The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN-PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA63, the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PA's ability to mediate toxicity at least 100-fold were identified in all four domains of PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2␤6 strand, the 2␤7 strand, and the 2␤10-2␤11 loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2␤6 and 2␤7 strands and the 2␤10-2␤11 loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure-function studies and for designing new antitoxic agents for treatment of anthrax.

Background:The cholesterol-dependent cytolysins (CDCs) undergo a complex set of structural transitions to form the homo-oligomeric pore complex. Results: Structural transitions are propagated between monomers of the oligomeric complex. Conclusion: Specific structural changes establish the geometry of the oligomeric pore complex and promote the completion of existing oligomers. Significance: CDCs use membrane binding and ordered intermolecular interactions to drive assembly of their ␤-barrel pore.

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin belonging to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule based on functional and sequence similarity with aerolysin, for which the crystal structure has been solved. In the present study we have substituted the entire primary structure of AT with alanine or cysteine in order to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain, but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane β-hairpin of the prepore oligomer.Clostridium septicum is a major cause of non-traumatic gas gangrene in humans, a fulminant form of myonecrosis that can progress to a fatal infection in less than 24 hours with reported mortality rates ranging from 67-100% (1-4). Of its many virulence factors, alpha toxin (AT) is the only lethal factor secreted by this organism (5) and it has recently been shown to be absolutely required for virulence of C. septicum (6). AT is classified as a pore-forming toxin and is a member of the aerolysin-like family of pore-forming toxins. AT and aerolysin share a great deal of structural and sequence similarity (72%) (5), which has allowed for the development of a molecular model of AT using the previously solved crystal structure of aerolysin ( Fig. 1) (7,8). Aerolysin is a two lobed protein in which the small lobe is comprised of domain 1 (D1) and the large lobe is comprised of domains 2-4 (D2-D4, Fig. 1). The primary structure of AT exhibits similarity with the large lobe of aerolysin but it lacks the small lobe structure of about 83 amino acids. Both toxins appear to follow a similar ordered path to form pores in cell membranes (5). Following secretion, they bind to receptors on the cell membrane where they are cleaved into their active form by cell surface proteases, usually furin. Once activated, the toxins oligomerize on the cell surface into a prepore complex followed by insertion of a transmembrane β-barrel into the membrane (8).

No abstract