Identification And Functional Verfication For Enterotoxigenic Escherichia Coli F4acR Genes In Piglet

Enterotoxigenic Escherichia coli (ETEC) F4ac is one of the most important pathogenic bacteria causing severe diarrhoea in piglets, whose fimbriae acting as ligands for specific carbohydrate receptors on the epithelial surface of the small intestine. Piglets lacking the receptor are resistant to infection by ETEC F4ac. The study were designed to screen the candidate genes of ETEC F4acR, to establish the model of ETEC F4ac adhesion to IPEC-J2 cells in vitro, to verify the function of candidate genes in cellular level and to investigate the molecular mechanisms of casual gene for swine diarrhea induced by Enterotoxigenic Escherichia coli F4ac.Experiment 1, four pairs of full-sibs were chosed for studying the pathogenesis of diarrhoea infected by ETEC F4ac strain. A 8-plex iTRAQ workflow involving HPLC (High Performance Liquid Chromatography) - MS/MS (tandem mass spectrometry) was adopted to profile the host differentially expressed proteins. The comparison of adhesive and non-adhesive showed a total of 245 proteins to be significantly altered. Out of these,117 (47.8%) were up-regulated and 128 (52.2%) were down-regulated. Analysis using PANTHER database was conducted to gain deeper insight into the pathway based on DEPs between " positive " and " negative " piglets. The only " Integrin signalling pathway:P00034 " was enriched and the small P value indicates that the result is nonrandom and potentially interesting. We found that ITGB5 is most likely to be the gene encoding the receptor protein of ETEC F4ac for its appropriate location, molecular function and protein molecular weight.Experiment 2, IPEC-J2 is a non-transformed, non-tumorigenic cell line that was isolated from neonatal piglet mid-jejunum intestinal tissues. This primary cell line has been used increasingly to characterize epithelial cell interactions with enteric bacteria and viruses. Quantitative real-time PCR was applyed to quantification of ETEC F4ac adhesion to IPEC-J2 cells. When the initial template concentration was in the range of 105-109 CFU/mL, a good linear relationship between bacterial cell counts and Ct value was expressed with the equation of y= - 0.3056x+13.418. We established the model of ETEC F4ac adhesion to IPEC-J2 cells that MOI= 200:1 for 4h. The quantitative real-time PCR method is a rapid, accurate and sensitive method for detecting and quantifying the adhering capacity of ETEC F4ac to the IPEC-J2 cells compared with colony counting method.Experiment 3, ITGB5, MUC4 and MUC13 were selected as candidate genes for controlling the expression of the receptor for ETEC F4ac, and a meaningless SLC12A8 gene was used as control. With the latest developed CRISPR/Cas9 gene knockout technique, we studied the molecular functions of candidate genes in response of IPEC-J2 cells to infection with F4ac ETEC. Result revealed the knockout of ITGB5 decreased bacterial adhesion in response to ETEC F4ac exposure. Meanwhile, the overexpression of ITGB5 gene would increased bacterial adhesion.These data provide preliminary foundation for further detecting of the gene function and pathways in which porcine ITGB5 gene involved.