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

Abstract

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.

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

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

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

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.