The bacterial strains and plasmids used in this study are shown in Table 1. Salmonella enterica serovar Enteritidis C50336 was the wild-type strain and used for constructing the ΔpipC mutant. The ΔpipC strain in this study was constructed following the λ-Red recombinase gene replacement method (Datsenko and Wanner, 2000). The primer sequences used for generating and confirming mutant strains are listed in Table 2. All bacterial strains were cultured on Luria-Bertani (LB) agar plates or in LB broth with necessary antibiotics at appropriate concentrations (for example, 100 μg/mL ampicillin and 34 μg/mL chloramphenicol) (Xiong et al., 2023).

Human epithelial Caco-2 BBE cells and mouse macrophage RAW264.7 cells used in this study were provided by BeNa Culture Collection (Shanghai, China). Both cell types were cultured in DMEM (Thermo Fisher Scientific Co., Ltd.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Co., Ltd.). Antibiotics were added as necessary, such as 50 μg/mL streptomycin and 50 U/mL penicillin, or 50 μg/mL gentamicin, in an incubator with 5% CO₂.

Kunming (KM) mice were obtained from Beijing Speifu Biotechnology Co., Ltd. Throughout the experiment, the mice were maintained in a sterile environment under standard housing conditions with an ambient temperature consistently kept at 22.0 ± 0.5°C and relative humidity maintained at 60 ± 10%. A 12-h light/dark cycle was established for the housing conditions. All animal experiments were conducted in full compliance with international ethical standards and the Experimental Animal Regulation Ordinances (HPDST 2020-17) stipulated by the Hebei Provincial Department of Science and Technology. The study protocol was reviewed and approved by the Animal Care and Use Committee of Hebei Normal University of Science and Technology.

The pipC gene deletion strain in this study was constructed using the λ homologous recombination method (Supplementary Figure 1). Briefly, the auxiliary plasmid pKD46 was introduced into C50336 through electroporation, which encodes the Gam, Exo, and Beta proteins required for λ homologous recombination. Using pKD3 as a template, the knockout fragments were amplified by P1 and P2. The knockout fragment was then introduced into C50336 containing pKD46 via electroporation, resulting in a primary recombinant strain with chloramphenicol resistance. This strain was selected using LB agar plates containing chloramphenicol and verified using primers P3 and P4. The positive strain obtained was named ΔpipC:cat. The pCP20 plasmid, which encodes the Flp recombinase, was able to excise the cat gene from the knockout fragment. The pCP20 plasmid was introduced into ΔpipC:cat by electroporation, resulting in a secondary recombinant strain. This strain was verified using primers P3 and P4. The positive strain obtained was named ΔpipC.

To construct the complement strain, the nucleic acids of C50336 were used as a template, and the complement fragment was amplified using P5 and P6. The complement fragment and pBR322 vector plasmid were digested with restriction endonucleases BamH I and Sal I, respectively. The two digested fragments were ligated using T4 DNA ligase. The recombinant vector was introduced into ΔpipC by electroporation and verified with P7 and P8. The positive strain was named ΔpipC + pipC.

To determine the genetic stability of the ΔpipC, it was serially passaged 40 times in LB medium, every 12 h. Nucleic acids from liquid cultures are extracted every other generation and PCR verification using P3 and P4.

The overnight cultures of C50336, ΔpipC, and ΔpipC + pipC were subcultured at a 1:100 ratio into 5 mL of LB liquid medium and incubated at 37°C in a shaking incubator. Growth was determined by monitoring the absorbance of bacterial cultures at 600 nm (OD₆₀₀ values). Growth curves were plotted based on the growth of each strain at different time points.

Overnight cultures of C50336, ΔpipC, and ΔpipC + pipC were washed three times with PBS and resuspended in the original volume. The bacterial counts before stress were determined using the traditional plate count method. The bacterial suspensions were exposed to acid stress (pH 3.5), alkaline stress (pH 10.0), and heat stress (42°C) for 1 h, as well as to oxidative stress (10 mmol/L H₂O₂) for 30 min. After stress exposure, the bacterial counts were determined. The survival rate of each strain under different conditions was calculated as follows: survival rate = (post-stress bacterial count)/(initial bacterial count).

The human epithelial cancer cell lines Caco-2 were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. The mouse macrophage RAW264.7 was cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin solution. When the cells reached 80% confluence, the monolayers were washed three times with PBS. The cells were then seeded in 12-well tissue culture plates at a density of 1 × 10⁶ cells/well. The plates were incubated at 37°C in an atmosphere containing 5% CO₂.

To investigate the impact of pipC gene deletion on the adhesion and invasion ability of SE, the overnight cultures of C50336, ΔpipC and ΔpipC + pipC were washed three times with PBS and subsequently resuspended. The number of bacteria per mL of bacterial suspension (number of infected bacteria) was measured. Following the washing of confluent cell monolayers with DMEM, C50336, ΔpipC and ΔpipC + pipC were inoculated, respectively, onto the Caco-2 cells at a multiplicity of infection (MOI) of 100:1 and were incubated for 1 h at 37°C under 5% CO₂ (Xiong et al., 2023).

For bacterial adhesion, the cells were washed, and then incubated with PBS containing Triton X-100 (0.5%) at 37°C for 10 min. The cell lysates were serially diluted and inoculated onto LB agar for counting. The number of bacteria per mL of cell lysate (number of adherent bacteria) was measured. The adhesion rate was calculated using the formula: Adhesion rate = (number of adherent bacteria/number of infected bacteria) × 100%.

For bacterial invasion, 1 h after bacterial colonization, the cells were incubated for an additional 1 h in DMEM with gentamicin (100 μg/mL), washed and incubated with PBS containing Triton X-100 (0.5%) at 37°C for 10 min. The cell lysates were serially diluted and inoculated onto LB agar for counting. The number of bacteria per mL of cell lysate (number of invading bacteria) was measured. Invasion rate = (number of invading bacteria/number of infected bacteria) × 100%.

To evaluate the survival rate of ΔpipC in phagocytic cells, after infecting the cells with bacteria for 2 h as described above, DMEM containing 100 μg/mL gentamicin was added and incubated for 1 h. One group of cells was then washed and lysed with 0.5% Triton X-100, and the intracellular bacteria were counted (intracellular bacteria at 3 h). Another group of cells was washed and incubated with DMEM containing 10 μg/mL gentamicin at 37°C for 20 h. These cells were also lysed with 0.5% Triton X-100, and the intracellular bacteria were counted (intracellular bacteria at 23 h). Intracellular survival rate = (intracellular bacteria at 23 h/intracellular bacteria at 3 h) × 100%.

A total of 75 six-week-old KM mice were randomly divided into 15 groups (n = 5). These groups were categorized into three sets: five groups for the ΔpipC, five groups for the C50336, and the remaining five groups designated as the ΔpipC + pipC group. Mice in the ΔpipC groups were intraperitoneally (i.p.) inoculated with ΔpipC containing of 1.68 × 10⁹, 1.68 × 10⁸, 1.68 × 10⁷, 1.68 × 10⁶ or 1.68 × 10⁵ CFU/mouse, respectively. Similarly, the C50336 groups were i.p. inoculated with C50336 containing of 2 × 10⁷, 2 × 10⁶, 2 × 10⁵, 2 × 10⁴ or 2 × 10³ CFU/mouse, respectivly. The ΔpipC + pipC groups were i.p. inoculated with ΔpipC + pipC containing of 2 × 10⁷, 2 × 10⁶, 2 × 10⁵, 2 × 10⁴ or 2 × 10³ CFU/mouse, respectively. Five additional mice were i.p. injected with the same volume of PBS as a negative control.

The number of dead mice was recorded for 14 days and LD₅₀ was calculated, which was calculated using the formula of log₁₀ [50% endpoint] = A + (B × C), where A = log₁₀ [infectious dose showing a mortality next below 50%], B = difference of logarithms = [50% − (mortality at infectious dose next below 50%)]/[(mortality next above 50%) − (mortality next below 50%)], and C = log₁₀ [difference between serial infectious doses used in challenge studies] (Park et al., 2022; Zhang et al., 2019).

In order to further investigate the effect of pipC gene deletion on the virulence of SE, qPCR was used to measure the expression levels of virulence genes in C50336, ΔpipC, and ΔpipC + pipC. In short, the bacteria were cultured to the logarithmic phase and total RNA was extracted using an RNA extraction kit (Aidlab, Beijing, China). Complementary DNA (cDNA) was synthesized using a reverse transcription kit (TOYOBO, Osaka, Japan). Primers were designed by referring to previous literature (Upadhyaya et al., 2013). The primer sequences used for qPCR are listed in Table 3. Using cDNA as a template, the relative gene expression was quantified using the comparative critical threshold (Ct) method through qPCR. Data were normalized to the endogenous control (16S rRNA), and the level of candidate gene expression between treated and control samples were determined. The qPCR thermal cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

Thirty mice were randomly divided into two groups (n = 15). One group was intraperitoneally injected with 1 × 10⁴ CFU/mouse of ΔpipC, while the other group received an equal dose of C50336. Additionally, three mice were injected with 200 μL of PBS as a negative control. At predetermined time points (3 days, 7 days, and 14 days), five mice from each group were randomly selected and euthanized. Their spleens, livers, and cecums were collected, homogenized in PBS, and subsequently serially diluted before being evenly spread on XLT4 agar. The number of CFUs for each sample was determined 12 h later. For this animal study, all mice were anesthetized using a 20% urethane (ethyl carbamate) solution, and every effort was made to ensure humane treatment of the animals.

Twenty-four female KM mice, aged 8 weeks, were randomly divided into two groups (n = 12): an immunization group and a control group. On day 0, the immunization group received an oral administration of 1 × 10⁶ CFU of ΔpipC, whereas the control group was orally administered 200 μL of PBS. A booster immunization with an identical dose was given to the immunization group on day 14 dpi, while the control group received an equivalent volume of PBS.

At 14 dpi and 28 dpi, 6 mice from each group were randomly euthanized, and spleens were collected for splenic lymphocyte stimulation test and detection of cytokine expression levels. Additionally, serum and feces were collected from 3 mice for IgG and SIgA detection. The vaccination regimen of this study is shown in Figure 1.

C50336 was cultivated overnight at 37°C, 180 rpm with shaking until it reached the logarithmic phase. The bacteria were centrifuged at a speed of 13,000 × g at 4°C, washed three times with PBS, and resuspended in PBS to a concentration of 1 × 10¹⁰ CFU/mL. Use SCIENTZ ultrasonic homogenizer (SCIENTZ-IID, Ningbo, China) to sonicate the cells and centrifuge the 13,000 × g sample at 4°C to granulate the fragments. The sample was sterilized through a 0.22 μm PES (polyethersulfone) filter (Merck, Darmstadt, Germany) (Ji et al., 2022).

Lymphocytes were isolated from each group of three spleen samples at 14 and 28 dpi. After Trypan blue dye exclusion testing, suspensions of splenic mononuclear cells (1 × 10⁷ cells/well) were cultured in Roswell Park Memorial Institute 1,640 medium (RPMI-1640) supplemented with 10% FBS and 100 μg/mL penicillin-streptomycin within 96-well tissue culture plates. These cultures were then incubated with 10 μg/mL soluble antigen or without any stimulant as a negative control, under conditions of 37°C and 5% CO₂ for a duration of 72 h. Lymphocyte proliferation was measured using an MTT kit (Beyotime, Shanghai, China). The cell proliferation was expressed as the stimulation index (SI), which was calculated using the equation: SI = (OD₅₇₀ of the antigen-stimulated cells)/(OD₅₇₀ of the unstimulated cells) (Kang et al., 2021).

Quantitative real-time PCR (qPCR) was employed to assess the mRNA expression levels of splenic cytokines, including IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ, at 14 and 28 days post-immunization (dpi). Total RNA was extracted from spleen tissues using Triquick Reagent (Solarbio, Beijing, China), and first-strand cDNA was synthesized with a cDNA synthesis kit (TOYOBO, Osaka, Japan). Samples were stored at −80°C until further use. qPCR was performed using the Ultra SYBR Green Mixture (CWBio, Jiangsu, China) on a Lepgen-96 Real-Time PCR System (LEpu, Lepgen-96, China). The primer sequences used for qPCR are listed in Table 4. Cytokine expression levels were normalized to the internal reference genes gapdh and β-actin, and relative expression was calculated using the 2^−ΔΔCt method (Kang et al., 2021). The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min.

To examine the antibody responses, serum and feces samples were collected from 3 mice per group at 14 and 28 dpi after inoculation with the initial and booster doses of ΔpipC. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the serum IgG and fecal IgA (SIgA) responses against the soluble antigen derived from SE. For serum extraction, whole blood samples were centrifuged for 10 min at 10,000 × g at 4°C. The resultant supernatant, containing the serum, was carefully separated from the pellet and preserved at −20°C until use. To obtain the fecal supernatant, the feces samples were weighed, followed by the addition of a 25% weight/volume (w/v) solution of fecal slurry comprised of 0.01% sodium azide, 1% protease inhibitor in PBS. The samples were homogenized by vortexing for 15 min. After centrifugation, collect the supernatant and store it at −80° C for IgA detection.

The ELISA plates were coated with Salmonella soluble antigen (1 μg/well) and incubated overnight at 4°C. Wells were blocked with 5% skim milk at 37°C for 2 h (200 μL/well), followed by washing three times with PBS containing 0.05% Tween-20 (PBST) (280 μL/well). Dilute the serum sample at 1:200 and add it to the well, then incubate at 4°C for 1 h (100 μL/well), and wash three times with PBST. The secondary Ab goat anti-mouse IgG-HRP diluted 1:10,000 was added (100 μL/well) (Applygen, Beijing, China). The Ab was allowed to interact with the samples for 35 min at 37°C, followed by washing three times with PBST. Add 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (100 μL/well) and incubate at 37°C for 10 min. The reaction was terminated by adding 50 μL of 2 M H₂SO₄ to each well, and the absorbance was read at 450 nm in plate reader (Tecan, Shanghai, China).

For IgA, the ELISA plates were coated with 1 μg Salmonella soluble antigen per well and incubated overnight. Wells were blocked with 5% skim milk at 37°C for 2 h, followed by washing three times with PBST. The undiluted fecal supernatant was added to the well (100 μL/well), followed by incubated at 4°C for 1 h. The secondary Ab goat anti-mouse IgA-HRP diluted 1:10,000 was added, and the rest of the protocol was performed as described above (Emerson et al., 2022).

To evaluate the immune protection of ΔpipC. Twenty 6-week-old female KM mice were randomly divided into 2 groups (n = 10), namely the vaccinated group and the unvaccinated group. The vaccinated group was orally immunized with 1 × 10⁶ CFU/mouse of ΔpipC, while the unvaccinated group was orally immunized with 200 μL of PBS. In addition, another 10 mice without vaccination and challenge were used as the control group. At 14 dpi, the vaccinated mice received the same dose of ΔpipC for enhanced immunity. At 28 dpi, the vaccinated group and unvaccinated group were challenged with 2 × 10⁷ CFU/mouse of C50336 by intraperitoneal injections. Deaths and clinical symptoms were recorded daily for 14 d post the challenge (dpc), and calculate the relative survival rate according to the formula: Relative survival rate = (mortality rate of the unvaccinated group − mortality rate of the vaccinated group)/mortality rate of the unvaccinated group × 100%.

Statistical analysis was conducted using GraphPad Prism 9 (GraphPad Software, CA, United States) and IBM SPSS (IBM Corporation, Armonk, NY, United States) software. Data are expressed as the mean ± standard error of the mean (SEM). All statistical analysis were two-way ANOVA and post-test. Differences between two samples were evaluated using Student’s t-test. Significant differences are indicated with an asterisk (*), where *: p < 0.05, **: 0.001 < p < 0.01, and ***: p < 0.001 are considered to represent statistically significant differences in mean values. ns means not significant (Kirthika et al., 2020).

We constructed a pipC gene deletion strain of SE by using the λ-Red homologous recombination method and constructed a complementation strain by using the pBR322 plasmid. The primers P3 and P4, P7 and P8 were used to identify ΔpipC and Δ pipC + pipC, respectively. As shown in Figures 2A,B, the pipC gene deletion strain and complementation strain have been successfully constructed.

The ΔpipC mutant was serially passaged 40 times in LB medium, and the presence of the pipC deletion was then assessed by PCR (Figure 2C). The pipC deletion was still detectable in the ΔpipC mutant strain, indicating that this strain has good genetic stability.

As shown in Figure 3A, the growth characteristics of C50336, ΔpipC and ΔpipC + pipC in LB medium did not differ greatly. This indicated that pipC deletion did not influence the growth characteristics of SE.

To investigate whether the deletion of the pipC gene affects the resistance of SE to environmental stress, the survival rates of C50336, ΔpipC, and ΔpipC + pipC were compared under acid stress (pH 3.5), alkaline stress (pH 10.0), oxidative stress (H₂O₂), and heat stress (42°C). The results shown in Figure 3B reveal that, compared to C50336, the survival rate of ΔpipC was significantly reduced under acidic, alkaline, and oxidative stress conditions, whereas no statistically significant difference was observed under heat stress conditions. Since pipC deletion did not affect the growth characteristics of SE, the reduced survival rate of the mutant strain can be attributed to the role of the pipC gene in enhancing resistance to acid, alkaline, and oxidative stress.

The adhesion rate and invasion rate in human epithelial Caco-2, and the intracellular survival rate in mouse macrophage RAW264.7 of ΔpipC and C50336 was determined. As shown in Figure 4A, the adhesion rate of bacteria was not different between C50336 and ΔpipC in Caco-2. However, the invasion rate in Caco-2 and the intracellular survival rate in RAW264.7 of ΔpipC, compared to of C50336, showed significantly reduced (Figures 4B,C). These results indicate that SE with pipC deletion shows attenuated virulence in vitro.

The virulence of the ΔpipC and C50336 strains was evaluated in 6-week-old KM mouse after i.p. challenge. As shown in Table 5, the LD₅₀ of ΔpipC was 2.99 × 10⁷ CFU, which was 47-fold higher than that of the wild-type C50336 (2.99 × 10⁷/6.32 × 10⁵ ≈ 47). The LD₅₀ of ΔpipC+pipC is 1 × 10⁶ CFU/mouse. The result indicated that the virulence of the ΔpipC was attenuated compared to the C50336.

To investigate the potential mechanism underlying the attenuation of SE virulence due to the deletion of the pipC gene, qPCR was employed to assess the expression levels of virulence factors. The results are presented in Figure 5. Compared to C50336, the expression levels of SPI-1 associated genes (invH, sipA, sipB, sipC, sopB, and sopE2) and SPI-2 associated genes (spvB, ssrA, orf245, ssaS, ssaT, ssaU, sseB, and sseD) in ΔpipC were significantly decreased. These findings suggest that pipC may influence the virulence of SE by modulating the expression levels of multiple virulence genes.

The results of bacteria colonization in the liver, spleen, and cecum are shown in Figure 6. All the liver, spleen and cecum samples from the blank control group were negative for Salmonella recovery. Bacteria could be isolated from the liver, spleen, and cecum at 3, 7, and 14 dpc. Compared to C50336, the counts of ΔpipC in the liver, spleen, and cecum were significantly lower at 3, 7, and 14 dpc, indicating that the colonization ability of ΔpipC in these organs was notably inferior to that of C50336. This suggests that the pipC gene can influence the colonization ability and virulence of SE.

To elucidate the specific immune responses to SE antigens following ΔpipC immunization, a splenic lymphocyte proliferation assay was performed using soluble antigens at 14 and 28 dpi. As shown in Figure 7A, the stimulation indices against the SE antigens for immunized group was 8.60 ± 0.34 at 14 dpi. Likewise, the stimulation indices against SE antigens for immunized group was 14.07 ± 0.51 at 28 dpi. The SI value of splenic lymphocytes in the immunized group was significantly higher than that in the control group, and the lymphocyte proliferation level further increased after enhanced immunization. These findings indicate that ΔpipC can induce strong specific immune responses.

To evaluate the humoral and mucosal immune responses in mice immunized with ΔpipC, the serum IgG and mucosal IgA responses against SE soluble antigens were measured by ELISA. At 14 dpi and 28 dpi, mice immunized with ΔpipC exhibited significantly enhanced serum IgG levels (Figure 7B) and secretory IgA (SIgA) levels (Figure 7C) compared to the control group, with further increases observed after booster immunization. These findings indicate that ΔpipC effectively induces robust specific humoral and mucosal immune responses, which are augmented with booster immunization.

We detected the expression of cytokines in spleen cells of immunized mice at 14 and 28 dpi. The results, shown in Figures 7D,E, revealed that at 14 dpi, the expression levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ in the ΔpipC group were significantly higher than those in the control group. At 28 dpi, the expression levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ in the ΔpipC group were notably higher than those in the control group. Additionally, the expression levels of IL-6 and TNF-α after the booster immunization were significantly higher than those after the primary immunization. The increased expression levels of cytokines highlight that ΔpipC can effectively induce a strong immune response in mice.

The survival percentages in the mice vaccinated orally with the ΔpipC followed by the challenge with the virulent SE are shown in Figure 8. The vaccinated group showed no mouse deaths and a 100% survival rate; among the 10 mice in the unvaccinated group, 8 mice died after the challenge, showing a survival rate of 20%. The clinical symptoms including anorexia, chills, diarrhea, emaciation, and depression in the vaccinated group were slight and temporary after challenged compared to the unvaccinated group. Therefore, immunization with 10⁶ CFU of ΔpipC provided full protection against SE challenge (see Figure 9).