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Neurobrucellosis is a serious central nervous system (CNS) inflammatory disorder caused by Brucella, and outer membrane protein-31 (Omp31) plays an important role in Brucella infection. This study aims to determine whether Omp31 can induce autophagy in BV-2 microglia. Another goal of the study is to further examine the effect of autophagy on the nuclear transcription factor κB (NF-κB) p65 signaling pathway. We observed that Omp31 stimulated autophagy by increasing microtubule-associated protein 1 light chain 3B (LC3B-II) levels and inducing autophagosome formation at 6 h and 12 h. Concomitantly, Omp31 induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression in a time-dependent manner but reduced the expression of TNF-α at 6 h. We utilized Omp31 with or without rapamycin or 3-methyladenine (3-MA) to treat BV-2 microglia, and it demonstrated further that Omp31 induced autophagy by promoting LC3B-II, Beclin-1 proteins expression and inhibiting the p62 protein levels. Furthermore, we explored the effects of autophagy on the NF-κB p65 pathway through western blot analysis, RT-qPCR assay, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The data suggest that Omp31 as well as rapamycin, the autophagy inducer, can decrease TNF-α levels through the inhibition of the NF-κB p65 signaling pathway. Taken together, Omp31 can function as a catalyst in both autophagy induction and NF-κB p65 signal inhibition. Furthermore, Omp31-induced autophagy may inhibit the expression of TNF-α by negatively regulating NF-κB p65 signaling pathway., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Brucellosis, caused by a facultative intracellular parasite Brucella species, is the most common bacterial zoonotic infection worldwide. Brucella can survive and proliferate in several phagocytic and non‑phagocytic cell types. Human brucellosis has similar clinical symptoms with systemic diseases, which may lead to delay of diagnosis and increasing of complications. Therefore, investigating the proliferation of Brucella in host cells is important to understand the pathogenesis of the disease. Dihydroartemisinin (DHA), a semi‑synthetic derivative of artemisinin, has been recommended by World Health Organization as an anti‑malarial drug. However, there have been few studies regarding its effectiveness against bacteria. In the present study, it was revealed that B. suis vaccine strain 2 (S2) grew in BV2 cells without significant cytotoxicity, and less than 20 µM DHA had no inhibitory effects on BV2 cells. Furthermore, DHA reduced B. suis S2 growth in BV2 cells, and increased the percentage of apoptosis and the expression of cleaved caspase‑3 in B. suis S2‑infected cells. Collectively, the present data indicated that DHA induced the caspase‑dependent apoptotic pathway to inhibit the intracellular B. suis S2 growth.

Objective To determine the influence of outer membrane protein 31 (Omp31) of B. melitensis on the formation of autophagosomes in BV-2 microglia cells. Methods BV-2 cells were treated for 6 hours with Omp31 at 0.17, 0.50, 1.50, 4.50, 13.50 μg/mL. Real-time quantitative PCR was used to detect the expression of microtubule associated protein 1 light chain 3B (LC3B) mRNA; Western blotting was applied to detect the expressions of LC3B II and LC3B proteins; transmission electron microscopy was performed to observe cell autophagy in each group. The levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-10 in the culture cell supernatant were determined with ELISA. Results Omp31 treatment resulted in an increase in the level of LC3B II protein, especially the most obvious increase in the ones treated by 0.5 μg/mL Omp31. The concentration of Omp31, being no more than 0.5 μg/mL, could promote the expression of LC3B mRNA, while more than 0.5 μg/mL Omp31, it could inhibit the expression of LC3B mRNA; 0.5 μg/mL Omp31 could induce the formation of more autophagosomes in BV-2 cells. Omp31 promoted the expressions of TNF-α and IL-6, and inhibited the expression of IL-10 in BV-2 cells. Conclusion Omp31 at the concentration of 0.5 μg/mL can significantly induce autophagy in BV-2 cells.

Neurobrucellosis is a chronic complication of human brucellosis that is caused by the presence of Brucella spp in the central nervous system (CNS) and the inflammation play a key role on the pathogenesis. Doxycycline (Dox) is a widely used antibiotic that induces apoptosis of bacteria-infected cells. However, the mechanisms of Brucella inhibition of microglial apoptosis and Dox induction of apoptosis are still poorly understood. In this study, we found that Brucella suis S2 strain (B. suis S2) increased calreticulin (CALR) protein levels and inhbited HMC3 cell apoptosis. Hence, we constructed two HMC3 cell line variants, one with stable overexpression (HMC3-CALR) and one with low expression of CALR (HMC3-sh-CALR). CALR was found to decrease levels of p-JNK and p-p53 proteins, as well as suppress apoptosis in HMC3 cells. These findings suggest that CALR suppresses apoptosis by inhibiting the JNK/p53 signaling pathway. Next, we treated HMC3, HMC3-CALR and HMC3-sh-CALR cell lines with B. suis S2 or Dox. Our results demonstrate that B. suis S2 restrains the JNK/p53 signaling pathway to inhibit HMC3 cell apoptosis via increasing CALR protein expression, while Dox plays the opposite role. Finally, we treated B. suis S2-infected HMC3 cells with Dox. Our results confirm that Dox induces JNK/p53-dependent apoptosis in B. suis S2-infected HMC3 cells through inhibition of CALR protein expression. Taken together, these results reveal that CALR and the JNK/p53 signaling pathway may serve as novel therapeutic targets for treatment of neurobrucellosis. [ABSTRACT FROM AUTHOR]

The ability to inhibit host macrophage apoptosis is one of the survival strategies of intracellular bacteria, including Brucella. In the present study the role of Mg2+/Mn2+ dependent protein phosphatase 1A (PPM1A) in the apoptosis of Brucella suis (B. suis) strain 2 vaccine-infected BV2 cells was investigated. Compared with control cells, the protein expression levels of cleaved caspase-3 were markedly increased in PPM1A short hairpin (sh)RNA-transfected BV2 cells. Flow cytometry analysis showed that treatment with JNK activator anisomycin significantly increased the rate of apoptosis in BV2 cells in comparison with the control cells. Furthermore, PPM1A shRNA significantly increased the levels of JNK phosphorylation and the levels of cleaved caspase-3 in BV2 cells infected with B. suis strain 2 in comparison with the control cells. DAPI staining showed nuclear condensation in B. suis infected BV2 cells transfected with PPM1A shRNA in comparison with the control shRNA cells. Flow cytometry analysis showed that PPM1A shRNA significantly increased the percentage of apoptotic BV2 cells infected with B. suis strain 2 compared with those transfected with control shRNA. Taken together, these data suggested that knockdown of PPM1A promotes apoptosis in B. suis strain 2-infected BV2 cells and that PPM1A may be a potential target in the development of treatments to inhibit intracellular growth of B. suis. [ABSTRACT FROM AUTHOR]