Binding mechanism of K88ab pili produced by enterotoxigenic Escherichia coli

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Virginia Polytechnic Institute and State University

Binding of K88ab pili by brush border membrane and mucus from pig small intestine was characterized by inhibition assay and Western blot. In Western blot, K88ab pili were bound by two major brush border membrane polypeptides with molecular weight of 61,500 and 57,000 in addition to numerous minor polypeptides and a major mucus polypeptide with molecular weight of 27,500. The results from Western blot assays with periodate oxidized and carbamylated brush border membrane and inhibition assay with brush border membrane glycopeptide suggest that amino groups (rather than carbohydrate) present on the protein moiety are a part of the recognition site for K88ab pili of receptor polypeptides in brush border membrane. Differences were obtained in the binding patterns of K88ab pili when brush border membranes were prepared from small intestines obtained from 2-, 21-, and 42-day-old piglets as well as adult hogs. Binding of K88ab pili by mucus polypeptides was greater when prepared from small intestines obtained from 2-day-old piglets than from piglets of other ages and adult hogs.

In inhibition assay, most fractions from sow milk and colostrum inhibited binding of K88ab pili. After gel filtration of colostral whey, fractions which contained IgG, IgA, and IgM produced the strongest inhibition of K88ab binding. Among fractions prepared from cow milk, casein and skim milk significantly inhibited binding of K88ab pili. In Western blot, αs1-casein, immunoglobulin chains, and MFGM polypeptides in sow milk and colostrum were shown to be able to bind K88ab pili. Additionally, αs1-casein was the major protein in bovine milk responsible for binding K88ab pili. In dot blot assay, IgG as well as brush border membrane could strongly bind K88ab pili. However, bovine αs1-casein showed only weak binding of K88ab pili. Binding of K88ab pili to these proteins and brush border membrane was inhibited by carbamylation and by addition of 100 mM D-galactosamine. The results suggest that the K88ab-binding proteins in milk and colostrum compete to bind K88ab pili with the receptors in the brush border membrane and that mechanisms involved in binding of K88ab pili by these proteins is similar to that by brush border membrane.