The bacterial toxin aerolysin kills cells by forming heptameric channels, of unknown structure, in the plasma membrane. Using disulfide trapping and cysteine scanning mutagenesis coupled to thiol-specific labeling on lipid bilayers, we identify a loop that lines the channel. This loop has an alternating pattern of charged and uncharged residues, suggesting that the transmembrane region has a b-barrel configuration, as observed for Staphylococcal a-toxin. Surprisingly, we found that the turn of the b-hairpin is composed of a stretch of five hydrophobic residues. We show that this hydrophobic turn drives membrane insertion of the developing channel and propose that, once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion. This rivet-like conformation was modeled and sequence alignments suggest that such channel riveting may operate for many other pore-forming toxins.

Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells. They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm. As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane. Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis. In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, i.e. it contains hydrophobic and amphipathic alpha-helices long enough to span a membrane. To render this 'membrane protein' water-soluble during the initial part of its life the hydrophobic helices are sheltered from the solvent by a barrel of amphipathic helices. In the second group of toxins the opposite strategy is adopted. The toxin is an intrinsically soluble protein and is composed mainly of beta-structure. These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic beta-sheet to generate sufficient hydrophobicity for membrane insertion to occur. Toxins from this latter group are thought to perforate the lipid bilayer as a beta-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal alpha-toxin. The two groups of toxins will be described in detail through the presentation of examples. Particular attention will be given to the beta-structure toxins, since four new structures have been solved over the past year: the staphyloccocal alpha-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin. Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed. Peptide toxins will not be covered in this review.

No abstract

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB 5 (the B subunit pentamer of cholera toxin) is investigated. When CtxB 5 was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzymelinked immunosorbent assay (t1 ⁄2 ϳ 8 min) and to migrate as SDS-stable pentamers (t1 ⁄2 ϳ 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of ϳ8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB 5 reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.Cholera toxin (Ctx) 1 and heat-labile enterotoxin (Etx) are the primary virulence factors produced by Vibrio cholerae and certain toxinogenic strains of Escherichia coli, respectively (1-3). Both toxins are heterooligomeric proteins comprising an A-subunit that exhibits ADP-ribosyltransferase activity and five Bsubunits that bind with high affinity to the glycolipid receptor, monosialoganglioside GM1, found in the plasma membranes of mammalian cells (4 -6). The B pentamer components of both cholera toxin (CtxB 5 ) and E. coli heat-labile enterotoxin (EtxB 5 ) are widely thought of as carrier molecules principally involved in delivering the toxin A-subunit into cells (3). However, more recent studies have revealed that these receptor binding moieties possess striking immunomodulatory properties that can down-regulate inflammatory immune reactions (7-9). Such findings have prompted renewed interest in the B-subunit pentamers and led to their testing as a potential therapeutic agents for the treatment of inflammatory allergic and autoimmune disorders (10 -13).Assembly of Ctx and Etx into AB 5 complexes occurs in the periplasmic compartment of the bacterial cell envelope (14 -16). Expression of either CtxB or EtxB in the absence of their corresponding A-subunits results in the formation of highly stable B-subunit pentamers that are devoid of enterotoxic activity. The in vivo pathway of B-subunit pentamerization is poorly understood, chiefly because of the difficulty of investigating such processes in the complex environment of the periplasmic space (17, 18). The use of in vitro conditions to study the disassembly and reassembly of the toxins was first reported by Finkelstein et ...