Refined candidate region for F4ab/ac enterotoxigenic Escherichia coli susceptibility situated proximal to MUC13 in pigs

Abstract

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100-144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.

Figure 1. Manhattan plot obtained from the P-values for F4ab/ac ETEC susceptibility in 120 pigs.

SNPs are plotted on the X-axis ordered by chromosomal position. Genome-wide -log(10) P-values adjusted to genomic control are plotted on the Y-axis.

Figure 2. Immunoblotting of F4ab/acR⁺ BBMVs with F4ac fimbriae (lane 2 and 4) or anti-MUC13 antibodies (lane 3 and 5).

Proteins were separated under reducing (A) and non-reducing (B) conditions. F4ac fimbriae bound to the F4-specific high molecular weight glycoproteins (only present in F4R⁺ pigs) and several non-specific F4-binding bands <50 kDa (present in F4R⁺ and F4R⁻ pigs) . Anti-MUC13 antibodies recognized BBMV protein bands of 55, 47, 34 and <25 kDa under reducing and non-reducing conditions, but bands of 200, 110 kDa only under reducing conditions. Lane 1  =  protein standards.

Figure 3. Absence of detection of intestinal MUC13 (lane 3) by anti-mucin 13 antibodies in the purified high molecular weight (MW) fraction of F4ab/acR⁺ BBMVs.

Purification occurred by anion exchange chromatography followed by gel filtration chromatography. Strong binding of F4ac fimbriae to the high MW glycoproteins can be seen in lane 2. Lane 1: protein standard.

Figure 4. Absence of F4ac fimbriae binding to immunoprecipitated intestinal MUC13.

Intestinal MUC13 was purified from 1 mg F4ab/acR⁺ brush border membrane vesicles (BBMVs) by immunoprecipitation with anti-MUC13 antibodies and protein A sepharose. The eluate (lane 2) and the non-precipitated fraction (lane 3) were immunoblotted with anti-MUC13 antibodies (A) or with F4ac fimbriae (B). Immunoprecipitation enriched a protein with a band of 110 kDa but this was not recognized by F4ac fimbriae. The F4-specific high MW glycoproteins were found in the non-precipitated fraction. Lane 1  =  protein standards. Arrows: position of the high MW glycoproteins.

Figure 5. Schematic representation showing the identified candidate region (chr13: 144,810,100-144,993,222) of F4ab/ac ETEC susceptibility between MARC0002946 (SNPa) and Indel MUC13 marker on chromosome 13 (chr13: 143,780,000-145,110,000).

(A) SNP1 (ASGA0089965), SNP2 (ASGA0091537) and SNP3 (ALGA0106330) are the most significant SNPs in the association study. MARC0002946 (SNPa) and ALGA0106230 (SNPb) are not associated with F4ab/ac ETEC susceptibility. The orange boxes represent all the annotated genes in the 1.33 Mb region of chromosome 13. The gray box represent the candidate region where no annotated genes were found during the in silico comparative mapping. (B) Schematic representation showing the genotypes of SNP1 (ASGA0089965), SNP2 (ASGA0091537) and SNP3 (ALGA0106330) of 68 strong adhesive (F4R⁺) and 52 non-adhesive (F4R⁻) for F4ab/ac ETEC. For SNP1 and SNP3, the dark green boxes represent CC genotype, light green boxes represent CT genotype and red boxes represent TT genotype. For SNP2, the dark green boxes represent TT genotype, light green boxes represent CT genotype and red boxes represent CC genotype. Pig 40 (F4aR⁺) and pig 3 (F4R⁻) show different genotypes for the markers than expected.