Diarrhea and edema disease in weaned piglets due to infection by Escherichia coli F18 is a leading cause of economic loss in the pig industry. Resistance to E. coli F18 depends on expression of receptors on intestinal epithelial cells, and individual immunity. This study was conducted in Sutai pig E. coli F18-resistant and -susceptible full sib-pair individuals, identified on the basis of resource populations and verification of adhesion assays. The molecular mechanism underlying E. coli F18 resistance was investigated through analysis of the expression of E. coli F18 receptor associated and innate immunity proteins, using proteomics and bioinformatics techniques. Two-dimensional electrophoresis analysis revealed a total of 20 differentially expressed proteins in E. coli F18-resistant and -susceptible groups (10 upregulated and 10 downregulated). A total of 16 differentially expressed proteins were identified by MALDI TOF/TOF mass spectral analysis. According to gene ontology and pathway analysis, differentially expressed proteins were mainly involved in cell adhesion, immune response and other biologically relevant functions. Network analysis of interactions between differentially expressed proteins indicated a likelihood of their involvement in E. coli F18 infection. The expression levels of several important proteins including actin beta (ACTB), vinculin (VCL), heat stress proteins (HSPs) and transferrin (TF) in E. coli F18-resistant and -susceptible individuals were verified by Western blotting, supporting the identification of ACTB, VCL, HSPs and TF as promising candidate proteins for association with E. coli F18 susceptibility.

Alpha-(1,2)-fucosyltransferase (FUT1) gene has some influence on economically important traits and disease resistance. DNA methylation plays an important role in human diseases but is relatively poorly studied in pigs by regulating the mRNA expression of genes. The aim of this study was to analyze the influence of promoter methylation on the expression of FUT1 gene. We used bisulfite sequencing PCR (BSP) and qPCR to analyze the methylation of the FUT1 5 0 -flanking region and FUT1 mRNA expression in the duodenum of Sutai piglets from newborn to weaning. FUT1 contains three CpG islands upstream of the start codon, of which two are located in the putative promoter region containing multiple promoter elements and transcription factor binding sites, such as CpG islands, a CAAT box, SP1, and EARLY-SEQ 1. The CpG island between nucleotides -1762 and -580 had a low degree of methylation, and its methylation level was significantly lower in 35-day-old piglets than 8-and 18-day-old piglets (P \ 0.05). FUT1 mRNA expression was significantly higher in 35-day-old piglets than 8-and 18-day-old piglets (P \ 0.05). Pearson's correlation analysis showed that the methylation of the CpG island between nucleotides -1762 and -580 of FUT1 was significantly, negatively correlated with FUT1 mRNA expression (P \ 0.05). These results demonstrate that differential methylation of CpG islands negatively regulates the expression of FUT1 in the porcine duodenum, suggesting a probable influence on the resistance of piglets to infection with ETEC F18.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.

No abstract

ABSTRACT. To assess the relationship between the expression of a(1,2)-fucosyltransferase (FUT1 and FUT2) genes and resistance to Escherichia coli F18 in weaned pigs, FUT1 and FUT2 expression levels in Large White, Meishan, and Sutai pigs (with resistance to E. coli F18) were determined using real-time PCR. The results revealed that FUT1 and FUT2 expression levels were higher in the liver, lungs, kidneys, stomach, duodenum, and jejunum than in the muscle and heart. Medium FUT2 expression levels were detected in the spleen, thymus, and lymph nodes. Intestinal FUT1 expression levels were higher in Sutai pigs than in Large White and Meishan pigs (P < 0.05). However, intestinal FUT2 expression levels were lower in Sutai pigs than in Large White and Meishan pigs (P < 0.05). FUT1 and FUT2 expression levels did not differ between Large White and Meishan pigs (P > 0.05). The results revealed that high FUT1 expression levels and low FUT2 expression levels in the intestines of Sutai pigs affected FUT1 and FUT2 enzymes, the synthesis of type 2 H and type 1 H antigens, and E. coli F18 adhesion. Moreover, low FUT2 expression levels conferred resistance to E. coli F18.