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Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

ABSTRACT: Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding.

SUBMITTER: Moonens K

PROVIDER: S-EPMC4337312 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

Moonens Kristof K Van den Broeck Imke I Okello Emmanuel E Pardon Els E De Kerpel Maia M Remaut Han H De Greve Henri H

Veterinary research 20150224

Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distan ...[more]

PMID: 25828907

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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:Fimbriae mediate the initial adherence of enterotoxigenic Escherichia coli (ETEC) to the piglet small intestine and play an important role in development of ETEC-driven postweaning diarrhea (PWD). PWD inflicts huge economic losses on the swine industry each year, making development of alternative treatment and prevention measures for PWD essential. Vaccine candidates that induce antifimbria antibodies that block the initial attachment and colonization of ETEC pathogens with fimbriae are one approach that could help prevent PWD. In this study, we constructed two multiepitope fusion antigens (MEFAs) that carried, expressed, and displayed representative epitopes of F4, F5, F6, F18, and F41 ETEC fimbriae. These MEFAs used either the F4 major subunit FaeG or the F18 adhesive subunit FedF as a backbone. To assess the potential of these MEFAs as antifimbria vaccine candidates that could help prevent PWD, we generated computational models of the MEFAs, constructed them, and then tested their immunogenicity by using them to immunize mice. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface. We found that coadministration of our MEFAs in mice successfully induced five fimbria-specific antibodies in accordance with the epitopes included in the MEFA constructs. Furthermore, the induced antibodies can significantly inhibit the ability of ETEC strains that express F4, F5, F6, F18, and F41 fimbriae to adhere to piglet small intestinal IPEC-1 and IPEC-J2 cells. Our findings indicate that the antifimbria antibodies induced by our FaeG-Fim41a-FanC-FasA and FedF-FasA-Fim41a-FanC fimbria MEFAs blocked adherence of five ETEC fimbriae, suggesting these multivalent fimbria MEFAs may be useful for developing broadly protective antifimbria vaccines against PWD caused by ETEC infections.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC)-associated postweaning diarrhea (PWD) is still a leading disease in recently weaned piglets. Vaccination is considered to be the most ideal and efficacious strategy for preventing PWD. Recently, a commercialized live monovalent F4 oral vaccine and a bivalent F4/F18 oral vaccine have been demonstrated to effectively protect piglets in the F4-positive (F4+) and F18+ ETEC challenge models. However, they will not provide cross-protection against F5+, F6+, or F41+ ETEC-associated PWD cases, as they lack all five fimbria antigens. Thus, a multivalent vaccine containing all five ETEC fimbriae would be more effective in preventing ETEC-driven PWD. In this study, we designed two fimbria-targeted MEFAs using the MEFA technology, and further study demonstrated that these coadministered MEFAs in mice can induce protective antibodies against the five fimbriae expressed by ETEC. These MEFAs could be used as an efficient PWD vaccine candidate; furthermore, MEFA-based structural technology provides an alternative and promising strategy for the development of vaccines against pathogens with heterogeneous virulence factors.

Dataset Information

Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

Publications

Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

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