Fusobacterium necrophorum (F. necrophorum) is a Gram-negative, non-spore-forming, rod-shaped anaerobic bacterium. It is one of the most common anaerobes isolated from abscesses and respiratory tract infections of animals [1,2]. F. necrophorum is an opportunistic pathogen that can cause numerous necrotic infections (necrobacillosis), among which bovine liver abscess and foot rot are significant concerns for the cattle industry [3]. The main virulence factors associated with pathogenesis include leucocytotoxin, endotoxin, hemolysin, hemagglutinin, proteases, and adhesins [3]. Among these pathogenic factors, leucocytotoxin and hemolysin are the two main virulence factors of F. necrophorum and are targets for F. necrophorum vaccines [4,5]. However, leucocytotoxin and hemolysin as vaccine antigens only partially protect from F. necrophorum infection [6,7]. Therefore, it is necessary to identify and evaluate other functional proteins of F. necrophorum as vaccine candidates.

Outer membrane proteins (OMPs), which are components of the cell wall of Gram-negative bacteria, play many roles in pathogenesis, resistance, and disease development by acting as a dynamic interface between the cell and its surroundings [8,9]. An important OMP of F. necrophorum, the 43 kDa outer membrane protein (43 K OMP), is a putative porin protein that is closely related to the OMPs of F. nucleatum and F. periodonticum [[10], [11], [12]]. This protein is widespread among bovine and ovine clinical isolates of F. necrophorum [10,13,14]. Furthermore, 43 K OMP of F. necrophorum can bind to the cell membrane of BHK cells, and bovine mammary epithelial cells [15,16]. These findings suggest a possible role for 43 K OMP in the pathogenesis of F. necrophorum, and such a role requires further investigation.

OMPs are major immunogenic proteins that confer bacteria host-fitness advantages including; immune evasion, stress tolerance, and resistance to antibiotics as well as anti-bacterials [17]. The OMPs of some Gram-negative bacteria have also been shown to be important virulence factors. The FomA porin of F. nucleatum, which has high homology to the 43 K OMP of F. necrophorum, is a Toll-Like receptor 2 agonist with immune adjuvant activity [18]. Whether 43 K OMP has a similar activity is unknown, but understanding the structure and function of 43 K OMP will facilitate the design of antimicrobial drugs and vaccines. While no immune stimulatory effects of 43 K OMP have been evaluated to date, evidence of its immunogenicity and the protective effect of anti-43 K OMP antibodies in F. necrophorum infection have been reported. For example, immunization of mice with recombinant 43 K OMP induced high levels of anti-43 K OMP IgG antibodies that were protective during F. necrophorum infection [19]. We have previously shown a total of 12 potential B-cell antigenic regions within 43 K OMP of F. necrophorum based on antigenic index, surface probability plot, hydrophilicity plot, flexible regions, and alpha regions [10,12]. Thus, 43 K OMP may be a candidate antigen for subunit vaccination to prevent F. necrophorum infection. Furthermore, the multi-component subunit vaccine composed of 43 K OMP, leucocytotoxin (PL4), and hemolysin (H2) provides better protection from liver abscess in mice than the single components [19]. Based on the structural and functional similarities between 43 K OMP and other Gram-negative bacterial porins, we have evaluated the biological function of 43 K OMP. Herein, the effect of the 43 K OMP of F. necrophorum on bovine mammary epithelial cell proteins was analyzed using Tandem Mass Tag (TMT)-based Proteomics. We investigated the ability of 43 K OMP to induce a macrophage immune response and analyzed the signaling pathway involved in 43 K OMP-induced inflammatory cytokine production as well as the role of 43 K OMP in the pathogenesis of F. necrophorum for host cells.