Related Experiment Videos
Recovery of intestinal membrane binding sites for K88 E. coli from pig mucosal organ cultures
Abstract:
Putative receptors for K88 + E. coli from piglet intestinal epithelium were released into the organ culture medium and were demonstrated by direct binding with K88 + E. coli through the utilization of an in vitro binding procedure or by immunoprecipitation with K88 antigen. Incorporation of 14C-glucosamine by newborn to day old and 3-week to 6-week old piglet jejunal and ileal mucosa, in organ culture, occurred throughout the 24 hr culture period. Uptake in both age groups and both areas of the intestine was similar with a somewhat greater incorporation by the older age group. Secretion of 14C-glucosamine-labeled components into the culture medium was demonstrated by gel filtration of the concentrated medium. Some large molecular weight components eluted in the void volume in excess of 2 X 10(6) daltons. A second peak of activity was spread from approximately 690K to 25K daltons. All eluted fractions demonstrated binding to K88 + E. coli. Antibodies to purified brush borders from susceptible pigs produced prominent precipitation bands following double diffusion with concentrated organ culture media which confirmed that the organ culture media contained labeled proteins of brush border origin. Immunoprecipitation of the intestinal mucosal organ culture media with K88 + pili and pilus antisera, followed by electrophoresis with SDS and reduced conditions, demonstrated a subunit of approximately 35K daltons.
Insights
Researchers identified K88+ E. coli receptors on piglet intestinal epithelium. These receptors, secreted into culture medium, bind to K88+ E. coli, revealing potential targets for controlling bacterial infections.
Area of Science:
- Microbiology
- Biochemistry
- Animal Science
Background:
- Enterotoxigenic Escherichia coli (K88+ E. coli) causes significant disease in piglets.
- Understanding the interaction between K88+ E. coli and the piglet intestinal epithelium is crucial for developing effective control strategies.
Purpose of the Study:
- To identify and characterize putative receptors for K88+ E. coli on piglet intestinal epithelium.
- To investigate the secretion and binding properties of these receptors in vitro.
Main Methods:
- Organ culture of piglet jejunal and ileal mucosa with 14C-glucosamine labeling.
- In vitro binding assays and immunoprecipitation to detect receptor-K88+ E. coli interactions.
- Gel filtration and SDS-PAGE to analyze secreted components and identify subunit molecular weights.
Main Results:
- Putative K88+ E. coli receptors were released into the organ culture medium.
- Labeled components, including high molecular weight species and a 35K dalton subunit, were secreted and bound to K88+ E. coli.
- Antibodies to brush borders confirmed the presence of labeled proteins of brush border origin in the medium.
Conclusions:
- Piglet intestinal epithelium secretes components that act as receptors for K88+ E. coli.
- These secreted receptors, potentially originating from brush borders, play a role in K88+ E. coli adhesion.