Role of Decay-Accelerating Factor Domains and Anchorage in Internalization of Dr-FimbriatedEscherichia coli

Abstract

Dr-fimbriatedEscherichia colicapable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr⁺E. coliwas characterized in a cell-cell interaction model. Binding of Dr⁺E. colito the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser¹⁶⁵by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr⁺E. colithrough a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-β-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr⁺E. coliwere morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr⁺E. coliand demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.