Porcine Milk Fat Globule Membrane Proteins that Bind to F4ac Fimbriae and prevent attachment of Escherichia coli to enterocytes

Abstract

Post-weaning diarrhea (PWD) in pigs is caused by enterotoxigenic Escherichia coli (ETEC) which is often controlled by antibiotic feed supplements with the potential risk of generation of antibiotic resistant bacteria. Prevention of attachment of F4ac positive ETEC to intestinal mucosa may potentially be used as one of the antibiotic-free strategies for the control of this disease. F4ac-positive ETEC has been reported to bind to fat globule membranes in porcine milk. In this study, we used an affinity chromatography technique to identify individual milk fat globule membrane (MFGM) proteins that bind to F4ac fimbriae. An affinity column with covalently coupled F4ac fimbriae to activated Sepharose was created and the following F4ac-binding proteins were isolated from the porcine MFGM: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, fatty acid binding protein 3, and xanthine dehydrogenase. Selected individual proteins of porcine MFGM, namely: xanthine dehydrogenase, butyrophilin, lactadherin, and fatty acid binding protein were isolated and tested for their inhibitory effects against attachment of F4ac positive ETEC and of F4ac fimbriae to either a small intestinal cell line (IPEC-J2) or primary porcine enterocytes by competitive ELISA. All of these proteins, except xanthine dehydrogenase, decreased attachment of F4ac positive E. coli and F4ac fimbriae to the intestinal cell line or primary enterocytes in a dose-dependent manner. Lactadherin demonstrated the strongest inhibitory effect on F4ac positive E. coli adherence to porcine enterocytes and the most potent interaction with F4ac fimbriae.