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The secretory proteins of Brucella mediate the expression of the bacterium in the host, thereby facilitating intracellular parasitism. With the exception of the recently reported BspJ, the Brucella nucleomodulin has not yet been characterized. We defined the Brucella nucleomodulin BspG and verified six proteins (PCBP1, KMT5C, NDUFS6, PCNA, CIAO2B, and SDHB) that interacted with BspG using a yeast two-hybrid assay and co-immunoprecipitation (CO-IP) screening. The deletion of BspG decreased the intracellular proliferation of B. abortus in both in vivo and in vitro experiments. The analysis found that these interacting proteins were related to energy generation, gene expression, and apoptosis of host cells. The crosstalk between B. abortus nucleomodulin BspG and host DNA replication/mitochondrial respiratory pathways promotes anti-apoptosis and infection, but the mechanism needs additional study., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Brucella abortus is a gram-negative intracellular parasite bacteria that causes serious health hazards in humans and animals. The type IV secretion system (T4SS), encoded by the virB promoter, has been identified as an important virulence factor for Brucella abortus , but its impact on Brucella abortus A19 remains unclear. In this study, the T4SS of Brucella abortus A19 was inactivated by deletion of the virB promoter, resulting in a mutant strain A19Δ virB . Real-time PCR and western blotting analysis demonstrated that T4SS-related proteins were not expressed after virB promoter deletion. Moreover, the survival rate of A19 in high-salt and strong acidic environments decreased after virB promoter deletion. Compared to the parental strain A19, the A19Δ virB mutant strain showed reduced growth rate in TSB, decreased invasion ability to macrophages and dendritic cells, and reduced virulence of the mutant strain in macrophages, dendritic cells, and mice. In addition, the A19Δ virB mutant strain showed enhanced autophagy in macrophages and dendritic cells compared with A19, and the A19Δ virB mutant strain was able to upregulate IL-6 and downregulate IL-10 in macrophages. These data help us to better understand the T4SS of the A19 vaccine strain and contribute to our efforts to improve Brucella vaccines.

Background: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified., Objectives: To verify the functions of nucleomodulin BspJ in Brucella 's intracellular infection cycles., Methods: Constructed Brucella abortus BspJ gene deletion strain ( B. abortus ΔBspJ) and complement strain ( B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro ., Results: BspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella -containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella ., Conclusions: BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella 's intracellular survival., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Korean Society of Veterinary Science.)

Brucella is a genus of Gram-negative intracellular pathogens that cause animal and human diseases. Brucella survival and replication inside immune cells is critical for the establishment of chronic infections. Protein modifications by small ubiquitin-related modifier proteins and the NF-κB pathway are involved in many cellular activities, playing major roles in regulating protein function that is essential for pathogenic bacteria during infection. However, the relationship between them in the intracellular survival of Brucella is still largely unknown. We demonstrated that Brucella abortus 2308 infection can activate the expression of small ubiquitin-related modifier-2 proteins in a time-dependent manner. We found the production of Th1 cytokines (IFN-γ and TNF-α) and the transcription of NF-κB/p65 were promoted by overexpression and inhibited by interference of small ubiquitin-related modifier-2. In addition, we showed that small ubiquitin-related modifier-2 can inhibit intracellular survival of Brucella abortus 2308 by regulating activation of the NF-κB pathway. Taken together, this work shows that small ubiquitin-related modifier-2 modification of NF-κB2/p65 is essential for the survival of Brucella abortus 2308 inside macrophages. This work may help to unravel the pathogenic mechanisms of Brucella infections.

To date, a variety of Brucella effector proteins have been found to mediate host cell secretion, autophagy, inflammation, and other signal pathways, but nuclear effector proteins have not yet been reported. We identified the first Brucella nucleomodulin, BspJ, and we screened out the BspJ interaction host proteins NME/NM23 nucleoside diphosphate kinase 2 (NME2) and creatine kinase B (CKB) through yeast two-hybrid and co-immunoprecipitation assays. These proteins are related to the host cell energy synthesis, metabolism, and apoptosis pathways. Brucella nucleomodulin BspJ will decrease the expression level of NME2 and CKB. In addition, BspJ gene deletion strains promoted the apoptosis of macrophages and reduced the intracellular survival of Brucella in host cells. In short, we found nucleomodulin BspJ may directly or indirectly regulate host cell apoptosis through the interaction with NME2 and CKB by mediating energy metabolism pathways in response to the intracellular circulation of Brucella infection, but the mechanism needs further study. [ABSTRACT FROM AUTHOR]