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. 2015 Apr;79(2):120-8.

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

Predrag Novakovic  1 , Yanyun Y Huang  1 , Betty Lockerbie  1 , Farshid Shahriar  1 , John Kelly  1 , John R Gordon  1 , Dorothy M Middleton  1 , Matthew E Loewen  1 , Beverly A Kidney  1 , Elemir Simko  1
Affiliations

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane

Predrag Novakovic et al. Can J Vet Res. 2015 Apr.

Abstract
in English, French

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.

Les Escherichia coli entérotoxinogénique (ETEC) positif pour F4ac doivent s’attacher à la muqueuse intestinale pour causer la diarrhée chez les porcelets. L’empêchement de l’attachement bactérien à la muqueuse intestinale est le moyen de défense le plus efficace contre la diarrhée induite par les ETEC. Les membranes de globules de gras de lait porcin (MFGM) ont été montré comme étant capable d’inhiber l’attachement des ETEC à la bordure en brosse intestinale; toutefois, les composantes spécifiques des MFGM porcines qui inhibaient l’attachement des ETEC aux entérocytes ne furent pas identifiées. Ainsi, le but de la présente étude était d’identifier les protéines des MFGM liant F4ac par immunobuvardage par superposition et chromatographie d’affinité. Le protéome des MFGM porcine fut caractérisé et les protéines liant F4as suivantes furent détectées par immunobuvardage par superposition et chromatographie d’affinité : lactadhérine, butyrophiline, adipophiline, acyl-CoA synthétase 3, et la protéine 3 liant les acides gras. La fonction biologique de ces protéines ne fut pas étudiée mais il est possible que leur interaction avec les fimbriae F4ac interfère avec l’attachement bactérien et la colonisation.(Traduit par Docteur Serge Messier).

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Figures

Figure 1
Figure 1
One dimensional (1D) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; lanes 1–3) and 1D overlay Western blot using purified F4ac as a probe (lanes 4–6). Porcine milk fat globule membranes (MFGM) proteins (lane 1), bovine serum albumin (BSA; lane 2), and purified porcine lactadherin (lane 3) were subjected to electrophoresis and stained with Coomassie blue. The proteins were transferred to polyvinylidene fluoride (PDVF) membrane and probed with purified F4ac. Using a chemiluminescent method, 4 positive bands (66 kDa, 47 kDa, 44 kDa, and 31 kDa) from porcine MFGM (lane 4) were detected. The negative control BSA (lane 5) did not react, while porcine lactadherin used as a positive control (lane 6) did react with F4ac fimbriae. When F4ac fimbriae were omitted, none of the samples showed signals (lane 7: porcine MFGM; lane 8: BSA; lane 9: porcine lactadherin).
Figure 2
Figure 2
A — Coomassie-stained porcine milk fat globule membranes (MFGM) proteins separated by 2D sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE); B — 2D SDS PAGE overlay Western blot of porcine MFGM proteins interacting with purified F4ac fimbria; C — F4ac-binding MFGM proteins isolated by affinity chromatography, separated by 2D SDS PAGE and visualized by Coomassie staining; and D — F4ac-binding MFGM proteins isolated by affinity chromatography, separated by 1D SDS PAGE and visualized by Coomassie staining. Major MFGM proteins (F4acfimbrial-binding proteins within closed ovals and other protein spots within closed squares) are indicated by number on the 2D SDS-PAGE (A) and these numbered proteins are identified by nanoLC-MS/MS (Table I). Several spots (dashed ovals and squares) were visible by Coomassie stain, but could not be identified by nanoLC-MS/MS. Some of these spots reacted with F4ac (dashed ovals), while others (dashed squares) did not. F4ac-binding MFGM proteins isolated by affinity chromatography and separated by 1D SDS-PAGE (D) were identified by nanoLC-MS/MS as a) xanthine dehydrogenase, b) acyl-CoA synthetase, c) butyrophilin, d) adipophilin, e) lactadherin, and f) fatty acid-binding protein.
Figure 3
Figure 3
F4ac-fimbriae isolated from enterotoxigenic E. coli were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blot using primary rabbit anti-F4ac-polyclonal antibodies and secondary anti-rabbit Cy5 antibodies (lane 1 — ECL Flex Rainbow molecular weight markers; lane 2 and 4 — bovine serum albumin as negative control; lane 3 and 5 — purified F4ac-fimbriae).
Figure 4
Figure 4
Immuno-dot-blot where (A) isolated and purified F4ac fimbriae as positive control; (B) F4ac fimbriae coupled to CNBr-activated Sepharose 4B affinity matrix; and (C) CNBr-activated Sepharose 4B without F4ac fimbriae used as negative control affinity matrix tested with primary rabbit anti-F4ac-polyclonal antibodies and secondary anti-rabbit Cy5 antibodies.
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References

    1. Hampson DJ, Gyles CL. Postweaning Escherichia coli diarrhoea in pigs. In: Gyles CL, editor. Escherichia coli in Domestic Animals and Humans. Wallingford, England: Cab International; 1994. pp. 171–191.
    1. Shahriar F, Ngeleka M, Gordon JR, Simko E. Identification by mass spectroscopy of F4ac-fimbrial-binding proteins in porcine milk and characterization of lactadherin as an inhibitor of F4ac-positive Escherichia coli attachment to intestinal villi in vitro. Dev Comp Immunol. 2006;30:723–734. - PubMed
    1. Vondruskova H, Slamova R, Trckova M, Zraly Z, Pavlik I. Alternatives to antibiotic growth promoters in prevention of diarrhoea in weaned piglets: A review. Vet Med. 2010;55:199–224.
    1. Choi BK, Schifferli DM. Characterization of FasG segments required for 987P fimbria-mediated binding to piglet glycoprotein receptors. Infect Immun. 2001;69:6625–6632. - PMC - PubMed
    1. Atroshi F, Alaviuhkola T, Schildt R, Sandholm M. Fat globule membrane of sow milk as a target for adhesion of K88-positive Escherichia coli. Comp Immunol Microbiol Infect Dis. 1983;6:235–245. - PubMed

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