S. enteritidis C50336 was isolated from the feces of a patient with diarrhea and purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). It was kept in the Key Laboratory of Preventive Veterinary Medicine, Hebei Province. All strains were cultured in Luria broth (LB) medium Haibo Biotechnology Co., Ltd., HB0128, China) at 37°C, and added ampicillin (100 μg/mL) or chloramphenicol (34 μg/mL) as required. The Caco-2 BBE cells and RAW264.7 cells were purchased from BeNa Culture Collection (Shanghai, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific Co., Ltd., TFS12491023, China) containing 10% fetal bovine serum (Thermo Fisher Scientific Co., Ltd., China) at 37°C in an incubator with 5% CO₂. The plasmids pKD3, pKD46, pBR322 and pCP20 for bacterial gene knockout were provided by Invitrogen.

Female Kunming mice aged 6–8 weeks were purchased from Beijing Speifu Biotechnology Co., Ltd. (Beijing, China).

The λ-Red homologous recombination technique was used to knock out the frdA gene (Zhang et al., 2020). Briefly, the chloramphenicol resistance cassette (cat) was amplified using long primers P1 and P2 (Table 1), which contain homologous fragments of the frdA gene, with plasmid pKD3 as a template. The purified PCR product was electrotransferred into the competent cell of the C50336 strain (harboring plasmid pKD46) to obtain a primary recombinant strain ΔfrdA::cat. The inserted cat gene was eliminated by further electrotransferring the pCP20 plasmid. The obtained strain was then placed in a water bath and treated at 42°C for 5–6 h to remove the temperature-sensitive plasmid pCP20. The frdA deletion was confirmed by PCR with primers P3 and P4 (Table 1). Subsequently, the purified PCR product was cloned into pMD-19T vector (Takara Biomedical Technology (Beijing) Co., Ltd.), and the recombinant vector was sent to Sangon Biotech (Shanghai) Co., Ltd. (China) for sequencing.

To generate the complemented strain, the whole ORF of frdA was amplified using primers P5 and P6 (Table 1) with the genome of the C50336 strain as a template. The PCR product was cloned into the pBR322 plasmid by using BamHI (NEB #R3136) and SalI (NEB #R3138) nucleic acid endonuclease (Takara Biomedical Technology (Beijing) Co., Ltd.,). The constructed pBR322-frdA plasmid was then transfered into the ΔfrdA mutant, with positive clones verified by PCR using primers P3 and P4, and named ΔfrdA+frdA. The frdA gene expression in ΔfrdA and ΔfrdA+frdA was confirmed by qPCR. Briefly, RNA of each strain was extracted using a bacterial RNA extraction kit (Beijing Aidlab Biotechnologies Co., Ltd., RN63, China), reverse transcribed into cDNA, and subjected to qPCR verification using primers P7 and P8 (Table 1) to assess the expression of the frdA gene.

Bacteria were cultured to the logarithmic growth phase and diluted to 10⁷ CFU/mL with saline. The bacterial suspension was separately incubated inacidic saline (pH 4.0) and alkaline saline (pH 10.0) at a ratio of 1:100, treated for 1 h, and then doubly diluted with phosphate-buffered saline (PBS) (Thermo Fisher Scientific Co., Ltd., TFS20012050, China) for bacteria counting. For heat stress, the bacterial suspension was incubated in PBS at a ratio of 1:100 and then treated under 42°C for 1 h, and for oxidative stress, the bacterial suspension was incubated in PBS containing 10 mmol/L H₂O₂ and treated for 10 min. The bacterial suspension after treatment under heat and oxidative stress were doubly diluted with PBS for bacteria counting. Bacteria were counted before and after the stress treatment, and the survival rate under stress conditions was calculated by dividing the bacteria count post-treatment by the bacteria count pre-treatment (Zhang et al., 2024).

The biofilm formation ability of each strain was determined using crystalline violet (CV) staining (Simm et al., 2014). Briefly, each bacterial strain was inoculated in LB and incubated statically at 30°C for 3 days. The bacterial culture was removed, and the formed biofilm was washed 3 times with PBS and fixed in methanol (Tianjin Fuyu Fine Chemical Industry Co., Ltd., 67-56-1, China) for 10 min, followed by staining using 2% CV (Shanghai Macklin Biochemical Co., Ltd., MFCD00011750, China) for 15 min for observing the thickness and color of the biofilm ring on the glass tubes. To quantify the biofilm formation, each strain was inoculated in a 96-well plate (Guangzhou Jet Bio-Filtration Co., Ltd., TCP011896, China) in 100 μL of LB and incubated statically at 30°C for 3 days. CV staining of the biofilm was carried out as above. The biofilm-bound CV was dissolved in 100 μL of ethanol (Tianjin Fuyu Fine Chemical Industry Co., Ltd., 64-17-5, China) and subjected to determination of the absorbance under 570 nm (OD₅₇₀). The assay was repeated 3 times.

To investigate the expression of the main components of biofilm, curli fimbriae and cellulose, we inoculated each strain on Congo red (Tianjin Damao Chemical Reagent Partnership Enterprise (Limited Partnership), AR3749, China) and Coomassie brilliant blue plate (Beijing Solarbio Science & Technology Co., Ltd., C8420, China) or LB agar containing Calcofluor White Stain (200 mg/L) without salt, respectively. As previously reported (Zhao et al., 2024), five μL of bacterial culture of each strain and inoculated it onto LB agar containing 160 mg/L Congo red and 10 mg/L Coomassie brilliant blue without salt and incubated at 30°C for 2 days, and the colony morphology and color were observed to assess the production of curli fimbriae. Then, five μL of bacterial culture of each strain was inoculated onto LB agar containing Calcofluor White Stain (200 mg/L) without salt and incubated at 30°C for 2 days, and the colony morphology was observed under ultraviolet (UV) light (366 nm) to determine the production of cellulose.

The motility assay of each strain was assayed by determining the bacterial range formed on semi-solid media. Five μL of bacterial culture of each strain was inoculated onto semi-solid LB plates containing 0.3% agar and incubated at 37°C for 6–8 h. The diameter of the bacterial zone was measured, and the assay was repeated 3 times.

The adhesion and invasion abilities of each strain were assayed via a Caco-2 cell model. The Caco-2 cells were seeded in a 6-well plate (Guangzhou Jet Bio-Filtration Co., Ltd., TCP001006, China), and the bacterial suspensions of logarithmic phase were added into the cell wells with a multiplicity of infection (MOI) of 100 (Zhou et al., 2025). The cell plates were centrifuged at 1,000 rpm for 5 min and then incubated for 1 h. The cells were washed with PBS three times to remove the free-standing bacteria. Then the cells were lysed with 1% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd., T8200, China) and the lysates were serially diluted for bacterial counting. The bacterial adhesion rate was calculated by dividing the number of adherent bacteria by that of the initially added bacteria (Zhang et al., 2020). For the invasion assay, preliminary operations were the same as the adhesion test, and at 1 h after infection, the cells were continually incubated in DMEM containing gentamicin (100 μg/mL) for 1 h to kill the extracellular bacteria. Then, the cells were lysed for bacteria counting. The invasion rate was calculated by dividing the number of invasive bacteria by that of the initially added bacteria (Zhang et al., 2020).

Intracellular survival assays were assayed in RAW264.7 cells. The former operation was the same as that of the invasion experiment. The RAW264.7 cells were seeded in a 6-well plate and infected with bacteria of logarithmic phase at an MOI of 100. The cells were cultured in DMEM containing gentamicin (100 μg/mL) after adhesion for 1 h and then lysed for bacteria counting at 3 and 23 h post-infection (hpi). Intracellular survival rate = (Number of bacteria inside cells at 23 hpi/Number of bacteria inside cells at 3 hpi) × 100% (Zhang et al., 2020).

Three biological replicates of C50336 strain and the frdA mutant were cultured to an OD₆₀₀ of 0.6 in LB medium. Total RNA of each strain was extracted using an RNA extraction kit (Beijing Aidlab Biotechnologies Co., Ltd., China) and the residual genomic DNA is removed by DNase treatment. The concentration, purity and integrity of RNA samples were quantified by a UV spectrophotometer and a Bioanalyzer instrument. RNA-seq assay was performed using the Majorbio Cloud platform (www.majorbio.com), and each sequencing library was generated using the TruSeqTM RNA sample preparation Kit (Illumina, Inc., CA). Differentially expressed genes (DEGs) were identified as the genes with a fold-change (treatment/control) of >2 or <0.5 and a corrected p-value <0.05.

qPCR was preformed to assess the expression of bacterial virulence gene. RNA was extracted using a bacterial RNA extraction kit (Beijing Aidlab Biotechnologies Co., Ltd., China) and treated with DNase I to remove genomic DNA. Then, RNA was taken as template to produce cDNA using a reverse transcription kit (Bohang Biotechnology Co., Ltd., China). The SYBR Green dye based qPCR was performed using this cDNA as template as described previously (Nguyen et al., 2021). The primers used were illustrated in Table 2.

Kunming (KM) mice were used for determine the LD₅₀ of each strain. One hundred and ten female KM mice were randomly divided into 11 groups with 10 mice per group. The five groups were intraperitoneally (i.p.) injected with C50336 at doses ranging from 2 × 10⁹ to 2 × 10⁵ CFU/mouse. Another five groups were i.p. injected with ΔfrdA at doses ranging from 1 × 10⁹ to 1 × 10⁵ CFU/mouse. And the left one group was intraperitoneally injected with an equal volume of PBS as a control. The mice were observed and recorded for abnormal performance and death for 14 days. The LD₅₀ value was calculated using the Modified Karber method (Park et al., 2020).

Forty-five female KM mice were divided into three groups, with 15 mice in each group. Each group was intraperitoneally injected with a bacterial suspension of C50336, ΔfrdA, or PBS at a dose of 1 × 10⁵ CFU/mouse. The mice were euthanized under anesthesia at different time points (3–14 days), and the spleen, liver, and lungs were aseptically picked and homogenized for bacteria counting on Salmonella-Shigella (SS) agar (Beijing Auboxing Biotechnology Co., Ltd., AUB02-003, China).

Thirty KM mice (6–8 weeks old) were randomly divided into three groups, named immunized, unimmunized, and control groups, respectively. For the immunized group, mice were i.p. injected with a dose of 1 × 10⁶ CFU/mouse of ΔfrdA (once on day 0 and once on day 14), and the other two groups were intraperitoneally injected with an equal amount of sterile PBS. At 28 days post-immunization, the mice of immunized and unimmunized were intraperitoneally injected with a lethal dose of strain C50336, at 2 × 10⁷ CFU/mice, whereas the control group received PBS. The mortalities were recorded every day for 14 days, and the relative percentage of survival (RPS) was calculated as [1 – (mortality in ΔfrdA immunization group/mortality in challenge group)] × 100% (Park et al., 2020).

Forty KM mice (6–8 weeks old) were randomly divided into two groups, named immunized and control groups, respectively. Immunization was carried out according to the method of 2.11; at the 0th, 7th, 14th, 21st, and 28th d of immunization, blood was collected from the tail tip of three mice in each group at random, and the blood was centrifuged at 3,000 rpm for 5 min. The serum antibody IgG levels of the two groups of mice were determined by indirect ELISA method (Zhao et al., 2025).

Thirty KM mouse (6–8 weeks old) were randomly divided into two groups, named immunized and control groups, respectively. Immunization was carried out according to the method of 2.11. At 3 d, 7 d, 14 d, and 21 d of immunization, three mice were randomly selected in each group, and the spleens of the mice were removed and weighed to calculate the spleen index. Spleen index = [spleen weight (g)/mouse body weight (g)] × 100 % (Arunima et al., 2020).

Mice (n = 3) from the control and immunized groups at 14 days post immunization were euthanized. The spleens were aseptically collected, and the lymphocytes were separated by homogenization and filtration via a 70 μm cell strainer. After cell counting, lymphocytes were seeded into 96-well tissue culture plate at 5 × 10⁵ cells/well. The cell wells were added with ConA (Concanavalin A) (final concentration of 5 μg/mL) (Shanghai Biyuntian Biological Co., Ltd., ST2062, China), C50336 bacterial antigen (final concentration of 5 μg/mL), and RPM1640 as control, respectively. The plate was incubated for 72 h at 37°C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL, Shanghai Biyuntian Biological Co., Ltd., ST316, China) was added, incubated for 4 h (Dong et al., 2011), followed by the addition of Formazan solvent (Shanghai Biyuntian Biological Co., Ltd., ST316, China), and incubated for another 4 h for measuring OD₅₇₀. The stimulation index (SI) was calculated according to the formula: [SI = (OD value of stimulated group – OD value of culture medium group)/(OD value of unstimulated group – OD value of culture medium group)] (Dong et al., 2011).

All animal experiments were conducted in full compliance with international ethical standards and the Experimental Animal Regulation Ordinances (HPDST 2020-17) as 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.

Statistical analyses were performed using GraphPad Prism version 9.5.0, with the one-way Analysis of Variance (ANOVA) followed by t-tests. Data were expressed as mean ± standard error. Significant differences were denoted with an asterisk (^*), where ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 are considered to represent statistically significant differences in mean values.

Using λ-Red recombination technology, a frdA gene deletion mutant of S. enteritidis C50336 and complemented strain were constructed. The frdA knockout mutant and the complemented were confirmed by PCR (Figure 1A).

To access the influence of frdA deletion on the growth of S. enteritidis, we examined the growth of each strain in LB medium. The data showed (Figure 1B) that all three stains displayed similar growth curves in LB, demonstrating that frdA deletion does not affect the growth of S. enteritidis in LB.

To investigate whether the frdA gene affects the resistance of S. enteritidis to various environmental stresses, we compared the survival of C50336, ΔfrdA, and ΔfrdA + frdA under conditions of acid solution, alkaline solution, heat stress and oxidative stress. The results showed a significantly decreased survival of the ΔfrdA strain under acidic (Figure 2A), alkaline (Figure 2B), heat stress (Figure 2C), and oxidative stress (Figure 2D) conditions as compared to the C50336 and ΔfrdA + frdA strains. These results suggest that the frdA gene plays important roles in the resistance of S. enteritidis to acid, alkali, heat stress, oxidative stress, and nitrification stress.

The biofilm formation ability of C50336, ΔfrdA, and ΔfrdA + frdA was examined in the present study, and the data showed that the ΔfrdA displayed apparently impaired biofilm formation compared with C50336 and ΔfrdA +frdA (Figure 3A). Quantitative results revealed that the biofilms formed by the ΔfrdA strains was significantly different at OD_570nm after staining and dissolution (Figure 3B). The ΔfrdA formed a colony with fewer wrinkles and a lighter color on the Congo red medium as compared with C50336 and ΔfrdA + frdA (Figure 3C), indicating that the deletion of frdA reduced curli production. The colony of ΔfrdA showed much weaker fluorescence under UV light (Figure 3D), suggesting the decreased production of cellulose. Taken together, all these results demonstrated that frdA is associated with the biofilm formation of S. enteritidis.

The motility of C50336, ΔfrdA, and ΔfrdA + frdA was assessed on semi-solid medium. The ΔfrdA showed much smaller bacterial zone on semi-solid plate as compared with C50336 and ΔfrdA +frdA in present study (Figure 4).

The data showed that the ΔfrdA displayed decreased adhesion and invasion to Caco-2 cells (Figure 5A) and impaired survival in macrophage RAW264.7 cells (Figure 5B). This indicates that the frdA gene is able to affect the adhesion, invasion, and intracellular survival ability of S. enteritidis.

The virulence of C50336 and ΔfrdA was assessed by the mouse model via intraperitoneal injection. The mice that received injections of both strains began to show symptoms such as trembling, arched backs, crusted eyes, disheveled fur, and even death. The mice of the control group showed no abnormality. The deaths of mice were recorded for LD₅₀ calculation. The LD₅₀ of ΔfrdA and C50336 were 7.94 × 10⁷ CFU and 1.26 × 10⁶CFU, respectively, with a 64-fold difference (Table 3).

To further reveal the mechanism underlying FrdA's effect on S. enteritidis, a transcriptomic approach was employed to screen and analyze the differentially expressed genes after frdA deletion. A total of 2,163 DEGs were determined, with 1,067 up-regulated genes and 1,096 down-regualted genes (Figure 6A).

Among these DEGs, we focused on the those associated with bacterial virulence, and confirmed the expression of these genes using qPCR method. The expression of csgD, prot6E, flgG, invH, sodC, ssrA, mrrl, pipB, ssaV, bscA, rfbH, spvB, sipA, xthA, or, f245 was significantly reduced (Figure 6B). This datas suggest that the frdA gene may regulate the virulence of S. enteritidis by modulating expression of multiple genes.

The bacterial burden of tissues and organs from C50336 and ΔfrdA infected mice was assayed here. Although mice were inoculated with equal doses of C50336 and frdA, the bacterial loads of ΔfrdA were significantly lower in the liver, spleen, and lungs than those of the wild-type strain at different time points (Figure 6C). To sum up above, all these results demonstrated that frdA deletion would lead to attenuation of S. enteritidis.

To determine the immunoprotective potential of ΔfrdA, mice were challenged with a lethal dose of C50336 at 28 days post immunization with ΔfrdA or PBS as control. The results showed (Figures 7A, B) that the mice that received ΔfrdA immunization displayed a survival rate of 100% with mild and transient depression, while the mice from the unimmunized group developed typical symptoms of S. enteritidis infection and final death with a 100% mortality.

Mice serum was collected at different days post immunization, and the results of antibody detection showed the ΔfrdA elicted IgG production at 7 days after immunization, and antibody levels were maintained at a high level after day 14 (Figure 7C), suggesting ΔfrdA could induce effective humoral immunity.

The spleen indices of the mice were measured at different time post immunization, and the results showed that the spleen indices of the immunized group of mice were much higher than those of the control group (Figure 7D). For cellular immunity, mice splenocytes were harvested for lymphocyte proliferation assays 14 days post immunization.The SI index of the immunized group of mice was much higher than that of the unimmunized group (Figure 7E).

Taken together, the ΔfrdA could elicit an effective immune response and provide promising protection against S. enteritidis.