Abstract
Enterotoxigenic Escherichia coli (ETEC) that expresses F4ac fimbriae is the major pathogenic microorganism responsible for bacterial diarrhea in neonatal piglets. The susceptibility of piglets to ETEC F4ac is determined by a specific receptor on the small intestinal epithelium surface. We performed an iTRAQ-labeled quantitative proteome analysis using a case-control design in which susceptible and resistant full-sib piglets were compared for the protein expression levels. Two thousand two hundred forty-nine proteins were identified, of which 245 were differentially expressed (fold change > 1.5, FDR-adjusted P < 0.05). The differentially expressed proteins fell into four functional classes: (I) cellular adhesion and binding, (II) metabolic process, (III) apoptosis and proliferation, and (IV) immune response. The integrin signaling pathway merited particular interest based on a pathway analysis using statistical overexpression and enrichment tests. Genomic locations of the integrin family genes were determined based on the most recent porcine genome sequence assembly (Sscrofa11.1). Only one gene, ITGB5, which encodes the integrin β5 subunit that assorts with the αv subunit to generate integrin αvβ5, was located within the SSC13q41 region between 13:133161078 and 13:139609422, where strong associations of markers with the ETEC F4ac susceptibility were found in our previous GWAS results. To identify whether integrin αvβ5 is the ETEC F4acR, we established an experimental model for bacterial adhesion using IPEC-J2 cells. Then, the ITGB5 gene was knocked out in IPEC-J2 cell lines using CRISPR/Cas9, resulting in a biallelic deletion cell line (ITGB5−/−). Disruption of ITGB5 significantly reduced ETEC F4ac adhesion to porcine intestinal epithelial cells. In contrast, overexpression of ITGB5 significantly enhanced the adhesion. A GST pull-down assay with purified FaeG and ITGB5 also showed that FaeG binds directly to ITGB5. Together, the results suggested that ITGB5 is a key factor affecting the susceptibility of piglets to ETEC F4ac.
Highlights
Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea is one of the major diseases in neonatal and weaned piglets, resulting in severe economic losses in the swine industry
We identified 18 SNPs through a genome-wide association study (GWAS), and these were strongly associated with the susceptibility of piglets to ETEC F4ac [9], and HEG1 and integrin subunit beta 5 (ITGB5) emerged as the most promising candidate gene for F4ac receptor (F4acR)
One hundred eighty-nine Large White piglets were examined for the adhesion phenotype by co-culturing epithelial cells from their jejunums with ETEC F4ac
Summary
Enterotoxigenic Escherichia coli (ETEC)-induced diarrhea is one of the major diseases in neonatal and weaned piglets, resulting in severe economic losses in the swine industry. It was refined to a 5.7 cm interval by using a meta-analysis [7], and it was further narrowed down to a 1.6 cm interval by using a pedigree disequilibrium test (PDT) [8]. Within this interval, we identified 18 SNPs through a genome-wide association study (GWAS), and these were strongly associated with the susceptibility of piglets to ETEC F4ac [9], and HEG1 and ITGB5 emerged as the most promising candidate gene for F4acR. Some further studies have been carried out to reveal the molecular basis of the susceptibility of piglets to ETEC F4ac [10, 11], the role of the F4acR protein and its encoding gene remain uncertain
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