Porcine Aminopeptidase N-a Receptor Protein For F4~+ Fimbriaed Enterotoxigenic Escherichia Coli (ETEC)

Neonatal and post-weaned piglets diarrhea is the most common gastrointestinal diseases affecting the development of swine industry worldwide. The colonization and infection of F4+ (K88+) Enterotoxigenic Escherichia coli (ETEC) strains in piglets intestines cause diarrheal disease and resulting in mixed, multiple and secondary infections, including yellow and white scour of piglets, edema disease of piglets, post-weaned piglets diarrhea etc. Up to now, ETEC F4 have three serotypes, namely ab, ac, ad, and the most common serotype is F4ac. These three serotypes have some differences in the binding of the intestinal cells, adhesion-mediated faeG gene and pathogenicity. It was reported that the binding of F4+E. coli and porcine intestinal epithelial cells depend on the presence of specific F4 receptors (F4Rs). Under F4+ E. coli infections, F4Rs positive piglets are sensitive to the pathogen bacteria and get infected, while F4Rs free piglets show their resistance. It means that interactions between F4ab, F4ac, F4ad fimbriae and specific receptors on the host intestinal mucosa are essential to initiate attachment, colonization, and infection for three serotypes.Since 1975, several F4 fimbriae related receptor genes and proteins have been reported, but it is hard to select and identify direct receptor genes or candidate proteins for the fimbriae. Even for the reported candidate receptors, there is still lacking effective method and persuasive evidence to prove it. Recently, porcine Aminopeptidase N (APN) was found to be a new receptor protein for F4 fimbriae, which involved in oral immune response, endocytosis and transcytosis of F4 fimbriae. Thus, we targeted APN protein and tried to verify its possibility and credibility as ETEC F4 direct receptor protein by a variety of methods. In addition to find out the feature combination domains of APN protein, we would like to obtain some target genes or proteins to develop the new and effective candidate vaccines and breeding some dominant species of pigs resistant against F4 infections. We also would like to investigate the pathogenic mechanisms and signaling pathways involved in the infections of three serotypes, and depending on these, we want to eliminate and control the diarrhea of piglets.A polymorphism mutation sites in the mucin 4 intron 7 has been linked to an importance percentage of F4 ETEC adhensive phenotype. We use the polymorphism of MUC4 intron 7 for primary selection of piglets from different breeds. After we checked the result of bacterial adhesion assay with brush border cells, we select our experimental target piglets and get the full length of APN gene from PCR by using the extracted jejunal RNA. We also got the polyclonal antiserum against APN after we expressed and purified APN protein in vitro. Then, we contructed the recombinant E. coli SE5000 strains carrying the fae operon gene clusters and three isogenic in-frame faeG gene deletion mutants, and find out the difference among them in the adherence and inhibition assay by compared with wild-type strains. Meanwhile, based on the IPEC-J2 cells, the stable transfected cell lines, including pEC129-APN IPEC-J2 cells and pcDNATM6.2-GW/miR-APN IPEC-J2 cells were cultured. Through protein biological activities assay, cell immunohistochemistry assay and some other experimental methods, we characterized the difference between the expressed APN proteins with the extracted APN proteins. Further, positive results from Y2H and pulldown assays both showed that there are a direct interaction between APN and FaeG proteins. In addition, we also make a series of experimental plans to explore whether modulating APN expression in IPEC-J2 cells could affect ETEC adherence, which sites are the functional binding domains of APN-FaeG proteins and the differences between the APN proteins with F4 three serotypes. Considering APN protein is a zinc-dependent exo-peptidase protein, we also would like to know the change of biological mechanism and the related signaling pathways under zinc ion treatement. Our studies can be divided into six parts in the following:1. Preliminary classification of the receptor for Enterotoxigenic Escherichia coli F4The receptor locus for ETEC F4 has been refined to a region on pig chromosome SSC13q41 area and closely related to MUC4 gene. According to the polymorphism mutation sites of MUC4 intron 7 with the Xba I restriction endonuclease, three adhesion phenotypes could be classified as susceptible homozygous SS, susceptible heterozygotes SR and resistance type RR. In this study, this biomarker feature of MUC4 gene polymorphism would be used for preliminary genotyping and in combination with adhesive typing results to select the experimental piglet samples. The piglet samples, Changbai 202 and Meishan 2877, from susceptible homozygote and susceptible heterozygote, were chosen for subsequent experiments. They both show a strong bacterial adhesion with the intestinal brash border cells.2. Cloning, expression systems construction and identification for Porcine Aminopeptidase N geneTo investigate APN as a candidate protein receptor for Enterotoxigenic Escherichia coli F4, we designed specific primers according to the GenBank data sequence on the porcine APN mRNA. The first-strand cDNA was synthesized from total RNA which was extracted from fresh Meishan susceptible piglet’s intestinal tissue. We got the full length of porcine APN gene depending on RT-PCR amplification and constructed both the eukaryotic expression and prokaryotic expression systems respectively. Recombinant plasmid sequencing and restriction enzyme digestion results revealed APN gene has been inserted correctly into both eukaryotic expression vector pcDNA3.1 and prokaryotic expression vector pET-28a(+), SDS-PAGE profile showed the recombinant expressing protein was inclusion body proteins with a single clear band of a relative molecular weight 110 KDa after purification. Construction of pcDNA3.1-APN and pET28a-APN expression systems for Porcine Aminopeptidase N gene and the APN protein expressed successfully laid the foundation and platform for further study about porcine APN as a candidate protein receptor for ETEC F4.3. FaeG directly involved in binding between F4+Enterotoxigenic Escherichia coli (ETEC) and porcine Aminopeptidase N (APN)The most prominent part of fimbriae is the FaeG subunit. This subunit was associated with glycoprotein or glycolipid receptor recognition and contributed to pathogen attachment to the host cells. In this study, we constructed the recombinant strain with the fae operon gene clusters and its △faeG mutants and sought to find out the interaction between APN and F4 fimbriae. The occurrence of agglutination between the purified APN protein and F4+ETEC strains suggested this protein can recognize and bind to the fimbriae of all three serotypes. The weak reaction of the isogenic AfaeG mutants with the APN protein and the direct interaction between the purified APN protein with the FaeG protein in the Y2H and pull-down assays conclude that the fimbrial FaeG subunit is the efficient part for adherence and act as the target site for APN protein as well.4. Influence of Porcine Aminopeptidase N on the adherent ability of F4+ Enterotoxigenic Escherichia coliIt was reported that APN promotes the endocytosis of F4ac fimbriae and enhances the mucosal immune response in intestinal epithelial cells. We found that APN protein have a direct interaction with the major FaeG subunit of F4 fimbriae. In this study, we used the stable transfected cell lines pcDNATM6.2-GW/miR-APN IPEC-J2/Caco-2 and pEC129-APN IPEC-J2/Caco-2 to detect the function of APN on the adherence with F4. The protype cell lines used as a control and the result demonstrate that RNAi interference significantly reduced the expression of APN, the expression of APN in pEC129-APN cells was higher than other two kinds of cells and modulating APN gene expression positively affected F4 bacterial adherence with the host cells.5. Research on the functional binding domain of porcine Aminopetidase NIn our previous study, we found a direct interaction between APN and FaeG subunit proteins exist, and modulating APN protein expressions in different cells tested have an effect on their adherence with bacteria. To verify the function of APN and its biological mechanism, we used overlapping peptide assays to map binding domains of APN-FaeG (F4ab/F4ac/F4ad) interactions. Combined with Pull-down, Western blot and other testing results, we eventually determine the functional binding domains for APN. Compared the APN-FaeG binding domain of three variants, the region of 148-160,199-217 aa for F4ab FaeG,149-161,176-188,200-218 aa for F4ac FaeG and 149-161,200-218 aa for F4ad FaeG are the main functional domains.6. The biological mechanism of porcine Aminopetidase NZinc ion (Zn2+) is an important trace metal elements for porcine growth, the lack of Zn2+ often leads to retarded growth of piglets. The high zinc dietary is not only conducive to the growth of piglets, but also efficient to reduce diarrhea. APN is a membrane-bound protein containing Zn2+ dependent exopeptidase, and also have a zinc activated site. In our previous study, we found that the higher concentration of Zn2+ decreased the biological activity of APN protein. Besides, the MAPK signaling pathways was reported to be involved with the change of APN gene expression and Zn2+ concentrations in the host. Thus, in this study, we focused on the biological mechanism of APN and Zn2+ in the progress of F4 E. coli induced diarrhea, and found that the concentrations of Zn2+ inhibit the adherence between F4 E.coli and the host cells, and also have effect on the phosphorylation of ERK, p38, JNK proteins and the expression of cytokines in cells.