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. Unless otherwise stated, the strains used in this study were cultured in Luria broth (LB) liquid medium(Haibo Biotechnology Co., Ltd., HB0128, China) (37 °C, 180 r/min). The cells used in this study, Caco-2BBE cells and RAW264.7 cells, were both maintained in the Key Laboratory of Preventive Veterinary Medicine of Hebei Province. They were cultured in a CO₂ incubator with 5% CO₂ using high-sucrose Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific Co., Ltd., USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Co., Ltd., China) and 1% Penicillin-Streptomycin Solution (Beijing Solarbio Science & Technology Co., Ltd., China). Gentamicin (100 μg/mL) was added to the medium when necessary. The plasmids pKD3, pKD46, pBR322 and pCP20 for bacterial gene knockout were provided by Invitrogen.

Kunming mice (6–8 weeks old) were purchased from Beijing Speifu Biotechnology Co., Ltd. (Beijing, China) and kept according to standard protocols for animal experiments.

The trxB gene-deleted strain was constructed using the λ-Red homologous recombination technique. The plasmid pKD3 served as a template to amplify the cat targeting gene fragment using P1/P2 primers ( Table 1 ). This fragment was then electrotransformed into C50336-pKD46 receptor cells. The strain was identified by PCR using P3/P4 primers ( Table 1 ). Subsequently, the strain was incubated in a water bath at 42°C for 5 to 6 hours to eliminate pKD46. Finally, the deletion strain C50336ΔtrxB::cat containing the cat gene was obtained. The extracted genome of the C50336 strain was used as a template to amplify the trxB gene fragment by PCR using P5/P6 primers ( Table 1 ). This fragment was cloned into the pBR322 plasmid to construct the recombinant plasmid pBR322-trxB. After sequencing confirmed the correct plasmid, it was electrotransformed into the ΔtrxB strain. The complementary strain, named ΔtrxB+trxB, was then constructed and verified by PCR and sequencing using P3/P4 primers. The purified PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. (China) for sequencing. The trxB gene expression in ΔtrxB and ΔtrxB+trxB 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 trxB gene.

The strains were cultured overnight, and then transferred into LB and M9 medium with a 1:50 dilution on the next day, and cultured at 37°C with shaking at 180 rpm. M9 medium consists of 17 g Na₂HPO₄·7H₂O, 3 g KH₂PO₄, 0.5 g NaCl and 1.0 g NH₄Cl. The OD₆₀₀ was measured every hour for 10 hours by sampling the culture. Finally, the growth curve was plotted to analyze the strains’s growth characteristics.

Bacterial motility was analyzed on semi-solid plates following the method described in literature (Duan et al., 2012). Five microliters of overnight culture was punctured into the center of the semi-solid medium plate and incubated at 37 °C for 5 h. The bacterial motility was assessed by measuring the diameter of growth on the semi-solid medium plate.

Referring to the literature (Dong et al., 2011), bacterial biofilms were measured using crystal violet staining for both observation and quantitative analysis. Each strain was inoculated into 5 mL of liquid medium and incubated at 30 °C for 3 days. The bacterial cultures were removed, and the biofilms attached to the tubes were washed three times with PBS. Then, they were stained with 2% crystal violet solution for 15 minutes. The crystal violet bound to the biofilm was dissolved in anhydrous methanol to determine OD₅₇₀ nm values for quantifying biofilm formation. This experiment was repeated three times independently.

Each strain was inoculated onto medium containing Congo red (160 mg/L) and Caulmers Brilliant Blue (10 mg/L), then incubated at 28°C for 48 hours. The morphology and color of the colonies of each strain were observed. Additionally, each strain was inoculated onto medium containing 200 mg/L fluorescent whitening agent and incubated at 28°C for 48 hours. The fluorescence intensity of the colonies was compared under D366 nm ultraviolet light to that of the fluorescent whitening agent alone, in order to analyze cellulose production in the biofilm.

The minimum inhibitory concentration was determined following the Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute, 2009). The studies on the relevance of Trx system and antibiotic resistance are very limited, thus the choice of antibiotics in this study was random. In this study, 12 antimicrobials commonly used in clinical practice, including penicillin, acetylmethquine, cefotaxime, cefuroxime, ampicillin, cotrimoxazole, norfloxacin, ciprofloxacin, enrofloxacin, ofloxacin, lincomycin, and fosfomycin (Shanghai Yuanye Bio-technology Co., Ltd., China), were selected to perform the MIC test. Each overnight culture was adjusted to the same concentration (1×10⁶ CFU/mL). Each antimicrobial was serially two-fold diluted in test tubes containing sterile Mueller Hinton (MH) medium. Then, equal amounts of the adjusted bacterial solution were added to the serially diluted antimicrobials. The mixtures were incubated at 37 °C for 16–18 hours. E. coli ATCC25922 was used as the control strain. The experiment was repeated three times.

Bacterial stress tolerance was assessed by measuring the survival of each strain under stress conditions as previously described (Bringer et al., 2005). Briefly, bacteria in the logarithmic growth phase were exposed to different stresses, including acidic stress (pH 3.5), alkaline stress (pH 10.5), heat stress at 42 °C, hypertonic stress (2.5 mol/L NaCl), and hypotonic stress induced by deionized water for 1 hour, as well as oxidative stress (10 mmol/L H₂O₂) for 10 minutes. After treatment, colonies were counted using serial dilution, and the survival rate of each strain was calculated as the ratio of colony-forming units (CFU) after stress exposure to the CFU before treatment. Three independent replicate experiments were used.

The Caco-2BBE cells line, derived from colon adenocarcinoma, was used to assess bacterial adhesion and invasion abilities (Kortman et al., 2012). Caco-2 cells were seeded in 6-well plates, and a bacterial suspension in the logarithmic growth phase was added to the wells at a multiplicity of infection (MOI) of 100. The cells were centrifuged at 1000 rpm for 5 min and then incubated at 37 °C for 1 h. Cells were washed three times with PBS, lysed with 1% Triton X-100, and the lysates were collected for bacteria counting. Adhesion rate = (number of adhered bacteria/number of bacteria in the inoculum per well) × 100%.

For the invasion assay, the infection method was the same as that of the adhesion assay. After incubation for 1 h at 37 °C in a 5% CO₂ incubator, the medium was replaced with DMEM containing gentamicin (100 μg/mL), and incubation continued for another hour. Then, cells were lysed by adding 1 mL of 1% Triton X-100. Invasion rate = (number of intracellular bacteria/number of bacteria in the inoculum per well) × 100%.

The bacterial intracellular survival in macrophage was determined using cell line RAW264.7, a mouse macrophage cell line. Similar to the adhesion and invasion assays, RAW264.7 cells were infected with bacteria at a MOI of 100 and cells were incubated for 1h and then the medium was switched to DMEM containing gentamicin (100 μg/mL) to continue incubation. The cells were lysed at 3 and 23 hours post-infection (hpi), and the lysates were collected for bacterial enumeration. Bacterial survival and proliferation within macrophages were determined by calculating the ratio of bacterial counts at 23 hpi to those at 3 hpi.

Fifty-five Kunming mice were randomly divided into 11 groups, with 5 mice in each group. Five groups were i.p. challenged with C50336 at concentration ranging from 2.1×10⁸ to 2.1×10⁴ CFU in 0.2 mL PBS per mouse. Another five groups were i.p. challenged with ΔtrxB strains at concentration ranging from 1.3×10¹⁰ to 1.3×10⁶ CFU. The remaining group was challenged with equal volumes of sterile PBS as a control. The onsets of disease and deaths were recorded for 14 consecutive days, and the LD₅₀ was calculated according to the method of Reed and Mutch (Park et al., 2020).

To determine the bacterial load in the primary target organs of S. Enteritidis, livers, spleens, and lungs were collected from infected mice for quantitative bacterial analysis. Twenty mice were randomly divided into 2 groups with 10 mice in each, and the mice from the 2 groups were inoculated with S. Enteritidis wildtype strain and trxB-knockout mutant at the dose of 10⁶ CFU per mouse respectively. At 6 h and 48 h post infection, Liver, spleen, and lung tissues were collected under sterile conditions, weighed and homogenized with 1 ml of sterile PBS. The homogenized solution was diluted in a gradient and spread on LB plates for bacterial counting.

The C50336 and ΔtrxB strains were scraped from the LB plate into LB liquid medium and incubated with shaking until the OD_600nm reached 1-2. Then, the bacteria were washed three times with sterile PBS. The bacterial precipitate was collected and immediately transferred to a liquid nitrogen tank for quick-freezing, followed by storage at -80 °C in an ultra-low-temperature refrigerator. Finally, they were sent to Shanghai Meiji Biological Company. RNA sequencing and bioinformatic analysis was performed using a cloud platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd., China) based on the data generated by the Illumina platform. The differentially expressed genes (DEGs) were identified as those showing a fold change (ΔtrxB vs. C50336) greater than 2 or less than 0.5, with a corrected p-value less than 0.05.

RNA was extracted using the Bacterial RNA Extraction Kit (Beijing Aide Biotechnology Co., Ltd., China). gDNA was eliminated during extraction using DNase I from the same company, and reverse transcription was performed using the Reverse Transcription Kit (Bohang Biotechnology Co., Ltd., China). RT-qPCR was performed using SYBR^® PreMix Ex Taq II (TaKaRa Biotechnology (Dalian) Co., Ltd., China). Relative gene expression was determined according to the relative critical threshold (Ct) method using a Stratagene Mx3000P system (Agilent Technologies, CA). Data were normalized using the 2^-ΔΔCt method to show the relative fold change of 12 virulence genes compared to C50336. The primers used for qPCR are listed in Table 2 .

Thirty Kunming mice were randomly divided into two groups: an immunized group and a control group. The immunized group received orally of the ΔtrxB strain, while the control group received the same dose of saline on days 0 and 14. Mice were received orally with ΔtrxB at a dose of 1.3 × 10⁸ CFU and received a booster immunization with the same dose 14 days later.

Serum-specific IgG assay: Indirect ELISA was employed to assess the level of S. Enteritidis-specific antibodies in the serum of immunized mice. The C50336 strain was cultured to logarithmic growth stage, collected for lysis and centrifugation, and the supernatant proteins were encapsulated at 1 µg per well. Blood was collected from the tail tip of three mice randomly taken from each group on days 0, 7, 14, 21 and 28 of immunization for serum isolation. The serum was subjected to an ELISA assay with a serum dilution of 1:400 and HRP labelled secondary antibody at a concentration of 1:10,000, and determined for OD_450nm after color development.

Spleen index determination: Three mice were randomly selected and weighed on the 7th, 14th, 21st, and 28th days after immunization, and the spleens were picked up and weighed after euthanasia for calculation of the spleen index. The spleen index was calculated by dividing the spleen weight by the body weight of the mouse as a percentage.

Lymphocyte proliferation assay: Three immunized or non-immunized mice were euthanized 14 days after immunization. The spleens of the immunized mice were aseptically isolated and homogenized. The homogenate was then filtered through a 70 μm cell strainer (Beijing Labgic Technology Co., Ltd.) to obtain spleen cells. Red blood cells were lysed using a red blood cell lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). The spleen lymphocytes were suspended in RPMI 1,640 Medium(Thermo Fisher Scientific Co., Ltd.) supplemented with 10% fetal bovine serum (FBS), 50 μg/mL penicillin, and 50 μg/mL streptomycin. Cell viability was assessed using the trypan blue exclusion test, and cells were counted using a haemocytometer. After cell counting, lymphocytes were seeded into 96-well tissue culture plate at 5×10⁵ cells each well. The lymphocytes were treated by adding bacterial supernatant antigen (7.5 μg/mL), ConA (2 μg/mL) and PBS of equal volume separately and furthered incubated at 37°Cwith 5% CO₂ for 72 h. Lymphocyte proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation and Cytotoxicity Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd.) according to the instructions. Lymphocyte proliferation capacity was evaluated by the stimulation index (SI), calculated as: SI = (OD₄₅₀ of stimulation group − OD₄₅₀ of medium-only group)/(OD₄₅₀ of unstimulated group − OD₄₅₀ of medium-only group).

Relative protection measurement: Twenty KM mice were randomly divided into two groups, each containing 10 mice. Group A was the immunization group, and Group B was the control group. Mice in Group A were received orally with 1.3 × 10⁸ CFU/mouse of ΔtrxB, followed by a booster immunization with the same dose at 14 dpi. Mice in Group B received an equal volume of PBS following the same immunization schedule. At 28 days post-immunization, mice of groups A and B were challenged with C50336 strain by i.p. injection at the dose of 2.1×10⁸ CFU. The morbidity and mortality of the mice were recorded for 14 days after challenge. The relative protection rate (RPS) was calculated using the formula: RPS = (the mortality of the control group − the mortality of the immunization group)/the mortality of the control group × 100%.

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.

The significance of the differences between C50336 and ΔtrxB strains in terms of motility, biofilm formation ability, environmental tolerance, invasiveness, adhesion rate, intracellular survival rate, bacterial load, virulence factor detection, and immune protection ability were determined by GraphPad Prism 9.5.0 software was used, along with one-way analysis of variance (ANOVA) combined with 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 trxB gene deletion mutant of S. Enteritidis C50336 (named ΔtrxB) and corresponding complemented strain (named ΔtrxB+trxB) were constructed. As shown in Figure 1A , the trxB knockout mutant and the complemented strain were confirmed by PCR. To access the influence of trxB deletion on the growth of S. Enteritidis, we examined the growth of each strain in LB medium and nutrient-poor M9 medium. The data showed ( Figures 1B, C ) that all three strains displayed similar growth curves in LB or M9 medium, demonstrating that trxB deletion does not affect the growth of S. Enteritidis.

The results of the motility skill assessment showed ( Figure 2A ) that the ΔtrxB strain formed a much smaller swarming zone on semi-solid plates compared with C50336 and ΔtrxB+trxB strains, and the motility of the strain after trxB gene restoration was restored compared to the trxB knockout strain, which was supported by the measurement of the diameters of the formed bacteria zones ( Figure 2B ). This result demonstrated that knockout of trxB could diminish the motility of S. Enteritidis.

The biofilm formation ability of C50336, ΔtrxB, and ΔtrxB+trxB strains was examined, and the data showed ( Figure 3A ) that much less biofilm formation of ΔtrxB strain was observed on tube surface compared to the C50336 and ΔtrxB+trxB strains, while the biofilm formation ability of ΔtrxB+trxB strains was restored compared to the trxB gene deletion strains via Crystal violet-staining. Quantitative results revealed that ( Figure 3B ) the trxB strain showed significantly lower absorbance values at OD_570nm.

In order to clarify which components of the biofilm were affected by trxB knockout, all three strains were inoculated into Congo red medium and fluorescent-containing medium, respectively. As shown in Figure 3C , the ΔtrxB strain formed smooth, pale pink colonies on Congo red medium, while the C50336 and ΔtrxB+trxB strain showed much darker color and more ruffled colonies, indicating that the deletion of trxB reduced curli production. The fluorescence luminescence intensity of ΔtrxB was significantly different compared to C50336, and the fluorescence intensity of the ΔtrxB+trxB strain was restored compared with the ΔtrxB strain ( Figure 3D ). All these results strongly indicate that the TrxB gene can influence the formation of S. Enteritidis biofilm.

The antibiotic sensitivity of the strains was assessed by MIC determination ( Table 3 ). Compared with the wild-type strain, the trxB gene deletion strain showed an 8-fold increase in susceptibility to penicillin and mequindox, and a 4-fold increase to cefotaxime, cefuroxime, ampicillin, cotrimoxazole, norfloxacin, ciprofloxacin, enrofloxacin, oxfloxacin, lincomycin, and fosfomycin. These results strongly suggest that TrxB plays an important role in the drug resistance capacity of S. Enteritidis.

The environmental tolerance results showed ( Figures 4A–F ) that the ΔtrxB strain had a significantly lower survival rate in acidic, alkaline, oxidative, thermal, hypertonic, and hypotonic stress environments compared with the C50336 strain. Furthermore, the survival rate of ΔtrxB+trxB strain in stressful environments was restored compared to ΔtrxB strains. These results indicates that TrxB plays a critical role in regulating the stress response of S. Enteritidis.

Using Caco-2 and RAW264.7 cell models, we tested the adhesion, invasion and intracellular survival abilities of the ΔtrxB mutant. The results showed ( Figures 5A, B ) that the adhesion and invasion rates of ΔtrxB were significantly lower than those of C50336, and the survival of ΔtrxB in cell-type macrophage of RAW264.7 lineage was significantly lower than that of C50336. The adhesion rate, invasion rate, and intracellular survival rate of the strain after trxB gene restoration were restored compared to the deletion strain. ( Figure 5C ). This indicates that TrxB affects the adhesion, invasion, and intracellular survival abilities of S. Enteritidis.

The pathogenicity results of the ΔtrxB strain results show ( Table 4 ) that the LD₅₀ value of the C50336 strain is 3.98 × 10⁶ CFU, while that of the ΔtrxB strain is 5.13 × 10⁸ CFU, representing a 129-fold increase. The results of bacterial burden in vivo showed ( Figure 6 ) that at 6 h post-infection, the bacterial loads in the liver, spleen and lungs from ΔtrxB group were similar to those of C50336 group. However, at 48 h post-infection, the ΔtrxB group showed significantly lower bacterial load in the liver, spleen and lungs compared to the C50336 strain. The above results suggest that deleting the trxB gene reduces virulence of S. Enteritidis.

RNA-seq analysis identified 1561 differentially expressed genes (DEGs) in C50336 strain after trxB knockout, including 847 up-regulated and 714 down-regulated genes ( Figure 7A ).

Among these DEGs, we selected virulence-related genes and confirmed their expression levels by PCR. The results showed ( Figure 7B ) that the expression of rpoS, csgD, bcsA, ompR, pipB, hflK, rfbH, spvB, flgG, ssrA, sodC and ssaV was significantly reduced. These results suggest that deletion of the trxB gene downregulates expression of multiple virulence genes in S. Enteritidis.

The mice were immunized with ΔtrxB and challenged with C50336 on day 28 ( Figure 8A ). After infection, their clinical signs and survival were monitored. The immune group mice remained in usual behavior and all survived the challenge. In contrast, control mice showed dishevelled hair, reduced appetite, and lethargy by day 2 post-challenge, with deaths beginning on day 3 and reaching 100% mortality by day 7 ( Figure 8B ). According to the formula for calculating immune protection rate, the RPS for ΔtrxB was 100%. This suggests that the ΔtrxB provides effective immunoprotection against C50336 attacks.

Mouse blood serum was collected at different times after immunization for the detection of IgG antibody levels. The results showed ( Figure 8C ) that ΔtrxB could induce antibody production on day 7 after immunization, and the antibody level was highest on day 14, indicating that trxB gene deletion induced humoral immune responses in mice, showing good immunogenicity.

Mouse spleens were collected at various time points after immunization to measure the splenic index. Results ( Figure 8D ) showed a significantly higher splenic index in immunized mice compared to controls, except at day 28 when no difference was observed. On day 21 post-immunization, splenocytes were collected for a lymphocyte proliferation assay. The data showed ( Figure 8E ) that the stimulation index (SI) index of the immunized group of mice was much higher than that of the unimmunized group.