Ronggai Sun

Document Type

Thesis - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School



Strains of enterotoxigenic Escherichia coli that express K88 fimbriae are among the most common causes of diarrhea in young pigs. Adhesion of bacteria to receptors on intestinal epithelial cells, mediated by K88 fimbriae, is the initial step in the establishment of infection. Three antigenic variants ofK88 fimbriae exist in nature: K88ab, K88ac, and K88ad. K88ac is the most prevalent, and may be the only variant of significance in swine disease. Each K88 fimbrial variant is composed of multiple antigenic determinants. Some of these determinants are shared among the three variants, and are referred to as conserved epitopes, whereas others are unique to a specific variant, and are referred to as variable epitopes. The objective ofthis study was to characterize the receptor-binding domain of K88ac fimbria of E. coli and identify individual amino acids that contribute to the receptor binding specificity of that fimbria. Monoclonal antibodies (MAbs) specific to either variable or conserved epitopes of K88ac fimbriae were produced. The specificity of each MAb was tested by ELISA and immunoblot. Fab fragments were prepared from these MAbs, and used to characterize the receptor binding domain of the fimbria by testing their ability to block the binding ofK88-positive bacteria and purified fimbriae to brush border vesicles prepared from porcine intestinal epithelial cells and purified K88 receptors, respectively. The purified receptors were intestinal mucin-type sialoglycoproteins (IMTGP) isolated from porcine enterocytes (A. K. Erickson, D.R. Baker, B. T. Bosworth, T. A. Casey, D. A. Benfield, and D. H. Francis. 1994. Infect. Immun. 62:5404-5410). Fab fragments prepared from MAbs specific for variable epitopes blocked the binding of bacteria to brush borders and fimbriae to IMTGP. However, those from MAbs specific for a conserved epitope did not. These observations indicate that the receptor binding domain of the K88ac fimbria is contained, at least in part, within the antigenically variable epitopes of that fimbria. It further supports the proposition that the pig intestinal glycoprotein IMTGP, which currently is under investigation, is a biologically relevant receptor for the K88ac variant. Epitope mapping for one of the K88ac-specific MAb, which is to a linear epitope, indicated that this MAb binds to a peptide stretch on the major fimbrial subunit of K88ac from amino acid No. 64 to amino acid No.107, suggesting that this variable region is involved in receptor binding. It contains two amino acids, Thr-74 and Val-94, which are unique to K88ac fimbria. Substitution by site-directed mutagenesis of either of the unique amino acids, Thr-74 or Val-94, in the identified receptor binding region of the K88ac fimbria, with the corresponding amino acids of the K88ad fimbria, Val-74 and Glu-94, respectively resulted in loss of the ability ofK88ac to bind to receptors on pig intestinal brush borders. It also resulted in loss of recognition by K88ac-specific neutralizing MAb. This suggests that both amino acids, Thr-74 and Val-94, are critical in forming the receptor-binding domain on the major fimbrial subunit of K88ac, and in determining the binding specificity for the K88ac fimbria. However, the substitution of these two amino acids with corresponding amino acids of the K88ad fimbria did not result in a switch in the receptor binding specificity from that of K88ac to that of K88ad. This suggests that amino acids at these two positions are each insufficient in detenning the binding specificity ofK88. Secondary structure prediction for the modified funbrial proteins indicated that substitution of either Thr-7 4 with Val-74 or Val-94 with Glu-94 altered the beta-sheet structure of the K88ac fimbrial protein. This structural change may have interrupted the receptor binding domain of K88ac fimbria, and resulted in the failure of the modified fimbriae to bind to porcine intestinal brush borders.

Library of Congress Subject Headings

Escherichia coli infections in swine
Intestines -- Infections
Bacteria -- Adhesion



Number of Pages



South Dakota State University