Structural characteristics of the plasmid-encoded toxin from enteroaggregative Escherichia coli.

The plasmid-encoded toxin (Pet)1 of enteroaggregative Escherichia coli is a serine protease autotransporter of the Enterobacteriaceae (SPATE). Its eukaryotic target is the actin-binding protein α-fodrin (1). When Pet cleaves α-fodrin in the target cell cytosol, the organization of the actin cytoskeleton is disrupted. The loss of actin filament structure then results in cell rounding and detachment from the substratum. In the intestinal epithelium of an intoxicated mammal, these effects compromise the integrity of the epithelial monolayer and produce a potentially life-threatening case of diarrhea (2, 3). Members of the SPATE family exhibit diverse functions, but all SPATEs contain the three characteristic domains of an autotransporter: the amino-terminal signal peptide, the mature protein, and the carboxy-terminal β-barrel structure (4). The signal peptide targets the nascent protein for delivery to the periplasmic space and is proteolytically removed from the toxin at this site. The mature protein (also called the passenger domain) contains a catalytic N-terminal domain that includes the canonical GDSGSPL serine protease motif of the SPATEs, followed by an extensive β-helix structural motif. The movement of the passenger domain through the bacterial outer membrane is mediated by the pore-forming β-barrel structure of the SPATE. Membrane-anchored passenger domains remain covalently linked to β-barrel structure, whereas secreted passenger domains are proteolytically nicked and separated from the extracellular face of the β-barrel. Few studies have considered how the structure of the secreted passenger domain affects host-toxin interactions. In this work we examined how the structure of the Pet passenger domain could influence its entry into the host cell cytosol. The 104 kDa passenger domain of Pet (hereafter simply referred to as Pet) is secreted into the extracellular milieu and enters the target eukaryotic cell by receptor-mediated, clathrin-dependent endocytosis (5). Internalized Pet then moves from the endosomes to the Golgi and from the Golgi to the endoplasmic reticulum (ER) by vesicle traffic (6, 7). Before entering the cytosol, ER-localized Pet associates with the Sec61 translocon pore (6). Sec61 is active in ER-associated degradation (ERAD), a quality control system that recognizes misfolded or misassembled proteins in the ER and exports them to the cytosol for disposal (8). ERAD also facilitates the ER-to-cytosol export of Pet, as mutant cell lines with aberrant ERAD activity display increased resistance to Pet intoxication (6). The structural feature(s) of Pet which engage the ERAD system are unknown. Like Pet, many AB toxins move from the cell surface to the ER and from the ER to the cytosol (9). These AB toxins, which include cholera toxin (CT), pertussis toxin (PT), and ricin, share a common structural organization that consists of an enzymatic A subunit and a cell-binding B subunit. Holotoxin disassembly occurs in the ER, and only the catalytic A subunit enters the cytosol. Thermal instability in the dissociated A chain generates an unfolded toxin conformation that most likely triggers its ERAD-mediated translocation to the cytosol (10-12). An absence or paucity of lysine residues in the toxin A chain protects it from the ubiquitin-dependent proteasomal degradation that usually accompanies ERAD-mediated export to the cytosol (13-15). Pet does not have an AB structural organization, and it enters the cytosol as an intact 104 kDa protein (6). The large size of Pet also distinguishes it from the 20-30 kDa toxin A chains, as does the numerous lysine residues in Pet. Because Pet is structurally distinct from the A chains of other ER-translocating toxins, we predicted that it activates ERAD by a process distinct from the AB toxin mechanism. Our data support this hypothesis and indicate that Pet can promote its ERAD-mediated translocation into the cytosol without the substantial loss of structure observed for some AB-type, ER-translocating toxins.