This proposal is designed to identify the developmental pattern of intestinal response to shigella toxin. We have recently identified an age-dependent, maturational pattern in the appearance of the glycolipid binding site, globotriaosylceramide (Gb3), for shigella toxin in rabbit small intestine, and have obtained preliminary data which suggest that the secretory effects of the toxin depend upon the appearance of Gb3. The major goals of this proposal are to characterize the developmental regulation of Gb3 and to confirm that Gb3 is the functional receptor in the small intestine. The relative resistence of human neonates to clinical shigellosis could be related to a developmental pattern of the receptors for shigella toxin in the small intestine. These experiments will include purification and quantitation of Gb3 from rabbit small intestine at various ages in order to establish age-dependent, quantitative difference in Gb3 as the basis for variations in functional responsiveness of the intestine to shigella toxin. Glycolipids will be extracted and purified on DEAE-Sephadex chromatography and Florisil or Unisil columns and quantitated by HPLC. The localization and distribution of Gb3 in proximal and distal small intestine, in crypt and villus cells and in subcellular fractions, will be studied at various ages. These experiments will delineate the specific membrane domain of the toxin receptor and will thus explain the observed variations in the response to the toxin at various levels of the small intestine. In order to confirm that Gb3 is the functional receptor involved in the toxin mediated process, Gb3 micelles will be inserted into the refractory intestine of newborn rabbits in order to render them sensitive to the secretory effects of the toxin, and Gb3 liposomes and monoclonal anti-Gb3 antibodies will be used to block the toxin- mediated fluid response in ligated ileal loops. The regulatory mechanisms involved in the developmental patter of Gb3 will be evaluated by measuring specific galactosyltransferase activity at various ages since preliminary data reveal decreased levels of Gb3 with high levels of its precursors in neonates. Hormonal modulation of the regulation of Gb3 synthesis will also be studied. The studies described will elucidate the developmental control and function of Gb3 in rabbits at various ages. These studies may provide new insights into the pathogenesis of shigella toxin mediated diarrhea, a major health problem in both developing and developed countries.
