Abstract:

This study was designed to study the evolutionary trend of structural genes of high pathogenicity island (HPI) of Escherichia coli (E. coli) and its effect on TNF/NF-κB pathway. In this study, MEGA and other software were used to analyze the genetic evolution of HPI structural genes, and R language function package was used to mine the gene expression omnibus (GEO) of E. coli infected cells, and differential gene enrichment analysis was performed. Molecular docking was used to predict the binding of HPI-Ybt to TNF/NF-κB pathway related proteins. E. coli and the previously constructed HPI deficient strain E. coliΔHPI were infected with IPEC-J2 cells, and qPCR, ELISA and Western blot were used to detect the key regulatory points in TNF/NF-κB pathway. Phylogenetic analysis showed that the intb gene of isolate YUNNAN001 was most closely related to GQ891729.1, but far from CQ903045.1. KEGG enrichment found that TNF and NF-κB pathways were both involved in E. coli infection, and there were 10 common genes. Volcano plot and heat map enrichment showed that E. coli infection significantly promoted the expression of PTGS2, NF-κB, TRAF6 and other proteins in TNF/NF-κB pathway (P < 0.05). Molecular docking showed that HPI-Ybt could bind to 10 proteins belonging to TNF/NF-κB pathway, among which MAP3K14 and TNFAIP3 were the strongest. The mRNA levels of NF-κB, PTGS2, TNF-α, TRAF6, TRIF, ATF4, cxcl2 and IL-6 in E. coli group were significantly higher than those in E. coliΔHPI group (P < 0.01), and compared with E. coliΔHPI group, E. coli group significantly activated the expression of TNF/NF-κB pathway proteins. In addition, E. coli infection significantly increased the levels of NF-κB and IL-6 compared with the E. coliΔHPI group (P < 0.01). The results suggest that E.coli-HPI activates TNF/NF-κB pathway by synthesizing Ybt and binding to key proteins in TNF/NF-κB pathway.

Key words: Escherichia coli, high pathogenicity island, Yersiniobactin, analysis of evolution, TNF/NF-κB pathway