Brucellosis is a disease caused by members of the Brucella genus, and it is considered one of the most serious and debilitating zoonotic diseases [1]. Brucella is a gram-negative, facultative intracellular pathogen with a spherical or short rod-shaped body without spores, capsule or exotoxins [2]. It is generally accepted that Brucella lacks the classical virulence factors such as exotoxins, capsules, plasmids, pili, and cytolysins. Its virulence factors are mainly related to the invasive and proliferative ability of Brucella, and include lipopolysaccharide (LPS), outer membrane proteins (OMPs), a type IV secretion system (T4SS), the BvrR/BvrS two-component system, and cyclic β-1,2-glucan, among others [3], [4]. Specific virulence factors present in Brucella are active in regulating and evading the host immune response, maintaining survival in the host and establishing a persistent infection [5], [6].

The bacterial flagellum is an elongated filament protruding from the bacterial cell, which allows bacteria to better adapt to their environment as a result of long-term adaptations in the evolutionary process [7]. It was previously thought that because Brucella lacked motility, they must not have flagella, but with the development of electron microscopy techniques, flagella were discovered on Brucella. DelVecchio et al [8] demonstrated that Brucella abortus had all the flagellar structural genes, but not the chemotaxis genes, which form three loci with extensive genetic homology to Mesorhizobium. Conversely, Fretin et al [9] observed that Brucella melitensis had 31 flagellar genes, among which 26 were structural and five were for motility, sufficient to assemble a complete flagellum. The disregard for the importance of the flagellum in Brucella’s virulence has often been because this function is not associated with its motility. Studies have demonstrated that mutations in genes that encode flagellins, such as fliF, flhA, flgI, motB and flgE, lead to reduced virulence of Brucella abortus [10], [11], [12]. Mutations in fliF and flgF attenuated virulence in infection models in mice and pregnant goats [12], [13]. The bacterial flagellin, FliK, is an essential protein in flagellar synthesis, controlling the length of the flagellar hook during assembly of the flagellum [14]. FliK is secreted from the bacterial cell during flagellar hook assembly, and stays in the cell by interacting with the secretory apparatus upon completion of hook assembly [15]. Previous research has suggested that FliK proteins are essential for virulence in Vibrio alginolyticus, Bacillus thuringiensis and Salmonella typhimurium, but few studies have looked at the effect of FliK on Brucella virulence [16], [17], [18].

While the mechanism by which the FliK regulates flagellar assembly has been extensively researched, its role in bacterial virulence and pathogenesis has received limited study. In this study, we investigated the effects of FliK on the biological properties of Brucella suis S2 and the inflammatory response of a murine macrophage cell line after infection by a Brucella suis S2 flik gene deletion mutant compared with the complemented and wild-type strains. In addition, recombinant FliK protein was obtained from a prokaryotic expression system. The LPS-mediated inflammatory response of RAW264.7 macrophages was established as a model, which further revealed the regulatory role of FliK in the response of macrophages to Brucella infection, as well as its correlation with the NF-κB signaling pathway and NLRP3 inflammasomes. Our study provides new evidence for the involvement of FliK in Brucella virulence and regulation of host cell inflammatory responses.