Evidence for clathrin mobilization during directed phagocytosis of Shigella flexneri by HEp2 cells

The enteroinvasive species Shigella flexneri expresses a plasmid-mediated capacity to penetrate epithelial cells by directed phagocytosis involving actin polymerization. In the present work, HEp2 cells were depleted of intracellular K+ in order to arrest receptor-mediated endocytosis and to evaluate the role of this endocytic pathway in the internalization of invasive microorganisms. Such a treatment, which efficiently inhibited diphtheria toxin endocytosis and dissociated clathrin coats of the cells, also totally prevented HEp2 cells internalizing Shigella. K2-depletion only weakly decreased actin polymerization induced by invasive Shigella, and rather increased the duration of this response. Double fluorescence staining of clathrin and filamentous actin in infected HEp2 cells showed accumulations of clathrin labelling underneath the region involved in actin polymerization. Such accumulations of clathrin-labelling could not be detected in K(+)-depleted cells. These results suggest a participation of clathrin in the internalization of S. flexneri into epithelial cells.