Research On FaeG Subunit Involved In F4~+ ETEC Infection And Receptor Binding

Enterotoxigenic Escherichia coli(ETEC)is an important zoonotic pathogen that causes diarrhea in human and young animals.Neonatal and post-weaned piglets diarrhea(PWD)induced by ETEC causes great economic losses due to high morbidity and high mortality in swine industry.PWD is mainly caused by ETEC expressing F4 or F18 adhesin.ETEC F4 have three serotypes,namely F4ab,F4ac,F4ad,the most common serotype is F4ac.A comparative analysis of the gene cluster between three serotypes showed that the major difference is on the faeG gene,and the FaeG subunit is also the major functional subunit of F4 fimbriae.It was reported that porcine Aminopeptidase N(APN)is a new direct receptor protein for F4 fimbriae.FaeG subunit is not only an effective adhesion subunit,but also a target to study the interplay between F4 fimbriae and APN.Therefore,we used cell and animal infection experiments first to clarify the role of FaeG subunit in bacteria infection.Then we targeted APN-FaeG functional binding domain to map APN-FaeG interplay sites by site-directed mutagenesis in key amino acid sites.In addition to figure out the differences in binding to receptor between three serotypes and the role of FaeG subunit playing in F4ac becoming a dominant serotype.The first part of the study,we try to.further clarify the role of FaeG subunit in activating the MAPK signaling pathway and animal infection experiments.It was reported that there is a direct interaction between APN and FaeG subunit proteins and the transcription of APN gene is closely related to MAPK signaling pathway.Thus,we focused on the activation of the MAPK signaling pathway and found that the faeG deletion mutant weaken the activation of the MAPK signaling pathway when stimulating the 1PEC-J2 cell lines.The result of infection experiments showed that the faeG deletion mutant also weaken the effect of piglets challenge experiments.The second part of the study continues focus on FaeG subunit.In order to further explore the combination differences with receptor protein between three serotypes,we targeted APN-FaeG functional binding domain,and conducted a functional verification of the nine selected peptides.By homology modeling of FaeG protien and the result of molecular docking analysis in APN-FaeG interplay,we could find out key amino acid sites of APN-FaeG interaction and then conducted site-directed mutagenesis in these key sites.The effects of the mutated peptides and FaeG protein were verified by laser confocal microscope analysis and pull-down assay.We eventually determine the functional interaction key amino acid.The result suggest that the amino acids V187 of F4ab,L206,N209,L212 and G213 of F4ac are the APN-bihding critical residues in FaeG.