| Summary: | Toll-like receptor 5 (TLR5) plays an important role in immune system. In this study, we performed transcriptome analysis of the duodenum in <i>E. coli</i> F18-resistant and -sensitive Sutai weaned piglets and analyzed the differential expression of <i>TLR5</i>. The cellular localization of TLR5 was investigated, and the effect of <i>TLR5</i> expression on <i>E. coli</i> invasion was evaluated after pig small intestinal epithelial cell lines (IPEC-J2) were stimulated by <i>E. coli</i>. The results showed that TLR5 expression level in duodenum and jejunum were significantly higher in <i>E. coli</i> F18-sensitive than in <i>E. coli</i> F18-resistant piglets. TLR5 protein was mainly expressed in the cytoplasm and cell membrane. The expression of genes associated with the TLR5 signaling pathway were significantly higher in <i>TLR5</i>-overexpressed cells than in control cells. Bacterial adhesion was higher in <i>TLR5</i>-overexpressed cells than in blank cells and lower in <i>TLR5</i> interference than in blank cells. The core promoter region of <i>TLR5</i> included two CpG islands and 16 acting elements. The methylation of the mC-6 site in the second CpG island of the promoter region had a regulatory effect on <i>TLR5</i> expression. Therefore, <i>TLR5</i> plays an important regulatory role on <i>E. coli</i> invasion. Low expression of <i>TLR5</i> inhibited the immune response and decreased cell damage, which was conducive to the resistance to <i>E. coli</i> stimulation. In conclusion, this study preliminarily revealed the molecular mechanism of <i>TLR5</i> gene regulating the resistance of piglets to <i>Escherichia coli</i>, and provided a new candidate gene for screening <i>Escherichia coli</i> resistance markers in pigs.
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