Investigation of the porcine MUC13 gene: isolation, expression, polymorphisms and strong association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac

Enterotoxigenic Escherichia coli (ETEC) F4ab and F4ac are major determinants of piglet diarrhoea. The locus for the ETEC F4ab/ac receptor has been mapped to SSC13q41. MUC13 is a transmembrane mucin expressed predominantly in the epithelial surface of the gastrointestinal tract and the MUC13 gene was assigned to SSC13q41, supporting it as a positional candidate gene for the ETEC F4ab/ac receptor. We herein determined the complete 2679-bp cDNA of pig MUC13, and proved that it was most highly expressed in the jejunum and moderately expressed in the trachea, stomach and liver. Furthermore, 13 MUC13 polymorphisms were identified in 19 founder animals of a White Duroc x Erhualian resource population, and a total of 727 F(2) animals with in vitro ETEC F4ab/ac adhesion phenotypes in this population were genotyped for three identified MUC13 polymorphisms including c.576C>T, c.908A>G and c.935A>C. The transmission disequilibrium test showed that the MUC13 alleles and haplotypes were significantly associated with susceptibility/resistance to ETEC F4ab/ac, especially between haplotype [C;G;A] and susceptibility to ETEC F4ac (P = 8.0e-18). Animals inheriting this haplotype were predominantly susceptible to ETEC F4ac (n = 291/303). Moreover, nearly all animals homozygous for haplotype [T;G;C] (n = 39/41) and a majority of those with the [C;A;A]/[T;G;C] haplotype pair (n = 79/88) were resistant to ETEC F4ab. Our results indicated that MUC13 is in strong linkage disequilibrium with the ETEC F4ab/ac receptor locus and provided potential markers for selection of ETEC F4ab/ac-resistant animals in the pig breeding scheme.     

Refined Candidate Region for F4ab/ac Enterotoxigenic Escherichia coli Susceptibility Situated Proximal to MUC13 in Pigs

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.     

The g.243A>G mutation in intron 17 of MUC4 is significantly associated with susceptibility/resistance to ETEC F4ab/ac infection in pigs

Using a porcine radiation hybrid panel, we assigned the mucin 4 (MUC4) gene to SSC13q41, which harbours the enterotoxigenic Escherichia coli (ETEC) F4ab/ac receptor locus. In addition, we identified two SNPs in intron 17 of MUC4 (DQ124298:g.243A>G and DQ124298:g.334A>G) in the parental population of a White Duroc x Erhualian cross. Association analysis showed that the MUC4 g.243A>G mutation was strongly associated with ETEC F4ab/ac, and especially with F4ac adhesion phenotypes in the White Duroc x Erhualian resource population, indicating that this polymorphism was in a significant linkage disequlibrium with the ETEC F4ab/ac receptor locus. Because of different linkage disequlibrium values between the ETEC F4ab and F4ac adhesion phenotypes and the MUC4 g.243A>G mutation, we argue that the inheritance of F4ab and F4ac receptors might be under the control of two closely linked loci.     

Gene Silencing of Porcine MUC13 and ITGB5: Candidate Genes towards Escherichia coli F4ac Adhesion

Background

Integrin beta-5 (ITGB5) and mucin 13 (MUC13) genes are highly expressed on the apical surface of intestinal epithelia and are thought to be candidate genes for controlling the expression of the receptor for enterotoxigenic Escherichia coli (ETEC) F4ac. Human MUC13 protein has an expected role in protecting intestinal mucosal surfaces and porcine ITGB5 is a newly identified potential receptor for ETEC F4ac.

Methodology/Principal Findings

To test the hypothesis that ITGB5 and MUC13 both play key roles in protection of the intestinal mucosa against pathogenic bacterium, porcine intestinal epithelial cells (IPEC-J2) were transfected with ITGB5-targeting, MUC13-targeting or negative control small interfering RNA (siRNA), respectively. Firstly, we measured mRNA expression levels of mucin genes (MUC4, MUC20), pro-inflammatory genes (IL8, IL1A, IL6, CXCL2), anti-inflammatory mediator SLPI, and PLAU after RNAi treatments with and without ETEC infection. Secondly, we compared the adhesions of ETEC to the pre- and post-knockdown IPEC-J2 cells of ITGB5 and MUC13, respectively. We found that ITGB5 and MUC13 knockdown both had small but significant effects in attenuating the inflammation induced by ETEC infection, and both increased bacterial adhesion in response to F4ac ETEC exposure.

Conclusions/Significance

Our current study first reported that ITGB5 and MUC13 are important adhesion molecules of mucosal epithelial signaling in response to Escherichia coli in pigs. These data suggest that both ITGB5 and MUC13 play key roles in defending the attachment and adhesion of ETEC to porcine jejunal cells and in maintaining epithelial barrier and immunity function.     

A genome-wide association analysis for susceptibility of pigs to enterotoxigenic Escherichia coli F41

Enterotoxigenic Escherichia coli (ETEC) is a type of pathogenic bacteria that cause diarrhea in piglets through colonizing pig small intestine epithelial cells by their surface fimbriae. Different fimbriae type of ETEC including F4, F18, K99 and F41 have been isolated from diarrheal pigs. In this study, we performed a genome-wide association study to map the loci associated with the susceptibility of pigs to ETEC F41 using 39454 single nucleotide polymorphisms (SNPs) in 667 F₂ pigs from a White Duroc×Erhualian F₂ cross. The most significant SNP (ALGA0022658, P=5.59×10⁻¹³) located at 6.95 Mb on chromosome 4. ALGA0022658 was in high linkage disequilibrium (r²>0.5) with surrounding SNPs that span a 1.21 Mb interval. Within this 1.21 Mb region, we investigated ZFAT as a positional candidate gene. We re-sequenced cDNA of ZFAT in four pigs with different susceptibility phenotypes, and identified seven coding variants. We genotyped these seven variants in 287 unrelated pigs from 15 diverse breeds that were measured with ETEC F41 susceptibility phenotype. Five variants showed nominal significant association (P<0.05) with ETEC F41 susceptibility phenotype in International commercial pigs. This study provided refined region associated with susceptibility of pigs to ETEC F41 than that reported previously. Further works are needed to uncover the underlying causal mutation(s).     

The receptor locus for Escherichia coli F4ab/F4ac in the pig maps distal to the MUC4�CLMLN region

Enterotoxigenic Escherichia coli (ETEC) with fimbriae of the F4 family are one of the major causes of diarrhea and death among neonatal and young piglets. Bacteria use the F4 fimbriae to adhere to specific receptors expressed on the surface of the enterocytes. F4 fimbriae exist in three different antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13. A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three?Cgeneration pedigree including 45 offspring was generated with the aim to use this recombination event to refine the localization of the F4bcR locus. All pigs were phenotyped using the microscopic adhesion test and genotyped for a total of 59 markers. The recombination event was mapped to a 220-kb region between a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest that the locus for F4bcR is located between the LMLN locus and microsatellite S0283.     

A genome-wide association study identifies two novel promising candidate genes affecting Escherichia coli F4ab/F4ac susceptibility in swine

Enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbria is the major pathogenic bacteria causing diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the susceptibility to ETEC F4ab/F4ac is an autosomal Mendelian dominant trait and the loci controlling the F4ab/F4ac receptor are located on SSC13q41, between markers SW207 and S0283. To pinpoint these loci and further validate previous findings, we performed a genome-wide association study (GWAS) using a two generation family-based population, consisting of 301 piglets with phenotypes of susceptibility to ETEC F4ab/F4ac by the vitro adhesion test. The DNA of all piglets and their parents was genotyped using the Illumina PorcineSNP60 BeadChip, and 50,972 and 50,483 SNPs were available for F4ab and F4ac susceptibility, respectively, in the association analysis after quality control. In summary, 28 and 18 significant SNPs (p<0.05) were detected associated with F4ab and F4ac susceptibility respectively at genome-wide significance level. From these significant findings, two novel candidate genes, HEG1 and ITGB5, were firstly identified as the most promising genes underlying F4ab/F4ac susceptibility in swine according to their functions and positions. Our findings herein provide a novel evidence for unravelling genetic mechanism of diarrhoea risk in piglets.     

Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion ( F4bcR ) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers ( MUC4gt and HSA125gt ) and an intronic SNP in MUC4 ( MUC4-8227 ) were used to create the linkage map. The region for F4bcR was refined to the interval SW207 – S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207 – MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.     

Association of B3GNT5 Polymorphisms with Susceptibility to ETEC F4ab/ac in the White Duroc?×?Erhualian Intercross and 15 Outbred Pig Breeds

The B3GNT5 gene is a candidate for the F4ab/ac receptor conferring susceptibility to enterotoxigenic Escherichia coli (ETEC) F4ab/ac in pigs. In this study, we screened mutations in the complete coding region of the porcine B3GNT5 gene and identified four SNPs in the 3' untranslated regions. We genotyped the four SNPs across a large-scale White Duroc × Chinese Erhualian F2 resource population (total F2 = 755) and 292 purebred piglets representing 15 Chinese and Western breeds. We found that the g.1476G→A locus and haplotypes [A;T;G;T] and [A;G;G;T] had significant association with susceptibility to ETEC F4ac in the resource population. None of the B3GNT5 polymorphisms and haplotypes was associated with susceptibility to ETEC F4ab/ac in outbred piglets. This result, together with other reports, supports the conclusion that B3GNT5 is not the responsible gene encoding the ETEC F4ab/ac receptors.     

Refined candidate region specified by haplotype sharing for Escherichia coli F4ab/F4ac susceptibility alleles in pigs

Infection of the small intestine by enterotoxigenic Escherichia coli F4ab/ac is a major welfare problem and financial burden for the pig industry. Natural resistance to this infection is inherited as a Mendelian recessive trait, and a polymorphism in the MUC4 gene segregating for susceptibility/resistance is presently used in a selection programme by the Danish pig breeding industry. To elucidate the genetic background involved in E. coli F4ab/ac susceptibility in pigs, a detailed haplotype map of the porcine candidate region was established. This region covers approximately 3.7 Mb. The material used for the study is a three generation family, where the founders are two Wild boars and eight Large White sows. All pigs have been phenotyped for susceptibility to F4ab/ac using an adhesion assay. Their haplotypes are known from segregation analysis using flanking markers. By a targeted approach, the candidate region was subjected to screening for polymorphisms, mainly focusing on intronic sequences. A total of 18 genes were partially sequenced, and polymorphisms were identified in GP5, CENTB2, APOD, PCYT1A, OSTalpha, ZDHHC19, TFRC, ACK1, MUC4, MUC20, KIAA0226, LRCH3 and MUC13 . Overall, 227 polymorphisms were discovered in the founder generation. The analysis revealed a large haplotype block, spanning at least 1.5 Mb around MUC4 , to be associated with F4ab/ac susceptibility.     

Analysis of Association Between the MUC4 g.8227C>G Polymorphism and Production Traits in Italian Heavy Pigs Using a Selective Genotyping Approach

In pigs, susceptibility to enterotoxigenic Escherichia coli (ETEC) K88 strains (locus F4bcR) is determined by a dominant allele, with the recessive allele determining resistance. The susceptible allele also appeared to be associated with higher growth rate even with discordant results. A single nucleotide polymorphism (SNP) in exon 7 of the mucin 4 (MUC4) gene (DQ848681:g.8227C>G), shown to be in close linkage disequilibrium with the F4bcR locus, has been used as marker to identify susceptible pigs, substituting invasive villous adhesion tests. We herein analyzed this SNP in Italian local breeds and applied a selective genotyping approach in Italian Large White, Italian Landrace, and Italian Duroc comparing allele frequency distribution in groups of pigs with extreme estimated breeding values (EBV) for average daily gain (ADG) and backfat thickness (BFT) to evaluate if this marker is associated with these traits. Allele G (associated with susceptibility to ETEC) was associated with higher ADG and BFT in Italian Large White (P = 6.66E-04 and P = 0.012, respectively) and higher ADG in Italian Landrace (P = 7.23E-12). This polymorphism was poorly informative in Italian Duroc. Antagonistic associations of the MUC4 g.8227C>G alleles on susceptibility to ETEC and growth performances evidence the complexity of applying marker assisted selection in pig breeding.     

MUC4 expression and localization in gastrointestinal tract and skin of human embryos

MUC4 is a heterodimeric membrane mucin, composed of two tightly linked subunits and implicated in the protection of wet-surfaced epithelia. Although human MUC4 and its rat analogue Muc4/sialomucin complex have been extensively studied in the adult human and in the adult and embryonic rat, respectively, there has been little attention paid to date to the human embryo. Based on studies with our monoclonal antibody 1G8 and commercial tissue arrays, we describe some unexpected features of the expression of MUC4 in human embryonic epithelia. In the human small intestine and colon, MUC4 appears at an earlier relative stage of development, compared to gestation time, than in the rat. Interestingly, MUC4 also appears in the embryo in the skin, then disappears late in gestation, consistent with its absence in adult skin. These results are consistent with an important protective role for MUC4 in the human embryo that is different from that in the rat or in the adult human.     

Detection of quantitative trait loci for porcine susceptibility to enterotoxigenic Escherichia coli F41 in a White Duroc × Chinese Erhualian resource population

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in neonatal and postweaning pigs. F41 is one of ETEC fimbriae that adhere to the small intestinal epithelium and lead to development of diarrhoea. The genetic architecture of susceptibility to ETEC F41 remains elusive in pigs. In this study, we determined the in vitro adhesion phenotypes of ETEC F41 in a total of 835 F2 animals from a White Duroc × Erhualian intercross, and performed a genome scan using both F2 and half-sib analyses with 183 microsatellite markers to detect quantitative trait loci (QTL) for porcine susceptibility to ETEC F41. The two analyses consistently revealed a 1% genome-wide significant QTL on pig chromosome 4. Moreover, we determined F41 adhesion phenotypes in 14 purebred Erhualian and 14 White Duroc pigs. The results showed that both the founder breeds are segregating for the F41 adhesion phenotype, while less percentage of Erhualian pigs were adhesive to ETEC F41 compared to White Duroc pigs.     

Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.     

The effect of mutation at M307 in <Emphasis Type="Italic">FUT1</Emphasis> gene on susceptibility of <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 and gene expression in Sutai piglets

Escherichia coli F18 (ECF18) is a common porcine enteric pathogen. The pathogenicity of ECF18 bacteria depends on the existence of ECF18 receptor in the brush border membranes of piglet’s small intestinal mucosa. Alpha (1) fucosyltransferase gene (FUT1) has been identified as the candidate gene controlling the adhesion to ECF18 receptor. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting resistant pigs. The piglets were divided into three groups, AA, AG and GG, according to the genotypes present at M307 of FUT1. Small intestinal epithelium cells of piglets with AA, AG and GG genotypes were selected to test the adhesion capability of the wild type E.coli expressing F18ab fimbriae, the recombinant E. coli expressing F18ac fimbriae or the recombinant E. coli secreting and surface-displaying the FedF subunit of F18ab fimbriae, respectively. Here, we examined the distribution and expression of porcine FUT1 mRNA in different tissues in Sutai pigs using real-time PCR. The results showed that piglets with AA genotype show resistance, whereas piglets with GG or AG genotypes are sensitive to the pathogenic E. coli F18 in Sutai piglets. FUT1 was expressed in all the tissues that were examined, and the gene’s expression was highest in the lungs. There was no significant difference in expression level among the three genotypes in the liver, lung, stomach and duodenum, where the gene expression was relatively high. The present analysis suggested that mutation at M307 in FUT1 gene determines susceptibility of small intestinal epithelium to E. coli F18 adhesion in Sutai piglet and the expression of FUT1 gene may be regulated by other factors or the mutation was likely to be in linkage disequilibrium with some cis-regulatory variants.     

大肠杆菌F18菌毛及其亚型的PCR鉴定

F18菌毛是产肠毒素大肠杆菌 (ETEC)与产vero细胞毒素大肠杆菌 (VTEC)的重要致病因子 ,可介导细菌对小肠细胞的黏附 ,并具有F18ab和F18ac 2个抗原亚型。根据已发表的F18ab菌毛A亚单位 (FedA ab)的基因 (fedA ab)设计 3条引物 ,建立了 2种聚合酶链式反应 (PCR)扩增方法。通过对F18ab 大肠杆菌、F18ac 大肠杆菌、K88 大肠杆菌、K99 大肠杆菌、987P 大肠杆菌、F4 1 大肠杆菌的试验 ,结果表明所建立的PCR方法可特异性鉴定F18 大肠杆菌并区别其亚型F18ab与F18ac     

Histomorphometric evaluation of intestinal cellular immune responses in pigs immunized with live oral F4ac+ non-enterotoxigenic E. coli vaccine against postweaning colibacillosis

Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac⁺ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac ⁺ non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac⁺ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immunophenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidinbiotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.Key words: adjuvanted/nonadjuvanted E. coli vaccine, intestinal immune cells, pigs.