levels, reduced ATP levels, and elevated mRNA expression levels of mtDNA2-6 after 48 h. Concurrently, the expression of nuclear genes POLG, SSBP1, and TOP1, which control mitochondrial replication, was consistent with mtDNA expression. These results indicate that B. abortus A19 infection causes mitochondrial dysfunction in bovine placental trophoblast cells.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) play crucial roles in chromatin structure modification and gene expression regulation. Previous studies have reported that microbial infections can affect the acetylation levels of host proteins [41]. SIRT2, as a key deacetylase in host cells, plays an important role in regulating various biological processes, including cell apoptosis, cell proliferation, oxidative stress, and host-pathogen interactions [42]. In this study, AGK2, a commonly used selective and specific SIRT2 inhibitor, was used to inhibit the deacetylase activity of SIRT2. This inhibitor has been applied in various cell models and has shown different effects, such as affecting cell cycle arrest, reducing cellular ATP levels, and inducing late apoptosis and necrosis [43]. As a well-established SIRT2 inhibitor, some studies using 10 µM AGK2 in mouse models have found no significant side effects [44]. In various bacterial infection models, such as Mycobacterium tuberculosis, Salmonella, and Listeria monocytogenes, AGK2 chemical inhibitors have been used to inhibit SIRT2 biological activity to explore the role of SIRT2 in bacterial pathogenic mechanisms. This study screened a non-toxic drug concentration of 10 µM for bovine placental trophoblast cells. At this concentration, the expression of SIRT2 protein in BTCs was significantly inhibited after 48 h, and this concentration did not affect the viability of Brucella itself. Therefore, 10 µM was chosen as the working concentration of AGK2.

SIRT2 plays a role in the apoptosis of various cell types. Studies have shown that down-regulation of SIRT2 can induce apoptosis in cancer cells and mediate oxidative stress-induced apoptosis [45, 46]. These findings indicate that SIRT2 has an anti-apoptotic function. However, the role of SIRT2 in apoptosis may be cell-type specific. Research has shown that SIRT2 does not participate in stress-induced apoptosis in human epithelial cancer cells HCT116 [47]. Moreover, SIRT family protein SIRT1 is also a crucial anti-apoptotic molecule in certain leukemia cells, indicating potential interactions between different SIRT proteins in regulating apoptosis [48]. Recent studies have found that a small amount of SIRT2 is localized in mitochondria and involved in regulating their function [49]. In bovine oocytes during meiosis, using a SIRT2 inhibitor led to mitochondrial dysfunction, increased ROS levels, and upregulation of the mitochondrial fission marker DRP1. This imbalance caused mitochondrial dynamics abnormalities in bovine oocytes during meiosis [50]. This study investigated the role of SIRT2 in Brucella-induced mitochondrial apoptosis in bovine placental trophoblast cells. The results showed that after Brucella infection and AGK2 treatment for 48 h, flow cytometry, and immunoblotting analysis revealed that AGK2 treatment further increased Brucella-induced apoptosis, and CFU counts showed a significant reduction in bacterial load. In the AGK2 treatment group, the mitochondrial membrane potential was significantly lower than in the control group, and ROS levels were significantly increased, indicating that SIRT2 inhibition affected mitochondrial function. To elucidate the mechanism by which SIRT2 mediates mitochondrial function, subsequent immunoblotting analysis was conducted to investigate the expression levels of key proteins involved in maintaining mitochondrial function. The results showed that SIRT2 inhibition significantly reduced the expression of the mitochondrial fission-related protein DRP1 in Brucella-infected BTCs.

There is a complex interaction between SIRT2 and P53. Studies have found that SIRT2 can interact with P53 and downregulate its activity [51]. Downregulation of SIRT2 can lead to P53 accumulation and apoptosis in cancer cells [45]. Therefore, we speculate that in the process of Brucella-induced apoptosis in BTCs, SIRT2 may participate in regulating P53 expression to inhibit apoptosis. Our results showed that in the infected group, SIRT2 inhibition for 48 h significantly reduced the total P53 protein expression, and acetylated P53 (K370) levels significantly increased, indicating that SIRT2 may inhibit Brucella-induced BTCs apoptosis by deacetylating P53 (K370). However, the specific molecular mechanisms by which SIRT2 regulates P53 require further investigation.

In summary, this study investigated the impact of SIRT2 on intracellular survival of Brucella and Brucella-induced mitochondrial apoptosis in trophoblast cells, revealing the role of SIRT2 in the pathogenic mechanism of Brucella. This provides a theoretical basis for exploring effective host-directed therapies for brucellosis, offering new insights into how Brucella evades immune clearance through host cell regulation mechanisms. However, this study faces challenges and unresolved questions. Firstly, the specific molecular mechanisms by which SIRT2 regulates mitochondrial function and cell apoptosis during Brucella infection require further investigation. Secondly, other signaling pathways and regulatory mechanisms induced by Brucella infection remain unclear; future research should focus on these aspects to comprehensively understand the complex network of interactions between Brucella and host cells. In conclusion, this study not only reveals the role of SIRT2 in regulating cell apoptosis during Brucella infection but also provides new directions for the development of targeted therapies against brucellosis.

Ethics approval and consent to participate

Not applicable. This study did not involve human participants or animals.

Consent for publication

Not applicable.

Availability of data and material

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the National Natural Science Foundation of China (32373016).

Authors' contributions

M. Z. and J.L. developed the conceptual framework and methodology. M.Z., N.Y., and M.H. handled software-related aspects. M.Z. and Y.Z. performed validation. M.Z. conducted the investigation. A.W. and D.Z. provided resources. A.W. and Y.J. managed data curation. M.Z. prepared the original draft of the manuscript. M.J. and Y.J. reviewed and edited the manuscript. A.W., W.L., and Y.J. oversaw the project. A.W. and Y.J. handled project administration and funding acquisition.All authors have read and agreed to the published version of the manuscript.

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SIRT2 Inhibition Enhances Mitochondrial Apoptosis in Brucella-Infected Bovine Placental Trophoblast Cells

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Biosafety Statement

2.2 Bacterial Strains and Mammalian Cells

2.3 BTCs Infection Assay and AGK2 Treatment

2.4 Flow Cytometry Analysis

2.5 Western Blotting

2.6 Assessment of Mitochondrial Membrane Potential Change

2.7 Cellular ATP Detection

2.8 Measurement of Reactive Oxygen Species Formation

2.9 RNA Isolation and Quantitative Real-Time PCR

2.10 Extraction of Nuclear and Cytoplasmic Proteins

2.11 Immunofluorescence

2.12 CFU Counting of Infected BTCs

2.13 Statistical Analysis

3 Results

3.1 Brucella infection induces apoptosis in BTCs

3.2 Brucella infection induces mitochondrial damage in BTCs

3.3 Brucella infection upregulates the expression of SIRT2 in BTCs

3.4 SIRT2 inhibits Brucella-induced mitochondrial apoptosis in BTCs.

3.5 SIRT2 Promotes Brucella Replication

4 Discussion

Declarations

References

Supplementary Files

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