Fig 1.
S. 4, [5], 12:i:- isolates demonstrating resistance to DDAB. (A) SEM images illustrate the morphological changes in the S. 4, [5], 12:i:- strain ZC055 following treatment with different concentrations of DDAB (0, 20, 100, 200, and 400 µg/mL). (B) A schematic representation of the IncHI1B plasmid map is provided. The outermost layer depicts COG functional annotation genes, with arrows in a clockwise direction indicating positive strand encoding. The subsequent layer displays plasmid sequence location coordinates. The third layer illustrates GC content, calculated using a 500 bp window and a 20 bp step size. Red areas indicate GC content lower than the average for the entire plasmid, while green areas denote higher GC content. The height of the peaks represents the magnitude of the difference from the average GC content. The innermost layer portrays GC skew, also calculated using a 500 bp window and a 20 bp step size. Pink areas signify lower G content compared to C, whereas light green areas indicate the opposite. (C) PCR was employed to detect the presence of sugE1, sugE2, and the IncHI1B plasmid in the S. 4, [5], 12:i:- isolates.
Fig 2.
Transcriptome analysis of the S.
4, [5], 12:i:- ZC055 after treatment with 100 µg/mL DDAB. (A) Distribution of differentially expressed genes: blue represents upregulated genes after DDAB treatment, orange represents downregulated genes after DDAB treatment, and gray represents genes unaffected by DDAB. (B) Transcriptional landscape of ZC055 after treatment with DDAB compared to ZC055 cultured in LB medium. Significantly upregulated genes after DDAB treatment are marked in green, significantly downregulated genes are marked in blue, and gray indicates genes with no significant changes between the two groups. The sugE1, sugE2, and SPI-1 genes are marked in purple or red, respectively. (C) Validation of sugE1 and sugE2 gene expression using quantitative real-time PCR in accordance with the RNA-seq data. (D) Expression of the sugE1 gene in ΔsugE2 compared to WT after treatment with 12.5 µg/mL or 25 µg/mL DDAB. (E) Expression of the sugE2 gene in ΔsugE1 compared to WT after treatment with 12.5 µg/mL or 25 µg/mL DDAB.
Table 1.
The MIC values of DDAB for the different strains.
Fig 3.
The efflux pump function of SugE1 and SugE2.
(A-B) The viability of bacteria after treatment with 0, 30, and 60 μM CCCP, followed by the addition of 50 µg/mL DDAB (A) or 100 µg/mL DDAB (B). (C) Accumulation kinetics and final fold change in accumulation of H33342 in ZC055, ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2. (D) The efflux kinetics and final efflux fold change of ethidium bromide (ETBR) was examined in ZC055, ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2.
Fig 4.
The adhesion and invasion ability of S.
4, [5], 12:i:- ZC055 and its derivate strains to IPEC-J2 cells. (A) The expression levels of T3SS1 in ZC055 treated with DDAB by RNA-seq. (B) qRT-PCR analysis of the expression levels of T3SS1 in ZC055 treated with 100 µg/mL DDAB. (C-D) The adhesion (C) and invasion (D) abilities of ZC055 treated with DDAB at concentrations of 1/2 MIC (100 µg/mL), 1/4 MIC (50 µg/mL), and 1/8 MIC (25 µg/mL) were compared to those of ZC055 cultured in LB medium in IPEC-J2 cells. (E-F) The infection assays were also performed using ZC055, ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2 in IPEC-J2 cells. The adhesion (E) and invasion (F) of ΔsugE1, ΔsugE2, and ΔsugE1ΔsugE2 to IPEC-J2 cells were compared to the adhesion and invasion of ZC055.
Fig 5.
SugE2 inhibits the virulence of S.
4, [5], 12:i:- in C57BL/6 mice. ZC055, ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2 were orally infected in C57BL/6 mice. (A) Kaplan-Meier survival curves of mice for 18 days after infection. The P value was determined using a log rank (Mantel-Cox) test. (B) Representative images of cecal lesions on day 3 post-infection and comparison of the body weight of ΔsugE1, ΔsugE2, and ΔsugE1ΔsugE2 infected mice with ZC055 infected mice on day 3 post-infection. (C) Bacterial load in the liver, spleen, and cecum of ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2 infected mice on day 3 post-infection. Organs were collected, homogenized, and the number of colony-forming units (CFU) was counted to quantify the bacterial load. The median values were plotted. A Mann-Whitney U test was used to compare the bacterial load in each organ of ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2 infected mice with ZC055 infected mice.
Fig 6.
Intestinal pathological and expression levels of tight junction proteins in mice intestine after infection with S.
4, [5], 12:i:- ZC055 and its derivate strains. (A) Representative images of histopathological sections from the mouse intestine collected on day 3 post-infection. The red arrows denote exfoliation of epithelial cells in the intestinal villi. The yellow arrows denote edema of the intestinal villi with detachment of the epithelium from the lamina propria. The blue arrows denote lymphocytic infiltration (magnification is 200 ×). (B) Expression levels of tight junction proteins, including Zona Occludens-1 (ZO-1), Claudin-3, and Occludin, in the intestines of WT, ΔsugE1, ΔsugE2 and ΔsugE1ΔsugE2 infected mice on day 3 post-infection were quantified using qRT-PCR. The expression levels were compared to those of tight junction proteins in the intestine of ZC055 infected mice, with the results presented as fold changes relative to the control group (PBS).
Fig 7.
SugE2 senses the intestinal content to inhibit the expression of SPI-1.
(A) The expression levels of the sugE1 and sugE2 genes in ZC055 cultured in LB medium supplemented with 50% mouse intestinal content or M9 medium. The results are presented as Log2foldchange relative to ZC055 cultured in LB medium. (B-E) The expression levels of the SPI-1 genes in ZC055 (B), ΔsugE1(C), ΔsugE2(D), andΔsugE1ΔsugE2 (E) cultured in LB medium supplemented with 50% mouse intestinal content were quantified using qRT-PCR. The results are presented as Log2foldchange relative to WT cultured in LB medium.
Fig 8.
SugE2 inhibits the expression of SPI-1 to affect the adhesion and invasion of S.
4, [5], 12:i:- ZC055. The adhesion (A) and invasion (B) abilities of ZC055, ΔsugE2, ΔsugE1ΔsugE2, ΔprgI, ΔprgIΔsugE2, and ΔprgIΔsugE1ΔsugE2 in IPEC-J2 cells. (C-E) The expression levels of the SPI-1 genes in ΔsugE1 (C), ΔsugE2 (D) and ΔsugE1ΔsugE2 (E) cultured in LB medium supplemented with 50% mouse intestinal content compared these strains cultured in LB medium.
Fig 9.
The contents of the mouse intestine (a) and quaternary ammonium compounds (b) can trigger the expression of the genes sugE1 and sugE2.
Subsequently, bacteria expel quaternary ammonium compounds via the SugE efflux pump (c). Concurrently, the expression of the sugE2 gene results in the downregulation of T3SS1 gene expression (d), which leads to a reduced ability of the bacteria to adhere and invade to IPEC-J2 cells (e), as well as their capacity to infect and colonize within the mouse host (f). Created in BioRender. Wen, Y. (2025) https://BioRender.com/w33f839.