This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Colonization factors (CF) promote the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes and are also responsible for their agglutination properties with human erythrocytes. In this project we used human A+ blood group erythrocyte membrane glycosphingolipid extract as a model to test the binding activities of CFA/I, CS1, CS2, CS3, CS14, CS17, and CS19 purified from human ETEC strains, as well as recombinant CS6. Using the overlay assay on high performance thin-layer chromatography (HPTLC) silica plates, we examined the CF-recognition pattern and identified a glycosphingolipid recognized by all of the biotinylated CF tested in the A+ blood group neutral glycosphingolipid extract. This glycosphingolipid was purified and analyzed using 1H-NMR, and shown to be a globotriaosylceramide (Gb3). To better understand the recognition specificity of the CF, we tested 11 neutral and acidic glycosphingolipids possessing galactosyl and N-acetylgalactosaminyl residues, and showed that only Lc2, Lc3, Lc4, Gb3, GA2 and GA1 were recognized. Based on the intensity of binding of the CF to Lc2, Lc3, Lc4, GA1 and GA2, we propose a new classification where CFA/I, CS3, CS6 and CS14 are in a low intensity recognition group while CS1, CS2, CS17 and CS19 are in a high intensity recognition group. A 3-dimensional structural interpretation for these affinities was proposed on the basis of results from molecular dynamics simulations.
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