Specific pathogen-free C57BL/6J mice (female, 6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Mice were raised in an environment with a light/dark cycle of 12 h, a constant temperature of 25°C and a humidity of 50%. A standard chow diet was provided for mice ad libitum throughout the experiments. All animal studies were conducted according to protocols approved by the Committee on the Ethics of Animal Experiments of Yangzhou University [Approval ID: SYXK (Su) 2017-0044].

Bacterial strains and plasmids used in the present study are shown in Supplementary Table 1 . The S. Enteritidis strain CMCC50041 (GenBank Accession No. CP013097.1) was used as the wild-type (WT) strain and for the construction of SPI-1 and SPI-2 mutants. SPI-1 and SPI-2 mutants of S. Enteritidis were previously constructed (Xiong et al., 2019). The ΔSPI-1/SPI-2 double-deficient strain in this study was constructed following the λ-Red recombinase gene replacement method (Datsenko and Wanner, 2000; Jiang et al., 2017). Primer sequences to generate and confirm the mutant strains are listed in Supplementary Table 2 . All bacterial strains were cultured on Luria-Bertani (LB) agar plates or in LB broth with necessary antibiotics at appropriate concentrations (e.g., 100 μg/mL ampicillin and 20 μg/mL chloramphenicol).

The murine RAW264.7 macrophage cell line was provided by the Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Waltham, MA, USA) at 37°C in a 5% CO₂ incubator.

The λ-Red system, an effective and widely used approach to inactivate the chromosomal genes in Escherichia coli and S. Typhimurium (Tischer et al., 2006), was used to produce SPI-1 and SPI-2 deficient mutants. ΔSPI-1/SPI-2 double mutant strain was constructed using ΔSPI-2 forward and reverse primers based on the ΔSPI-1 deficient strain. All gene deletions were verified by PCR analysis and sequencing. All primers used for amplification of certain regions in SPI-1 and SPI-2 of S. Enteritidis are shown in Supplementary Table 2 . Growth curves of S. Enteritidis C50041 WT, ΔSPI-1, ΔSPI-2, and ΔSPI-1/SPI-2 deletion mutants were determined to evaluate the influence of SPI deficiency on Salmonella growth. Biochemical tests were performed using an API 20E identification kit (bioMerieux SA, Lyon, France) in accordance with the manufacturer’s protocol.

S. Enteritidis colonies were cultured overnight at 37°C with agitation (180 rpm) in LB liquid broth. Log-phase bacteria were obtained by diluting the stationary-phase bacteria to fresh LB at a ratio of 1: 40 and subculturing to the mid-log-phase (3 h). Bacteria were pelleted, washed three times with cold phosphate-buffered saline (PBS), and resuspended with DMEM. The bacterial concentration was estimated based on the optical density at 600 nm, and the CFUs were calculated by serial dilution on agar plates. Before infection, RAW264.7 macrophages were seeded in 24‐well plates at a density of 2 × 10⁵ cells/well overnight in DMEM containing 10% FBS without antibiotics to obtain 80% confluence. Next, Salmonella was added at a multiplicity of infection of 100:1. Plates were centrifuged at 1,000 × g for 8 min to synchronize infection and allowed to invade for 1 h. After washing cells twice with DPBS, DMEM supplemented with 100 μg/mL gentamicin was used to kill the extracellular bacteria for 1 h.

For invasion assays, RAW264.7 cells continued to be infected for 0.5 h, washed twice with DPBS, and lysed with 1% Triton X-100. Subsequently CFU numbers of intracellular bacteria were calculated to determine the bacterial invasion by plating on LB agar. For the proliferation assay, cells continued to be infected for 0.5 h, washed twice with DPBS, and continued to be maintained in culture medium containing 10 μg/mL gentamicin for 2, 4, 6, or 8 h to quantify bacterial proliferation.

Murine RAW264.7 macrophages were seeded in 24-well plates at a density of 2 × 10⁵ cells per well and infected with Salmonella WT and mutants as described above. Cells cultured in DMEM were set as the negative control group, which was not infected with bacteria. After Salmonella infection for 2 h, culture supernatants were harvested for the quantification of lactate dehydrogenase (LDH) with an LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China). The absorbance value of the DMEM control group was subtracted from all treatment groups according to the manufacturer’s instructions. In addition, cells were collected and centrifuged at 1,000 × g for 10 min and resuspended in 400 μL of binding buffer. Early and late apoptosis among different groups were conducted using an Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China). Percentages of apoptotic cells were analyzed using a FACS Aria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FlowJo_v10 software (FowJo, Ashland, OR, USA).

Female C57BL/6J mice were randomly divided into 5 groups (n = 13 per group, n = 5 for tissue collection and n = 8 for survival monitoring). For oral infections, water and food were withdrawn for 4 h before C57BL/6J mice were infected intragastrically with 10⁶ CFU of S. Enteritidis C50041 WT or the indicated ΔSPI-1, ΔSPI-2, or ΔSPI-1/SPI-2-deficient strains in a 200-μL volume. Bacteria were prepared and CFU numbers were determined as described above. Drinking water and food were offered at 2 h post infection. To evaluate the cytokine levels in livers and spleens of mice, tissues were homogenized using a Precellys 24 homogenizer (Rockville, MD, USA). Cytokine expression levels in organs were measured using qRT-PCR. To evaluate the activation of MAPK and JAK-STAT signaling pathways in the tissues, homogenized specimens were centrifuged for 10 min at 12,000 × g at 4°C. p-ERK and p-STAT2 levels in collected supernatants were measured by Western blotting. For analysis of the bacterial burden in tissues, infected mice were sacrificed 4 days post infection. Next, mouse cecums, livers and spleens were aseptically collected, homogenized in sterile ice-cold PBS, serially ten‐fold diluted, and plated on LB plates for CFU determination. Before homogenization, the cecums were aseptically removed and rinsed three times with sterile PBS. Meanwhile, tissue samples from the spleen and liver were removed for pathological analysis. The mortality of mice in different infection groups was recorded daily and monitored for 2 weeks.

Cells were collected and lysed in Cell Lysis Buffer for Western (Beyotime) followed by centrifugation at 12,000 × g at 4°C for 5 min. The supernatant was harvested and the protein concentration was determined by BCA Protein Assay Kit (Beyotime). Protein samples were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the polyvinylidene fluoride membrane was used for protein transfer. The membrane was blocked with 1% bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 h at room temperature, followed by primary antibodies and horseradish peroxidase (HRP)-conjugated IgG secondary antibodies (1:5000 dilution). Antibodies used in this study were listed below: rabbit anti-p-STAT2 (Y690) (Abcam, Cambridge, UK), rabbit monoclonal anti-p-ERK1&2 (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-β-actin (C4) (Santa Cruz Biotechnology, Dallas, TX, USA), HRP-conjugated goat anti-rabbit IgG H&L (Abcam), and goat anti-mouse IgG H&L-HRP (Calbiochem, San Diego, CA, USA). The immunoblotting was conducted with the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured with the Amersham™ Imager 600 System (GE Healthcare, Chicago, IL, USA). Band densities were analyzed using ImageJ software (https://imagej.nih.gov/ij/) and normalized to the corresponding internal marker β-actin.

Total mRNA preparations of RAW264.7 macrophage and mouse tissues were extracted with a Total RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). For preparation of tissue mRNA, mouse livers and spleens were harvested and stored using liquid nitrogen. The frozen tissues were homogenized in RLT buffer before extraction. RNA concentrations were measured using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized using a PrimeScript RT Reagent Kit (Takara, Dalian, China). qRT-PCR was performed with a QuantStudio 6 Real-Time PCR System (Applied Biosystems, Foster, CA, USA). The total volume of PCR reaction was 20 μL, and it was conducted in triplicate using the Fast Start Universal SYBR Green Master (Roche, Basel, Switzerland) according to the manufacturer’s protocols. The real-time PCR efficiencies (E) for the reference gene and target genes were determined using the diluted cDNA templates, and calculated based on the equation: E = 10^[–1/slope] (Rasmussen, 2001). Normalized levels of gene expression were calculated following the Pfaffl method based on E and the CT deviation of the treatment sample versus the control, and expressed in comparison to the reference gene (Pfaffl, 2001). β-actin gene was used as the reference control. Primer sequences for the qRT-PCR are shown in Supplementary Table 3 .

Mouse livers and spleens were collected, formalin-fixed, and embedded in paraffin. The sections were deparaffinized, antigen-retrieved with sodium citrate buffer, permeabilized in Triton X-100 (0.2%) for 0.5 h, and then blocked with PBS containing 5% BSA for 1 h. Mouse p-ERK1&2 and p-STAT2 were stained using rabbit monoclonal anti-p-ERK1&2 and rabbit anti-p-STAT2 (Y690) antibodies with the blocking buffer overnight at 4°C. Cy3-conjugated goat anti-rabbit secondary antibody (Abcam) was diluted in blocking buffer and incubated for 1 h at room temperature according to the manufacturer’s instructions. The nuclear DNA was stained with 6-Diamidino-2-phenylindole (DAPI). The sections were mounted with the coverslips and Prolong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were captured using the fluorescence microscope (Leica, Wetzlar, Germany).

Mouse tissues were fixed with 10% neutral formalin for 24 h, then embedded with paraffin, cut into sections (5 μm), and stained using hematoxylin and eosin (H&E) by standard techniques. Histology sections were observed using an Eclipse Ci-L microscope (Nikon, Tokyo, Japan). Pathological assessment of livers and spleens were performed by two pathologists in a blinded manner using the criteria described in Supplementary Table 4 . The sum of the individual scores for each tissue sample was determined as the combined pathological score.

Data were comparatively analyzed with the unpaired Student’s t test and were shown as mean ± SEM. All statistical analysis was performed with GraphPad Prism (version 8.0.2, GraphPad Software, San Diego, CA, USA). The significant differences were determined when the P-value was ≤ 0.05 (*), 0.01 (**), and 0.001 (***).

Salmonella ΔSPI-1, ΔSPI-2, and ΔSPI-1/SPI-2-deficient strains were constructed to identify roles for each SPI in bacterial virulence of macrophages and a mouse model. Using the λ-Red system, SPI-1 (39 kb) and SPI-2 (40 kb) were deleted to generate ΔSPI-1 and ΔSPI-2, respectively, while both gene cassettes were deleted to generate the ΔSPI-1/SPI-2-deficient strain ( Figure 1A ). Each strain was confirmed by PCR analysis and sequencing, which showed that mutants were constructed correctly ( Figure 1B ). Our assessment of growth rates confirmed that bacterial growth was unaffected by deletion of SPI-1 or SPI-2 ( Figure 1C ). In addition, mutant strains were biochemically characterized using an API 20E identification kit, which revealed no differences between mutant strains relative to the WT strain ( Figure 1D ).

To study the effects of SPI-1 and SPI-2 on bacterial invasion and proliferation in vitro, the RAW264.7 macrophage cells were infected with C50041 WT, ΔSPI-1, ΔSPI-2, or ΔSPI-1/SPI-2 strains. Mutants showed significantly decreased abilities to invade macrophages relative to WT ( Figure 2A ). In addition, intracellular proliferation of WT S. Enteritidis was substantially increased 2 h following infection compared with the three mutants ( Figure 2B ). Compared with WT S. Enteritidis, LDH release caused by mutant strains in macrophages was significantly decreased ( Figure 2C ). In addition, both SPIs independently resulted in the apoptosis of murine macrophages following infection ( Figure 2D ).

The effects of SPI-1 and SPI-2 on activation of inflammatory pathways were evaluated in murine macrophages following Salmonella infection. The real-time PCR efficiencies for the reference gene and target genes were 2.04 for β-actin, 2.07 for IL-1β, 2.02 for IL-6, 2.15 for IFN-β, 2.08 for ISG15, 2.16 for IFIT1, 1.96 for IFIT2, 1.98 for IFIT3b and 2.05 for CXCL2 ( Supplementary Figure 1 ). The results show that SPI-1 and SPI-2 of S. Enteritidis induced multiple inflammatory responses, including MAPK (ERK-mediated) and JAK-STAT (STAT2-mediated) pathways ( Figures 3A, B ). Moreover, S. Enteritidis SPI-1 and SPI-2 contributed to robust production of inflammatory cytokines such as interleukin (IL)-1β and IL-6, and interferon-stimulated genes (ISGs) such as IFN-β, ISG15, IFIT1, and IFIT3b ( Figure 3C ). Inflammatory responses 3 h after infection were stronger compared with those at 1 h. Collectively, these results demonstrate that both SPI-1 and SPI-2 of S. Enteritidis are necessary to induce inflammation in macrophages.

To evaluate the contribution of SPI-1 and SPI-2 to the virulence of S. Enteritidis in vivo, immune responses following Salmonella infection were determined in a mouse model. The results show that deficiency of the two SPIs, especially SPI-2, significantly abolished the production of inflammatory cytokines and various ISGs in liver and spleen ( Figures 4A, B ). In addition, infection with WT Salmonella resulted in robust phosphorylation of ERK and STAT2. However, the SPI-1 mutant displayed reduced p-ERK and p-STAT2 levels. Notably, ΔSPI-2 and ΔSPI-1/SPI-2-deficient strains completely abolished the activation of the two inflammatory pathways ( Figures 4C, D ).

To further characterize the evoked immune response, we performed immunofluorescence assays to examine activation of inflammation-related signaling pathways in the liver and spleen of mice following Salmonella infection. Expression of p-ERK1&2 and p-STAT2 was evaluated as markers of the activation of MAPK and STAT signaling pathways. The results show that WT S. Enteritidis induced the most pronounced activation of p-ERK1&2 and p-STAT2 in liver and spleen tissues of infected mice, while the SPI-1 mutant induced moderate activation of inflammatory signaling. However, neither ΔSPI-2- or ΔSPI-1/SPI-2-infected mice displayed obvious inflammation activation, showing similar results to the PBS control group ( Figure 5 ).

Pro-inflammatory cytokines activate inflammatory processes that result in macrophage recruitment and polymorphonuclear neutrophil infiltration, which lead to cellular injury in the host. Characteristic signs of this process include strong multifocal neutrophilic infiltration, multifocal necrosis, severe diffusion, and cell swelling. To investigate the impact of SPI on pathological manifestations of liver and spleen injury, we performed histopathologic analyses of S. Enteritidis‐infected mice. As shown in Figure 6 , liver and spleen tissues from mice infected with WT S. Enteritidis C50041 exhibited severe inflammation and damage on day 4 post infection. The average pathological scores for liver and spleen of WT-infected mice were 5 and 3.7 with severe neutrophil infiltration and necrosis. S. Enteritidis ΔSPI-1-infected mice demonstrated moderate histopathological changes in liver and spleen tissues with the average pathological scores of 2.3 and 1.7 respectively as a result of infection. However, tissues from mice infected with ΔSPI-2 and ΔSPI-1/SPI-2 mutants displayed slight damage and inflammation similar to that observed in control mice. The average pathological scores for liver and spleen of ΔSPI-2-infected mice were 0.7 and 0.3 with mild neutrophil infiltration. Slight inflammation and even no lesions were observed in ΔSPI-1/SPI-2-infected mice with the average pathological score of 0.3 for the spleen.

To determine the effects of SPI-1 and SPI-2 on the virulence of S. Enteritidis, bacterial localization and mouse survival were monitored following Salmonella infection. The results show that numbers of bacteria were significantly decreased in liver, spleen and cecum tissues 4 days after challenge with the SPI-1-deficient strain, suggesting that the SPI-1 mutant maintained a moderate level of virulence. However, bacteria could not be detected in ΔSPI-2- or ΔSPI-1/SPI-2-infected mice, suggesting that strains lacking SPI-2 or SPI-1/SPI-2 were avirulent ( Figures 7A–C ). Furthermore, no deaths were observed when mice were challenged with ΔSPI-2 or ΔSPI-1/SPI-2-deficient strains. In contrast, 20% of animals infected with the same dose of S. Enteritidis ΔSPI-1 died on the sixth day post‐infection and 60% of mice succumbed to infection by the ninth day ( Figure 7D ). These results showed that SPI-1 and SPI-2 enhanced S. Enteritidis virulence by causing cytokine storm and promoting bacterial colonization ( Figure 8 ).