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• Deletion of the flik gene did not affect the main biological properties of Brucella. • Deletion of the flik gene enhances the inflammatory response to Brucella infection in RAW264.7 cells. • FliK inhibits the inflammatory response in RAW264.7 cells through the NF-κB pathway and NLRP3 inflammasome pathway. The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella , but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response. [ABSTRACT FROM AUTHOR]

• In this study, we continue our exploration of the alr gene, investigating its role in the interaction between Brucella and the host through murine and cellular experiments. • Our findings indicate that the extracellular substances produced by Δ alr effectively activate the TLR4-NF-κB-NLRP3 pathway in macrophages, inducing a more robust inflammatory response and subsequently reducing bacterial survival within cells. • Furthermore, Δ alr exhibits lower toxicity and enhanced immunogenicity in mice. • Importantly, we further elucidate the pivotal role played by alr in immune evasion during the interaction between Brucella and the host, providing insights for both complementing alr function and advancing Brucella vaccine development. Brucella is an intracellular parasitic bacterium lacking typical virulence factors, and its pathogenicity primarily relies on replication within host cells. In this study, we observed a significant increase in spleen weight in mice immunized with a Brucella strain deleted of the gene for alanine racemase (Alr), the enzyme responsible for alanine racemization (Δ alr). However, the bacterial load in the spleen markedly decreased in the mutant strain. Concurrently, the ratio of white pulp to red pulp in the spleen was increased, serum IgG levels were elevated, but no significant damage to other organs was observed. In addition, the inflammatory response was potentiated and the NF-κB-NLRP3 signaling pathway was activated in macrophages (RAW264.7 Cells and Bone Marrow-Derived Cells) infect ed with the Δ alr mutant. Further investigation revealed that the Δ alr mutant released substantial amounts of protein in a simulated intracellular environment which resulted in heightened inflammation and activation of the TLR4-NF-κB-NLRP3 pathway in macrophages. The consequent cytoplasmic exocytosis reduced intracellular Brucella survival. In summary, cytoplasmic exocytosis products resulting from infection with a Brucella strain deleted of the alr gene effectively activated the TLR4-NFκB-NLRP3 pathway, triggered a robust inflammatory response, and reduced bacterial survival within host cells. Moreover, the Δ alr strain exhibits lower toxicity and stronger immunogenicity in mice. [ABSTRACT FROM AUTHOR]

Brucella species are infectious facultative intracellular pathogens. They have evolved multiple strategies to thwart immune responses and replicate in macrophages for chronic persistence in the host. As a Brucella effector, BtpB is transferred into target cells through the type IV secretion system. BtpB, a Toll/interleukin-1 receptor domain-containing protein, blocks host innate immune responses by interfering with Toll-like receptor signaling. However, the intracellular targets and their activated downstream pathways remain unclear. In this study, we constructed a strain of Brucella suis S2 with a deletion in the gene for BtpB, Δ btpB , and the complemented strain, C-Δ btpB with a restored copy of the btpB gene. The bacterial growth curves and stress resistance results showed that BtpB did not affect B. suis S2 growth. Infection of alveolar macrophages with WT and Δ btpB strains showed that BtpB inhibited TLR2 and TLR4 expression and attenuated NLRP3 inflammasome activation. BtpB also attenuated secretion of the Brucella -induced proinflammatory cytokines, IL-1β, IL-6, and TNF-α, in alveolar macrophages while up-regulating IL-10 expression. In general, the results confirmed that BtpB specifically inhibits TLR2/TLR4 and disrupts NLRP3 signaling pathways to inhibit host immune responses in early Brucella infections. • BtpB inhibits Brucella -induced innate inflammatory responses. • BtpB inhibits TLR2 and TLR4 expression, and nuclear translocation of NF-κB during Brucella infection. • BtpB suppresses the formation of cellular ROS during Brucella infection. • BtpB inhibits NLRP3 inflammasome activation during early Brucella infection. [ABSTRACT FROM AUTHOR]