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

Veterinary Research
Pengpeng XiaGuoqiang Zhu

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.

References

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Apr 8, 2015·Journal of Basic Microbiology·Pengpeng XiaGuoqiang Zhu
Feb 10, 2016·Veterinary Research·Pengpeng XiaGuoqiang Zhu
Nov 2, 2016·Current Protocols in Protein Science·Benjamin Webb, Andrej Sali

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Datasets Mentioned

BETA
KF280271

Methods Mentioned

BETA
PCR
enzyme-linked immunosorbent assay
ELISA
pull-down
confocal microscopy
Confocal
glycosylation
two-hybrid

Software Mentioned

SPSS
X Protein – Protein Docking Web Server
PyMoL1
GraphPad
GRAMM
TotalLab
GraphPad Prism
Modeller

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