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Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.

ABSTRACT: F4(+) enterotoxigenic Escherichia coli (ETEC) strains cause diarrheal disease in neonatal and post-weaned piglets. Several different host receptors for F4 fimbriae have been described, with porcine aminopeptidase N (APN) reported most recently. The FaeG subunit is essential for the binding of the three F4 variants to host cells. Here we show in both yeast two-hybrid and pulldown assays that APN binds directly to FaeG, the major subunit of F4 fimbriae, from three serotypes of F4(+) ETEC. Modulating APN gene expression in IPEC-J2 cells affected ETEC adherence. Antibodies raised against APN or F4 fimbriae both reduced ETEC adherence. Thus, APN mediates the attachment of F4(+) E. coli to intestinal epithelial cells.

SUBMITTER: Xia P

PROVIDER: S-EPMC4746772 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.

Xia Pengpeng P Wang Yiting Y Zhu Congrui C Zou Yajie Y Yang Ying Y Liu Wei W Hardwidge Philip R PR Zhu Guoqiang G

Veterinary research 20160209

F4(+) enterotoxigenic Escherichia coli (ETEC) strains cause diarrheal disease in neonatal and post-weaned piglets. Several different host receptors for F4 fimbriae have been described, with porcine aminopeptidase N (APN) reported most recently. The FaeG subunit is essential for the binding of the three F4 variants to host cells. Here we show in both yeast two-hybrid and pulldown assays that APN binds directly to FaeG, the major subunit of F4 fimbriae, from three serotypes of F4(+) ETEC. Modulati ...[more]

PMID: 26857562

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Project description:Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO(3)-3Galß1Cer), sulf-lactosylceramide (SO(3)-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).

Project description:BackgroundEnterotoxigenic Escherichia coli (ETEC) F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses. Although Bacillus licheniformis (B. licheniformis) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of B. licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line (IPEC-J2) when stimulated with ETEC F4.MethodsIn the present study, the effects of B. licheniformis PF9 on the release of pro-inflammation cytokines, cell integrity and nuclear factor-κB (NF-κB) activation were evaluated in ETEC F4-induced IPEC-J2 cells.ResultsB. licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-6 during ETEC F4 infection. Furthermore, the gene expression of Toll-like receptor 4 (TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited. These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B. licheniformis PF9 treatment. The immunofluorescence and western blotting analysis revealed that B. licheniformis PF9 increased the expression levels of zona occludens 1 (ZO-1) and occludin (OCLN) in ETEC F4-infected IPEC-J2 cells. Meanwhile, the B. licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance (TEER) and cell permeability assay. Interestingly, B. licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors (including luxS, estA, estB, and elt) in ETEC F4.ConclusionsCollectively, our results suggest that B. licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways. Besides, B. licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.

Dataset Information

Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.

Publications

Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.

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