Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic Escherichia coli K88ac+ Fimbriae

Abstract

An affinity chromatography technique was utilized to isolate and purify the receptors of Escherichia coli K88ac؉ fimbriae from the mucus of the small intestines of newborn piglets. Purified K88ac؉ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B). The immobilized fimbriae were used for the affinity purification of the K88ac؉ receptors. Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound receptor was washed exhaustively with a buffer containing a high concentration of salt and a detergent. The receptors were eluted as a single component at a low pH. The isolated proteins were then subjected to enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot (immunoblot) analyses. The two proteins were of high purity, were responsible for nearly all of the fimbrial binding capacity of the crude mucus, and had molecular masses of 26 and 41 kDa. The method for isolation of E. coli binding proteins is simple and yields purified intestinal receptors in a single chromatographic run. The intestinal mucus of different piglets has different proportions of the two receptor proteins.Enterotoxigenic Escherichia coli (ETEC) strains bearing K88 fimbriae can specifically adhere to receptors in the mucus of the small intestines of piglets, causing diarrhea (14, 15). A layer of mucus covers the epithelial cells in the mammalian small intestine, is secreted by specialized goblet cells, and contains receptors that recognize specific adhesion proteins (2, 5, 15, 16). K88 fimbriae are filamentous surface appendages that enable ETEC to bind to the receptor in the mucus layer of the small intestine. This binding prevents the removal of the bacteria by intestinal peristalsis and is a prerequisite for virulence (6,10,18,19,20). Neonatal piglets are extremely sensitive to infection by ETEC but can be protected by specific anti-K88 egg yolk antibodies (11,14,17). Piglets are commonly infected by E. coli strains expressing several serological types of the K88 fimbrial antigens. The K88 fimbrial antigens are classified into three sets designated K88ab, K88ac, and K88ad. Each variant shares a common antigen (a) and expresses one of three variant-specific antigens (b, c, or d, respectively) (6, 7, 9, 12). It is also well documented that genetics play a significant role in the susceptibility of piglets to infectious diarrhea. Piglets are resistant to infection if they are genetically defective in their ability to express K88-specific brush border receptors (9, 18); furthermore, the presence of K88-specific receptors in porcine ileal mucus is age dependent (4, 5). The receptors of porcine small intestine mucus that bind to K88ab fimbriae have been isolated and identified by using gel filtration chromatography and 3 Hlabeled E. coli (15), while the K88ac fimbrial receptors of porcine intestinal brush border have been identified using by 35 SO 4 -labeled E. coli (3, 6). However, little information is av...

Potential probioticLactobacillus strains for piglets from an arid coast

“…These results showed the highest percentage of adhesive strains in the caecum and intestine compared with faeces. Pathogens as Escherichia coli that inhabit the gastrointestinal tract of pigs present surface proteins that recognise in a specific manner receptors in the mucus (Fang et al, 2000). Moreover these results support the previously observed by Namba et al (2007) who reported that intestinal bacteria which colonise the surface of intestinal mucosa, showed a high-adhesive capability to fish intestinal mucus.…”

Inhibition of Adhesion ofEscherichia coliK88ac Fimbria to Its Receptor, Intestinal Mucin-Type Glycoproteins, by a Monoclonal Antibody Directed against a Variable Domain of the Fimbria

“…In addition, the receptor to which it binds must be somewhere other than in brush borders, because MAb 36/41 blocks the binding of K88ac to porcine enterocyte brush borders of phenotypes A and B, the only brush borders to which K88ac binds (1). It has been suggested by other investigators that the mucus covering the intestinal epithelium contains glycoproteins to which K88 fimbriae bind and that these glycoproteins may serve as receptors for the adherence of bacteria to that mucus (6,10). The mucus glycoproteins identified were different from the receptors previously identified on porcine enterocyte brush border membranes (10).…”

“…It has been suggested by other investigators that the mucus covering the intestinal epithelium contains glycoproteins to which K88 fimbriae bind and that these glycoproteins may serve as receptors for the adherence of bacteria to that mucus (6,10). The mucus glycoproteins identified were different from the receptors previously identified on porcine enterocyte brush border membranes (10). If the fimbrial tip subunit is adhesive, it may bind to mucus glycoproteins or perhaps to receptors found on the epithelium in another part of the gastrointestinal tract or in an alternative host.…”

Intestinal Adherence of Vibrio cholerae Involves a Coordinated Interaction between Colonization Factor GbpA and Mucin

“…The intestinal mucus secretion assay was performed by a previously described method (15), with modifications. Briefly, adult BALB/c mice that were kept without food for 24 h were orally incubated with 1 ml of overnight cultures of V. cholerae N16961, N1RB3, or N1RC2.…”