Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (K d ϭ 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of Յ5.2. CNF1 is the first bacterial toxin described that uses both a clathrinindependent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol. INTRODUCTIONBacterial protein toxins are among the most powerful virulence factors produced by pathogenic microorganisms. In general, these toxins are divided into three groups according to their mechanism of action (Boquet and Gill, 1991). Group I toxins such as the heat-stable Escherichia coli enterotoxin (reviewed by Sears and Kaper, 1996) act on cell surface where they induce transmembrane signaling. Group II toxins or "pore-forming toxins" (e.g., perfringolysin O, Staphylococcus aureus alpha toxin, and aerolysin [reviewed by Bhakdi et al., 1996]) act by disrupting the integrity of the plasma membrane. Finally, group III toxins, such as diphtheria, cholera, or tetanus toxins, transfer an enzymatically active domain into the cytosol and modify a eukaryotic target, which gives rise to toxicity (reviewed by Montecucco et al., 1994). The latter group is clearly the most potent, because it is believed that one or a few copies of the toxin enzymatic domain, for instance of the diphtheria toxin (DT) fragment A, introduced into the cytosol can kill a cell within 2 d (Yamaizumi et al., 1978). Group III toxins are also known as A-B toxins on the basis of their structure-function relationships, which involve a catalytic domain (the A subunit) and cell binding and membrane translocation domains (the B subunit).Translocation of the catalytic domain of the type III toxins into the eukaryotic cytosol appears to be accomplished by either one or two mechanisms (reviewed by Johannes and Goud, 1998;Lord and Roberts, 1998;Montecucco, 1998). In the first instance, for which the paradigm is DT (Sandvig and Olsnes, 1991), the toxin first binds to its cell surface and is ...

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.