Epithelial glucosphingolipid expression as a determinant of bacterial adherence and cytokine production

Abstract

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is a structural analog of ceramide that inhibits glucosylation of this molecule and thus of glucosphingolipid (GSL) expression by living cells. In this study, we used PDMP to slow the synthesis of the globoseries of GSLs (globo- GSLs) (derived from the precursor Galα1-4Galβ1-4Glc-ceramide) by cultured human kidney and large intestinal epithelial cells. The aim was to deplete the cells of receptors for P-fimbriated Escherichia coli and to examine the effects on the bacterially induced cytokine response. The mammalian cells (A498, HT-29, and Caco2) were cultured in the presence of PDMP in order to deplete them of GSLs. The cells were then subjected to GSL analysis or used to test bacterial adherence and cytokine production. The globo-GSLs were identified by thin-layer chromatography. Bacterial adherence was quantitated by microscopy, and interleukin-6 secretion was quantitated by the B9 bioassay. The interaction of bacteria with the globo-GSLs was studied by using E. coli strains and recombinant clones expressing P fimbriae. E. coli strains expressing type 1 fimbriae binding to mannose-containing glycoproteins were used as controls. PDMP treatment was found to reduce the content of the globo-GSLs in mammalian cells and the adherence of P- fimbriated E. coli to these cells. In contrast, PDMP treatment had no effect on the adherence of type 1-fimbriated E. coli or their activation of cytokine production by A498 cells. P-fimbriated E. coli elicited an interleukin-6 response in the A498 cells; this response was reduced after treatment with PDMP. The results emphasize the role of GSLs as receptors for P-fimbriated E. coli and for the cytokine response elicited by attaching bacteria.