Binding determinants in the interplay between porcine aminopeptidase N and enterotoxigenic Escherichia coli F4 fimbriae

Abstract

The binding of F4+ enterotoxigenic Escherichia coli (ETEC) and the specific receptor on porcine intestinal epithelial cells is the initial step in F4+ ETEC infection. Porcine aminopeptidase N (APN) is a newly discovered receptor for F4 fimbriae that binds directly to FaeG adhesin, which is the major subunit of the F4 fimbriae variants F4ab, F4ac, and F4ad. We used overlapping peptide assays to map the APN-FaeG binding sites, which has facilitated in the identifying the APN-binding amino acids that are located in the same region of FaeG variants, thereby limiting the major binding regions of APN to 13 peptides. To determine the core sequence motif, a panel of FaeG peptides with point mutations and FaeG mutants were constructed. Pull-down and binding reactivity assays using piglet intestines determined that the amino acids G159 of F4ab, N209 and L212 of F4ac, and A200 of F4ad were the critical residues for APN binding of FaeG. We further show using ELISA and confocal microscopy assay that amino acids 553–568, and 652–670 of the APN comprise the linear epitope for FaeG binding in all three F4 fimbriae variants.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0519-9) contains supplementary material, which is available to authorized users.

Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

“…As we know, F4ac infections mostly occur in the piglets with the presence of F4ac receptors, and porcine aminopeptidase N (APN) is a newly found F4ac fimbrial receptor in our and other labs' previous studies (Melkebeek et al 2012;Xia et al 2018a). In the later experiment, we proved that the amino acids 149-161, 176-188, and 200-218 are the determinant epitopes for F4ac FaeG to interact with APN directly, and further determined N209 and L212 are the critical sites of F4ac FaeG in binding to the jejunum of piglets (Xia et al 2018a(Xia et al , 2016. That is to say, FaeG is the target for receptor recognition and can control receptor-mediated binding capacity as well.…”

“…Nine of 25-day-old Landrace and Large White 2-way crossbred Pigs were screened according to our previous studies (Xia et al 2018a , 2015a ). These piglets are susceptible to F4ac + E. coli and randomized into three groups: the control group without ETEC infection, F4ac infected group and F4acΔ faeG infected group.…”

Zinc uptake system ZnuACB is essential for maintaining pathogenic phenotype of F4ac+ enterotoxigenic E. coli (ETEC) under a zinc restricted environment

“…Based on our in vitro results, we can infer that the ZnuACB system contributes in colonizing ETEC C83902 in a zinc-limited gut. Our previous research has shown that FaeG, the major subunit of F4 fimbriae, can bind to the specific porcine aminopeptidase N (APN) receptor and tightly adheres to the porcine cell lines of the IPEC-J2 cell [ 38 ]. This means that FaeG is the most important and direct factor to evaluate the adherence ability for F4 E. coli .…”

Zinc is an important inter-kingdom signal between the host and microbe

“…In our previous study, we observed that APN directly interacted with the major FaeG subunit of F4 fimbriae and affected F4 bacterial adherence to host cells [92]. The binding determinants of the APN-FaeG interaction contain residues important for zinc binding, and in a certain concentration range, the change in Zn 2+ concentrations affects the biological activity of the APN protein as well as the adherence between E. coli F4 and host intestinal epithelial cells [90,93]. In addition to cleaving N-terminal amino acids from small oligopeptides on the apical surface of intestinal epithelial cells, APN participates in regulating the MAPK/ ERK1/2 signalling pathway in monocytes, while its zincbinding site is blocked by inhibitory antibodies [91,94].…”