The bacteria strains and plasmids used in this study are listed in Table 1 . The pathogenic strain C48-1 (A:1) of P. multocida was isolated from chicken with fowl cholera in Jiangsu, China, in 1953 and was stored in our laboratory. P. multocida C48-1 strain (A:1) was cultured in tryptic soy broth (TSB; Difco Laboratories, USA) or tryptic soy broth agar (TSA) medium containing 5% fetal bovine serum (FBS; Sigma-Aldrich, USA) at 37°C. Escherichia coli DH5α and BL21 (DE3) were cultured in the lysogeny broth (LB; Difco Laboratories, USA) medium at 37°C. Mouse macrophages RAW264.7 were cultured in the Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, USA) containing 10% FBS and under 5% CO₂. Wild-type C57BL/6N, TLR2,^−/− and TLR4^−/− mice were injected intraperitoneally with 4% thioglycolate broth (Sigma-Aldrich, USA). After 3 days of induction, the peritoneal macrophages were collected and cultured in the Roswell Park Memorial Institute (RPMI; Sigma-Aldrich, USA) 1640 medium with 10% FBS under 5% CO₂ for 2 h. The C57BL/6N mice were purchased from the Charles River Laboratories (Beijing, China). TLR2^−/− and TLR4^−/− with a C57BL/6N background were purchased from Cyagen Biosciences (Suzhou, China). Mice were maintained under specific pathogen-free (SPF) conditions in a standard animal care facility (Laboratory Animal Center, Harbin Veterinary Research Institute, Harbin, China). All animal experiments were performed according to the animal protocols approved by the Laboratory Animal Ethical Commission of Harbin Veterinary Research Institute, CAAS (HVRI-IACUC-2019-214).

The following antibodies were used: antibodies against ERK1/2, p38, jun N-terminal kinase (JNK), p65, p-ERK1/2, p-p38, p-JNK, and p-p65 were purchased from Cell Signaling Technology (USA); β-actin was purchased from Yeasen (Shanghai, China). Antibodies against TLR2 and TLR4 were purchased from Novus Biological (USA). Mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kits used were purchased from Neobioscience (China). The p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059), JNK inhibitor (SP600125), and NF-κB inhibitor (BAY 11-7082) were purchased from Sigma-Aldrich (USA).

The PMCNA_RS00975 amino acid sequences of P. multocida strain C48-1 (GenBank Accession numbers: GCA_004286945.1) were aligned with the sequences of 17 homologous proteins from other bacteria ( Supplementary Table S1 ) using the Basic Local Alignment Search Tool (BLAST). These protein sequences were aligned using the ClustalW program using the Molecular Evolution Genetics Analysis (MEGA) 11 software (10.1093/molbev/msab120). The phylogenetic tree of these proteins was generated using a neighbor-joining method.

The upstream and downstream sequences of the PMCNA_RS00975 gene (999 bp) (GenBank accession number: OAZ06802.1) of the C48-1 strain were amplified by PCR using primers PPm00975-up-F/R and PPm00975-down-F/R ( Table 2 ). Based on the plasmid pUC-4K, the Kanamycin (Kan) resistance gene expression cassettes were amplified using the Kan-F/R primers ( Table 2 ) and digested with EcoR I and BamH I. Afterward, the Kan resistance gene expression cassette was ligated to the vector pBluescript-SK (+) with the same digestion treatment to construct the recombinant plasmid pBC-PMCNA_RS00975M.

The isogenic PMCNA_RS00975 gene mutant was constructed using the method previously described (Aranda et al., 2010). Briefly, the plasmid pBC-PMCNA_RS00975M was electrotransformed into cells of the strain C48-1, and spread onto TSA containing 100 μg/mL Kan and incubated for 72 h at 37°C to select recombination single-crossover. The selected Kan mutant was spread on the chloromycetin (Cm)- and Kan-resistant media, and the Cm-sensitive but Kan-resistant mutant was selected to be the homologous recombination double-crossover. The primers PPm00975-JD-F/R and Kan-F/R ( Table 2 ) were used to identify the double-crossover mutant. The successfully identified double-crossover mutant was termed C48-ΔPMCNA_RS00975 and identified using PCR with primers KMT1T7/KMT1SP6. The identified correctly C48-ΔPMCNA_RS00975 mutant strain was subcultured for 20 generations, and the Kan resistance gene was detected every five generations to identify its genetic stability.

For producing a complemented strain of the mutant, spectinomycin (Spec) resistance gene expression cassettes were amplified using the Spec-F/R primers ( Table 2 ) and inserted into the endonuclease Nde I of plasmid pAL99 to generate the plasmid pAL99spec ^R (Harper et al., 2003; Jiang et al., 2015). Simultaneously, the PMCNA_RS00975 gene expression cassettes comprising a 134-bp upstream segment, a 178-bp downstream segment, and 999-bp the PMCNA_RS00975 were amplified from the genomic DNA of the wild-type strain using PCR using the primers PPm00975-EF/R ( Table 2 ) and digested with BamH I and Xho I. Afterward, they were cloned into the shuttle plasmid pAL99spec ^R for constructing the recombinant plasmid pAL99spec ^R-PMCNA_RS00975. Further, the recombined plasmid pAL99spec ^R-PMCNA_RS00975 was electrotransformed into the C48-ΔPMCNA_RS00975 mutant under the same conditions as described above. Next, the pAL99spec ^R-PMCNA_RS00975 plasmids were transformed into the C48-ΔPMCNA_RS00975 mutant strain to generate the complemented strain C48-ΔPMCNA_RS00975C. Next, the C48-1 wild-type, C48-ΔPMCNA_RS00975 mutant, and complemented strain C48-ΔPMCNA_RS00975C were cultured in cultured in TSB medium containing 5% fetal bovine serum with or without 100μM 2,2’-dipyridyl (iron-restrictive medium) at 37°C for 8 h, following which the OD_600nm values were measured. The sequences of the primers synthesized by Sangon (Shanghai, China) are shown in Table 2 .

To assess the function of PMCNA_RS00975 in the virulence of P. multocida, C57BL/6N mice (6–8-week-old) were infected with the wild-type, mutant, or complemented strains via intraperitoneal injection at a dose of 5 × 10¹ to 5 × 10⁵ CFUs (n = 10 per group). The virulence of the wild-type, mutant, and complemented strains was compared using the survival curve of mice and mortality on the 7th day post-infection. Mice showing severe clinical signs were considered moribund and were humanely killed.

Next, the function of PMCNA_RS00975 in the virulence of P. multocida was confirmed by incubating the wild-type C48-1, C48-ΔPMCNA_RS00975 mutant, and complemented strain C48-ΔPMCNA_RS00975C with RAW264.7 cells for 3 h, 6 h, 9 h, and 12 h at a dose of 10 CFUs. The expression of IL-6 from cell supernatant was collected and detected using a mouse IL-6 ELISA kit.

The PMCNA_RS00975 gene was amplified using PPm00975-EF/R and cloned into plasmid pPRO-EX-HTb to construct a recombinant plasmid pPRO-EXHTb-PMCNA_RS00975. The recombinant plasmid pPRO-EXHTb-PMCNA_RS00975 was transformed into Escherichia coli BL21 (DE3) competent cells with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Tiangen, China) at 16°C. The soluble protein was purified according to the manufacturer’s protocol of the Ni-NTA His-Bind Resin Affinity Kit and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the purified recombinant PMCNA_RS00975 (rPMCNA_RS00975) proteins were dialyzed in PBS and analyzed using the anti-His monoclonal antibody (Thermo, USA) or the anti-rPMCNA_RS00975 mouse serum. The anti-rPMCNA_RS00975 mouse serum was prepared according to the previous method (Xu et al., 2023).

To determine if the expression of PMCNA_RS00975 protein by P. multocida C48-1 strain elicited the production of antibodies during infection, we performed western blotting. The rabbit antiserum of P. multocida C48-1, produced in our laboratory, was used as the primary antibody to analyze the immunogenicity of the PMCNA_RS00975 protein produced by infection with P. multocida C48-1.

For western blotting analysis, the rPMCNA_RS00975 protein was transferred onto polyvinylidene fluoride membranes (Merck, Millipore, USA) and blocked with 5% skim milk overnight at 4°C. Anti-rPMCNA_RS00975 serum from mice or anti-P. multocida C48-1 serum from rabbits was used as the primary antibody, and the IRdye800CW-conjugated goat anti-mouse IgG antibody or IRdye800CW-conjugated goat anti-rabbit IgG antibody was used as the secondary antibody. Subsequently, membranes were scanned and visualized by the LI-COR Odyssey CLx imaging system (LI-COR Biotechnology, USA).

The subcellular location of PMCNA_RS00975 protein was located using a previously described method (Bose and Taylor, 2005). Briefly, the C48-1 strain was cultured in the TSB medium containing 5% bovine serum at 37°C for 8 to 10 h and treated with polymyxin B sulfate (10 mg/mL) in PBS for 10 min on ice to release periplasmic contents. The residual cells in culture supernatants were removed through a 0.45 μm-pore size membrane. Spheroplasts were separated from the soluble periplasmic fraction by centrifugation at 10,000× g for 10 min. The spheroplast fraction was resuspended in 1× SDS-PAGE loading buffer, and the supernatant containing the periplasmic contents was diluted with 2× SDS-PAGE loading buffer. To obtain total membranes, the spheroplast fraction was resuspended in 10 mM Tris-Cl (pH 8.0) and sonicated to break open the cells. After low-speed centrifugation at 10,000× g at room temperature (RT) for 10 min to remove any unbroken cells, the lysate was centrifuged at 100,000×g at 4°C for 30 min to pellet the membrane fraction. The inner and outer membranes were differentially solubilized as follows. The membrane pellet was resuspended in 10 mM Tris-Cl–100 mM NaCl containing 2.5% Sarkosyl, held at RT for 30 min, and centrifuged at 200,000× g at RT for 1 h. The supernatant was retained as the inner membrane fraction, and the pellet containing the outer membrane was resuspended in 200 μL of 10 mM Tris-Cl (pH 8.0). Proteins were estimated using the Bicinchoninic Acid (BCA) Protein Assay Kit (Tiangen Biotechnology, China). Equal protein amounts (10 μg) from each fraction were subjected to western blotting using the anti-rPMCNA_RS00975 mouse serum, and IRdye800CW-conjugated goat anti-mouse IgG antibody (LI-COR Biotechnology, USA) was used as the secondary antibody. Subsequently, the membranes were scanned and visualized by the LI-COR Odyssey CLx imaging system (LI-COR Biotechnology, USA).

The peritoneal macrophages of TLR2^−/− and TLR4^−/− with a C57BL/6N mice background were isolated using the method described previously (Xu et al., 2023). The peritoneal macrophages were seeded into 24-well microplates at a density of 3 × 10⁵ cells/well and incubated at 37°C with 5% CO₂.

RAW264.7 cells or isolated peritoneal macrophages were seeded into 24-well plates at a density of 2 × 10⁵ cells/well and stimulated with the rPMCNA_RS00975 protein or LPS (Sigma-Aldrich, USA) for 6 h. To confirm whether the purified protein was contaminated by LPS, the rPMCNA_RS00975 protein was boiled to ensure no residual LPS remains in the sample. In experiments designed to block the MAPK and NF-κB signaling, RAW264.7 cells were pretreated for 30 min at 37°C with inhibitors of p38 (20 μM), ERK1/2 (20 μM), JNK (20 μM), or NF-κB (20 μM) before the rPMCNA_RS00975 protein exposure as described above. In experiments designed to block the TLR signaling, peritoneal macrophages were pretreated for 1 h at 37°C with an antibody against TLR2 (5 μg/mL) or TLR4 (5 μg/mL). Simultaneously, peritoneal macrophages isolated from TLR2^-/-, TLR4^−/−, and wild-type mice were incubated with the rPMCNA_RS00975 protein. In the experiments designed to verify the neutralizing activity of the anti-rPMCNA_RS00975 mouse serum, the rPMCNA_RS00975 protein was pre-incubated with anti-rPMCNA_RS00975 mouse serum at 37°C for 1 h, then the mixture was exposed to peritoneal macrophages isolated from TLR2^-/-, TLR4^−/−, and wild-type mice for 6 h. Next, the cell culture supernatants were collected, and the expression of IL-6 was measured using the ELISA Kit.

The extraction/quantification of RNA from RAW264.7 cells was performed according to the previous method (Xu et al., 2023). RAW264.7 macrophages were incubated with the rPMCNA_RS00975 protein for 3 h. In the experiments designed to identify the TLRs involved in the induction of IL-6 cytokine by the rPMCNA_RS00975 protein, the rPMCNA_RS00975 protein was pre-incubated with mouse anti-serum at 37°C for 1 h, then the mixture was exposed to RAW264.7 cells for 3 h. Next, RNA levels of the analyzed genes were normalized to the amount of GAPDH present in each sample. The sequences of the qRT-PCR primers are listed in the Table 2 .

Western blot analysis was performed using the previous method established in our laboratory (Xu et al., 2023). Briefly, RAW264.7 cells were seeded into 12-well plates at a density of 5 × 10⁵ cells/well. The cells were incubated at 37°C with 5% CO₂ and stimulated with the purified rPMCNA_RS00975 for various time intervals or various doses. Next, the stimulated cells were lysed and centrifuged to collect the supernatant. Equal protein concentrations were separated by SDS-PAGE and severally detected with the primary antibodies against p-ERK1/2, p-p65, and β-actin. Subsequently, the IRdye800CW-conjugated anti-rabbit IgG (LI-COR Biotechnology, USA) or anti-mouse IgG antibodies were used as the secondary antibodies to visualize by the LI-COR Odyssey CLx imaging system. The signal intensity values in western blotting were quantified with the ImageJ software (National Institutes of Health, USA).

Statistical analysis was carried out using GraphPad version Prism 6.0 (GraphPad Software, USA). All data from experiments repeated was represented at least thrice are presented as the mean ± standard (SD) deviation. Differences in the groups were analyzed by corresponding t-test based on the Gaussian distribution and variance analysis. The p-value < 0.05 was considered significant.

Amino acid sequences of these homologous genes exhibited more than 50% identity with that of the PMCNA_RS00975 from P. multocida. These homologous genes are mostly annotated as a putative encapsulin protein, also annotated as the DUF6260 family protein and encapsulating for peroxidase. The phylogenetic tree based on the deduced amino acid sequences of homologous genes revealed that the PMCNA_RS00975 protein belonged to the big branch neighbors, a putative encapsulating protein amino acid from M. pernigra and G. parasuis, and identity was 73.49% and 74.70%, respectively ( Figure 1A ). These results suggested that the PMCNA_RS00975 protein expressed from these homologous genes belonging to the clade and played a role in the same manner as P. multocida encapsulin and might be involved in iron metabolism. Additionally, the C48-ΔPMCNA_RS00975 mutant showed growth retardation (2.5~3.5 h) compared to the wild-type strain in iron-restricted medium, which further indicates that the PMCNA_RS00975 protein is involved in the iron metabolism of P. multocida ( Figure 1B ).

To evaluate the function of PMCNA_RS00975 in the virulence of P. multocida, the isogenic PMCNA_RS00975 mutant C48-ΔPMCNA_RS00975 was constructed based on positive selection techniques ( Supplementary Figure S1A ). Further, the PMCNA_RS00975 gene of the 20th generation mutant was identified using PCR and DNA sequencing (data not shown). The recombinant plasmid pAL99spec ^R-PMCNA_RS00975 was used to electrotransform the mutant to obtain the complemented strain, which was identified using PCR ( Supplementary Figure S1B ). Further, the C48-ΔPMCNA_RS00975 mutant exhibited no growth difference rate compared with the wild-type strain at cells/1 h to cells/8 h ( Supplementary Figure S1C ).

All mice infected with the wild-type strain C48-1 (50 CFUs/mice) died within 24 h, whereas the C48-ΔPMCNA_RS00975 mutant (5 × 10³ CFUs/mice to 5 × 10⁵ CFUs/mice) could not cause any death during the trial on the seventh day of post-infection. The survival ratios (100%) of mice infected with the C48-ΔPMCNA_RS00975 mutant were significantly increased (5 × 10⁵ CFUs/mice), and those of mice infected with the complemented strain C48-ΔPMCNA_RS00975C were partially regained to 60% (5 × 10⁴ CFUs/mice) and 50% (5 × 10⁵ CFUs/mice) ( Figure 2A ). In addition, all mice in the control group were injected with PBS and survived during the 7 consecutive observation days. Furthermore, a significant reduction was observed in IL-6 secretion by RAW264.7 cells ( Figure 2B ) exposed to the C48-ΔPMCNA_RS00975 mutant strain compared with those exposed to wild-type and complemented strains 3 to 12 h post-infection. These results suggested that lack of PMCNA_RS00975 markedly attenuated the virulence of P. multocida C48-1 strain.

DNA sequencing confirmed the successful construction of the pPRO-EXHTb-PMCNA_RS00975 recombinant expression vector. The rPMCNA_RS00975 protein expression induced with 0.5 mmol/L IPTG was largely present in the supernatant at 16°C ( Supplementary Figure S2A ). Further, the purified rPMCNA_RS00975 protein was confirmed by western blotting using an anti-His monoclonal antibody ( Supplementary Figure S2B ) or anti-P. multocida rabbit serum ( Supplementary Figure S2C ). These results indicated that rPMCNA_RS00975 protein is immunogenetic and expressed during P. multocida infection.

To determine the localization of the PMCNA_RS00975 protein in P. multocida C48-1, cell fractions were isolated and analyzed by western blotting using the anti-rPMCNA_RS00975 mouse serum. The rPMCNA_RS00975 protein could be identified by the anti-rPMCNA_RS00975 mouse serum ( Figure 3A ). Additionally, the PMCNA_RS00975 protein was detected largely in the whole-cell lysate, cell outer membrane fraction, and the weak bands in the cell culture supernatant ( Figure 3B ), indicating that the PMCNA_RS00975 protein is located at the outer membrane of P. multocida.

To investigate the function of the recombinant rPMCNA_RS00975 protein in inducing pro-inflammatory cytokines, the activation of the MAPK or NF-κB signaling pathways was analyzed by western blotting. Furthermore, the phosphorylation status of ERK1/2 and p65 was detected after the rPMCNA_RS00975 protein (10 μg/mL) was exposed to RAW264.7 cells. The results demonstrated the rPMCNA_RS00975 protein induced the phosphorylation status of ERK1/2 and p65 in RAW264.7 cells after 30 to 120 min ( Figure 4A ). The amount of p-p65 decreased over time, whereas the amount of p-p65 reached its peak at 30 min; the amount of p-ERK1/2 increased over time, whereas the amount of p-ERK1/2 reached their peak at 120 min ( Figure 4B ). These results indicated that the rPMCNA_RS00975 protein activated the NF-κB and ERK1/2 signaling pathways in macrophages. As expected, the IL-6 by LPS and Pam3CSK4 activities were significantly decreased in RAW264.7 cells. Furthermore, the expression of IL-6 was significantly inhibited by the inhibitors of p65 (BAY 11-7082) and ERK (U0126) but not by those of p38 (SB202190) and JNK (SP600125) ( Figure 4C ). These results proved that rPMCNA_RS00975 induced the secretion of IL-6 via the NF-κB and MAPK signaling pathways.

rPMCNA_RS00975 protein induced IL-6 to promote the identification of the signaling for this protein and trigger the secretion of pro-inflammatory cytokines. The mRNAs of TLR2 but not those of TLR4 were upregulated after stimulating the peritoneal macrophages with rPMCNA_RS00975 ( Figure 5A ). Further, RAW264.7 cells were pretreated with or without blocking antibodies against TLR2, TLR4, or an isotype control antibody for 1 h before stimulation with rPMCNA_RS00975, LPS, or Pam3CSK4. LPS and Pam3CSK4 were used as positive controls to demonstrate the blocking effects of anti-TLR4 and anti-TLR2 antibodies. The rPMCNA_RS00975 protein-induced production of IL-6 in RAW264.7 cells was inhibited by specific antibodies against TLR2, but not by specific antibodies against TLR4 ( Figure 5B ). In addition, compared with the release of IL-6 in peritoneal macrophages isolated from C57BL/6N mice, the release of IL-6 was inhibited in those isolated from TlR2^−/− mice, but not in those isolated from TLR4^−/− mice ( Figure 5C ). These results indicated that rPMCNA_RS00975 protein-induced IL-6 proinflammatory cytokines were mediated via the TLR2 signaling.