Role of decay-accelerating factor domains and anchorage in internalizationof Dr-fimbriated Escherichia coli

Dr-fimbriated Escherichia coli capable of invading epithelial cells recogni zes human decay-accelerating factor (DAF) as its cellular receptor. The rol e of extracellular domains and the glycosylphosphatidylinositol anchor of D AF in the process of internalization of Dr(+) E. coli was characterized in a cell-cell interaction model. Binding of Dr(+) E. coli to the short consen sus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was cri tical for internalization to occur. Deletion of short consensus repeat 3 do main or replacement of Ser(165) by Leu in this domain, or the use of a mono clonal antibody to this region abolished internalization. Replacing the gly cosylphosphatidylinositol anchor of DAF with the transmembrane anchor of me mbrane cofactor protein or HLA-B44 resulted in abolition or reduction of in ternalization respectively. Cells expressing glycosylphosphatidylinositol-a nchored DAF but not the transmembrane-anchored DAF internalized Dr(+) E. co li through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-beta-cyclodextrin, a sterol-chelating agent. Elect ron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr(+) E. coli were morphologically distinct between the a nchor variants of DAF. The cells expressing glycosylphosphatidylinositol-an chored DAF contained a single bacterium in tight-fitting vacuoles, while th e cells expressing transmembrane-anchored DAF contained multiple (two or th ree) bacteria in spacious phagosomes. This finding suggests that distinct p ostendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr(+) E . coil and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.