S. Typhimurium strains (ATCC14028) purchased from the Guangdong Engineering and Technology Research and Development Center of Microbial Food Safety (Guangzhou, China) was used as a wild type (WT). HtpG mutant strains (ΔhtpG) were in-frame deletion mutant strains constructed using the λ-RED homologous recombination method (Datsenko and Wanner, 2000). The complement strains denoted as CΔhtpG were constructed by amplifying the HtpG gene fragment followed by connected to pBR-322 plasmid and electrotransformed into the ΔhtpG strains. All the strains, WT, ΔhtpG, and CΔhtpG were cultured in lysogeny broth (LB, Huankai, China) at 37°C, 200 r/min on a shaker.

The single colonies of the WT strains (n = 4) and ΔhtpG strains (n = 4) were respectively incubated in fresh LB medium at 37°C, 180 r/min in an air shaker overnight. The next day, the bacterial suspension was added to the fresh LB at a ratio of 1:100 and cultured on an air shaker until OD600 = 1. The supernatant was discarded by centrifugation, and the bacteria were washed twice with sterile PBS, followed by storage of the bacterial pellet at −80°C. The samples were sent to Novogene Bioinformatics Technology Co., Ltd (Beijing, China) to perform RNA sequencing and analysis.

The Agilent 2100 bioanalyzer was utilized to detect the total amount and integrity of RNA. The mRNA is randomly interrupted in the Fragmentation buffer, fragmented mRNA as a template, used random primers to synthesize the first strand of cDNA in M-MuLV reverse transcriptase, and synthesized the second strand under the action of DNA polymerase I. AMPure XP beads were applied to screen cDNA of about 70–420 bp, and PCR amplification was performed. AMPure XP beads were used again to purify the PCR products to obtain the library. After the library passed the quality inspection, Illumina sequencing was performed. The image data of the sequenced fragments measured by the high-throughput sequencer were converted into sequence data (reads) by CASAVA base recognition. Filter the original data to obtain clean data, including removing reads with adapters, removing reads with unidentifiable base information, and removing low-quality reads. The reference genome and annotation file were obtained in NCBI. Bowtie2 software was used to locate and analyze clean data. Gene expression analysis used HTSeq v0.6.1 to calculate the reading of each gene, calculate the FPKM of each gene based on the gene length, and map it to the reading of the gene by technology. DESeq2 R package (1.20.0) was used for differential expression analysis. The method of Benjamini and Hochberg was used to adjust the p-value to control the false discovery rate. Genes with adjusted p-values less than 0.05 found by DESeq2 were designated as expression differences. The GO enrichment analysis of differentially expressed genes was realized by GOseq R package software, and the KOBAS software analyzed the statistical enrichment of differentially expressed genes in the KEGG pathway.

The swimming motility of S. Typhimurium WT or ΔhtpG strains was determined on plates of LB medium solidified with Agar (Huankai, China) at a concentration of 0.5%. One microliter of fresh bacterial suspension was pipetted with OD600 = 1 into a plate, followed by incubation for 12 h at 37°C to record the diameter of growth circle.

Biofilm formation by S. Typhimurium WT or ΔhtpG strains in the wells of microtiter plates after 24 h of incubation was evaluated by crystal violet staining. Cultures were grown overnight at 37°C with shaking and then diluted 1:100 in fresh LB medium. Aliquots of 200 μl of these cultures were dispensed into the wells of 96-well microtiter plates and cultured at 30°C for 24 h. After discarding the medium, the adhering bacteria were washed three times with sterile PBS and stained with 0.5% crystal violet solution for 30 min, followed by washing with sterile PBS and drying. Thereafter, the stained biofilm was dissolved with glacial acetic acid and the absorbance was measured at OD570.

IPEC-J2 and RAW 264.7 cell lines were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, USA) supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum (FBS, Gibco, USA) at 37°C 5% CO₂.

IPEC-J2 was used for adhesion and invasion assay; IPEC-J2 and RAW 264.7 were used for intracellular proliferation assay. The cells were colonized with an equal number of the indicated WT, ΔhtpG, and CΔhtpG strains for 1 h (multiplicity of infection, MOI ≈ 100). For adhesion assay, the cells were washed three times with sterile PBS and then incubated for 10 min with PBS containing 0.5% Triton X-100 (v/v). For invasion assay, the cells were incubated for another 30 min in DMEM with gentamicin (100 μg/ml), washed, and incubated with PBS containing 0.5% Triton X-100 for 10 min; for intracellular proliferation and cell infection assay, the cells were incubated for an additional 30 min in DMEM with gentamicin (100 μg/ml), washed and cultured in DMEM containing 50 μg/ml gentamicin, followed by collection of cell lysates at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h. Serial 10-fold dilutions of cell lysates were plated on LB agar and incubated for 16 h to count the UFC of bacteria.

Eighteen 6-week-old male BALB/c mice with similar body weight were randomly divided into three groups and used for the S. Typhimurium challenge test. The mice were fasted for 12 h before the challenge, and mice were subsequently gavaged with 100 μl of PBS containing 1×10⁸ CFU S. Typhimurium WT strains or ΔhtpG strains (treatment group) or the same amount of PBS (NC group), re-feeding after half an hour of gavage. Four days after infection with S. Typhimurium, the mice were sacrificed by cervical dislocation, and the serum was collected. Liver, spleen, and thymus were completely stripped and weighed. Mouse liver, spleen, and ileum tissues were collected and stored at −80°C for subsequent experiments. The animal study was reviewed and approved by the Animal Care and Use Committee of the South China Agricultural University (SCAU2019B142).

Total RNA was extracted from S. Typhimurium, mice, and cells with Eastep Super RNA extraction kit (Promega, Shanghai, China). Approximately 2 μg of total RNA was reverse-transcribed using a M-MLV reverse transcriptase kit (Promega, Shanghai, China). The primers were synthesized by Tsingke (Guangzhou, China). Quantitative PCR was performed in 20μL reaction system with specific primers and AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The amplification was operated on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, California, USA). The relative expression of pig and mouse mRNA was normalized to GAPDH, and the mRNA of S. Typhimurium was normalized to gyrA. The expression of genes was analyzed by the method of 2^−ΔΔCt. The sequence information of all the primers involved in this study is shown in Table 1 .

The cell culture medium was collected after the strains infected the cells for 12 h. The ileal and spleen tissues were homogenized with PBS, the supernatant was then collected after centrifugation at 4000 r/min for 5 min. Cytokines were quantified with Enzyme-Linked Immunosorbent (ELISA) kits (Neobioscience, Shenzhen, China) according to the respective instructions. Briefly, the samples were added to the test well and incubated at 37°C for 90 min to bind the antibody. Then, the biotinylated antibody and avidin HRP were sequentially added to the test wells. Tetramethylbenzidine substrate solution is used for color development and stop solution to stop color development. Read the value at 450 nm with a microplate reader (Bio-Rad, California, USA).

HtpG recombinant proteins containing endogenous His, S, and Trx tags derived from the pET-32a cloning vector (Novagen, Germany) were purified using a Ni-affinity column (Sangon, China). Briefly, double digestion of pET-32a with restriction enzymes NcoI and XhoI amplified the HtpG gene that repeats 20 nt with the restriction site of the vector; Trelief™ SoSoo Cloning Kit (Tsingke, Beijing) was applied to ligate the linearized plasmid with the amplified fragment. Then, the constructed plasmid was transformed into E. coli BL21 strain (Tsingke, Beijing). BL21 strain was grown in 1 L of LB broth within ampicillin (50 mg/ml) at 37°C for 3 h, and IPTG (1 mM) was used to induce at 20°C for 16 h. After enzymatic digestion consisting of 0.2 mg/ml lysozyme, 20 μg/ml DNase, 1 mM MgCl₂, and 1 mM phenylmethylsulfonyl fluoride (PMSF), the supernatants were purified by Ni-column. The Toxin-Eraser Endotoxin Removal Kit (Genscript, USA) was used to remove lipopolysaccharide (LPS), and the Toxin-Sensor Chromogenic LAL Endotoxin Assay Kit (Genscript, USA) was used to detect the residual LPS. Ultrafiltration tube (Millipore, USA) was used for concentration and desalting. The protein concentrations were measured by Bradford assay (Thermo Fisher, USA). The purity was tested by SDS-PAGE (10%, acrylamide).

Cell proliferation was determined using Cell Counting kit-8 (CCK-8, Dojindo, Japan). Add 100 μl of suspension containing about 1×10⁴ IPEC-J2 cells to a 96-well cell plate and culture it for 6 h. Then, add different concentrations of HtpG recombinant protein and culture it for 24 h. Add 10 μl of CCK-8, incubate for 1 h, and measure the absorbance at 450 nm. The LDH-assay kit (Nanjingjiancheng, China) was used to assess the cell toxicity.

All data are expressed as the means ± standard error (SE). Data analysis was performed using the SPSS 25.0 software (IBM), and one-way analysis of variance with Dunnett’s test was used. p < 0.05 was considered statistically significant. Comparison between the two sets of data was used with independent sample t-test, and differences were considered significant when *p < 0.05 and **p < 0.01 were obtained.

To study the regulation of HtpG gene in S. Typhimurium, HtpG mutant strains (ΔhtpG) were constructed by RED homologous recombination system. Based on the mutant strains, the pBR-322 plasmid was used to construct complement strains (CΔhtpG). The results of qPCR showed that the relative expression of HtpG mRNA in mutant strains was significantly reduced (p < 0.01), while that of supplemented strains was significantly increased (p < 0.01) ( Figure 1 ).

To reveal the regulatory mechanism of HtpG, transcriptome sequencing and analysis of S. Typhimurium wild strains and HtpG-mutant strains were conducted. Overall, 6.45–8.18 million pieces of clean data and 1.0–1.2 Gb of RNA-seq data in wild-type strains and HtpG-mutant strains were obtained ( Table S1 ). The data were submitted to the NCBI Sequence Reads Archive database (PRJNA746115). The reads obtained by sequencing were mapped to the S. Typhimurium genome (NC_003197.2) in the NCBI database. The results are shown in Table S2 , the total mapped comparison rate of each sample is above 99%, and the multiple mapped comparison rate is above 95%. The analysis of gene expression differences was performed using DESeq2 software, and the screening criteria were p < 0.05. Compared with the WT strains, the ΔhtpG strains had a total of 383 differentially expressed genes, including 182 upregulated genes and 201 downregulated genes. ( Figures 2A, B ). According to KEGG pathway analysis in Figure 2C , the upregulated genes were mainly enriched in the ribosomal pathway, while the downregulated genes were mainly enriched in the Salmonella infection, flagellar assembly, bacterial chemotaxis, and other pathways.

Five differential genes enriched in Salmonella infection pathway were randomly selected for verification of the transcriptome result. Compared with the WT strains, the relative expression of PrgJ, SipB, SopB, FliC, and SipA mRNA of the ΔhtpG strains was significantly downregulated (p < 0.05), which was consistent with the results of transcriptomics ( Figure 2D ).

RNA-seq results showed that flagellar assembly-related genes of S. Typhimurium were downregulated after HtpG mutation, which was then validated by determining the mobility of Salmonella, as represented by the size of the bacterial swimming circle. The results showed that the diameter of the swimming circle of ΔhtpG strains was significantly smaller than that of WT strains and CΔhtpG strains (p < 0.05) ( Figure 3A ).

The effect of HtpG mutation of S. Typhimurium on its biofilm formation ability was tested ( Figure 3B ). The results showed that the amount of biofilm formed by the ΔhtpG strains was significantly lower than that of the WT strains and CΔhtpG strains (p < 0.05).

Adhesion to intestinal epithelial cells is a key step of S. Typhimurium infecting the host (Wagner and Hensel, 2011). Thereby, IPEC-J2 cells were used as the object to detect the difference in adhesion ability of WT strains, ΔhtpG strains, and CΔhtpG strains. After 1 h of incubation, the number of adhering and invading S. Typhimurium was counted. The results showed that the adhesion and invasion ability of the ΔhtpG strains were both significantly lower than that of the WT strains and CΔhtpG strains (p < 0.05) ( Figures 3C, D ).

S. Typhimurium with MOI ≈ 100 was added to IPEC-J2 cells, followed by detection of the number of S. Typhimurium within the cells at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h of infection. Compared with the WT strains and the CΔhtpG strains, the number of S. Typhimurium in the cells of the ΔhtpG strains at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h of incubation was extremely significantly reduced (p < 0.01) ( Figure 3E ), which suggested that the deletion of HtpG can reduce the intracellular proliferation ability of S. Typhimurium.

Similar results were found in the RAW264.7 cell experiment. Two hours after infection, the number of S. Typhimurium in RAW264.7 cells in the ΔhtpG infection group was significantly lower than that in the WT infection group and the CΔhtpG infection group (p < 0.05). After 6 h, compared with the WT and CΔhtpG infection groups, the number of bacteria in the cells of the ΔhtpG infection group was significantly reduced (p < 0.01), while the number of bacteria in the CΔhtpG infection group was significantly higher than that in the WT infection group (p < 0.05) ( Figure 3F ), indicating that mutating the HtpG gene of S. Typhimurium can reduce its ability to proliferate in intestinal epithelial cells and macrophages.

Transcriptomics revealed that HtpG mutation led to downregulation of Salmonella infection pathway gene expression; we thus analyzed the host inflammatory response induced by HtpG mutant strains during the infection process through an in vitro infection model.

The final body weight of mice challenged with WT and ΔhtpG strains was both significantly lower than the mice in the NC group (p < 0.01); however, the final body weight of mice in the ΔhtpG strain group was significantly higher than the mice in the NC group (p < 0.05) ( Table 2 ). This indicated that mutant of the HtpG gene can alleviate the weight loss of mice caused by S. Typhimurium. Compared with the NC group, the spleen and liver weights of the ΔhtpG group and the WT group were significantly increased (p < 0.05), and the weight of the thymus was significantly decreased (p < 0.05), indicating that the oral administration of S. Typhimurium caused huge damage to the mouse immune system. However, the ΔhtpG group exhibited a reduction (p < 0.05) of spleen weight along with an increase (p < 0.05) in final body weight relative to of the WT group ( Table 2 ), which suggested that mutant of HtpG may reduce the toxicity of S. Typhimurium, probably attenuating host acute inflammation caused by its infection. To confirm this speculation, we then quantified inflammatory cytokines in mice after infection.

As shown in Figure 5A , compared with the gavage with WT strains, the gavage with ΔhtpG strains decreased the relative expression of spleen IL-1β, IL-18, TNFα, and IFNγ of mice (p < 0.05). The relative expression of ileal IL-1β (p < 0.01) and IL-18 (p < 0.01) as well as IFNγ (p < 0.05) of mice gavaged with ΔhtpG strains was significantly lower than that gavaged with WT strains ( Figure 5B ). Regarding the inflammatory cytokine contents in mice after infection, the contents of TNFα, IFNγ, IL-1β, and IL-18 in serum, spleen, and ileum ( Figures 5C–E ) of mice challenged with ΔhtpG strains were significantly decreased (p < 0.05) as compared with mice challenged with WT strains. Overall, the mutation of HtpG gene could alleviate acute inflammatory injury in mice infected by S. Typhimurium.

To further validate the role of HtpG in S. Typhimurium infection, a recombinant HtpG protein (rHtpG) derived from S. Typhimurium was constructed. It could be seen from Figure 6A that the addition of rHtpG had no significant effect on the proliferation of S. Typhimurium in IPEC-J2 cells at 1 h and 2 h of infection (p > 0.05). At 8 h, 12 h, and 24 h, the number of S. Typhimurium in the cells added with rHtpG was significantly higher than that in the control group (p < 0.05) ( Figure 6A ).

The number of S. Typhimurium in RAW 264.7 cells was detected 6 h after infection ( Figure 6B ). Compared with the control group, the number of S. Typhimurium in the rHtpG treatment group was significantly increased (p < 0.05).

The LDH content in the IPEC-J2 cell culture medium after adding rHtpG for 12 h was detected ( Figure 6C ). Compared with the control group, the LDH content in the rHtpG group did not increase significantly (p > 0.05), suggesting that the rHtpG would not cause cell damage. CCK-8 was used to detect cell proliferation for 12 h. As shown in Figure 6D , the absorbance of IPEC-J2 cells treated with different concentrations of rHtpG (1, 10, 100, 500, and 1000 μg/ml) did not change significantly compared with the control group, which revealed that rHtpG did not affect cell proliferation.

After incubating cells with S. Typhimurium and rHtpG for 12 h, we detected the relative mRNA expression and secretion level of inflammatory cytokines. As shown in Figure 6E , rHtpG significantly increased the relative expression of IL-8 and TNFα in IPEC-J2 cells (p < 0.05) but had no significant effect on IL-18 (p > 0.05) expression. In the case of S. Typhimurium infection, the addition of rHtpG could significantly increase the relative expression of TNFα, IL-8, and IL-18 (p < 0.05). Meanwhile, rHtpG significantly increased the secretion of IL-8 within cells (p < 0.05) ( Figure 6F ).