The p21 Rho-activating Toxin Cytotoxic Necrotizing Factor 1 Is Endocytosed by a Clathrin-independent Mechanism and Enters the Cytosol by an Acidic-dependent Membrane Translocation Step

Abstract

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 ...

Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function

“…Bar≈10 µm. , mediating clathrin-independent endocytosis requiring transient membrane acidification (Contamin et al, 2000).…”

37-kDa Laminin Receptor Precursor Modulates Cytotoxic Necrotizing Factor 1–mediated RhoA Activation and Bacterial Uptake

“…It is described as AB toxins, such as diphtheria, cholera, and tetanus toxins, which are comprised of catalytic domain, cell binding domain, and membrane translocation domain (22). Entry of CNF1 into eukaryotic cells includes binding to a host receptor and internalization via an endocytic mechanism (16). Endocytosed CNF1 is routed to the degradative pathway, i.e.…”

“…Endocytosed CNF1 is routed to the degradative pathway, i.e. fusion with the late endosome compartment and, its catalytic activity is translocated into the cytosol in an acid-dependent manner (16). Previous competition experiments suggested the presence of the receptor for CNF1 and the specific interaction between CNF1 and the cellular receptor.…”

“…However, it is unclear how CNF1 enters the eukaryotic cells and activates Rho GTPases. CNF1 has been suggested to be internalized via receptor-mediated endocytosis upon binding to a cell surface receptor (15,16), but the identity and characteristics of the CNF1 receptor are not known.…”

Cytotoxic Necrotizing Factor 1 Prevents Apoptosis via the Akt/IκB Kinase Pathway: Role of Nuclear Factor-κB and Bcl-2

“…In eukaryotic cells, CNF1 binds to its receptor, reported to be the receptor of laminin , and it is endocytosed and released into the cytoplasm by an acidic-dependent mechanism (Contamin et al, 2000). Once in the cytoplasm, CNF1 exerts its enzymatic activity that is represented by deamidation of a pivotal glutamine residue of the guanosine triphosphate (GTP)-binding proteins Rho, Rac, and Cdc42 (glutamine 63 of Rho or glutamine 61 of Rac and Cdc42), giving rise to a glutamic acid (Flatau et al, 1997;Schmidt et al, 1997;Lerm et al, 1999).…”