The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.

Screening peptide libraries is a proven strategy for identifying inhibitors of protein-ligand interactions. Compounds identified in these screens often bind to their targets with low affinities. When the target protein is present at a high density on the surface of cells or other biological surfaces, it is sometimes possible to increase the biological activity of a weakly binding ligand by presenting multiple copies of it on the same molecule. We isolated a peptide from a phage display library that binds weakly to the heptameric cell-binding subunit of anthrax toxin and prevents the interaction between cell-binding and enzymatic moieties. A molecule consisting of multiple copies of this nonnatural peptide, covalently linked to a flexible backbone, prevented assembly of the toxin complex in vitro and blocked toxin action in an animal model. This result demonstrates that protein-protein interactions can be inhibited by a synthetic, polymeric, polyvalent inhibitor in vivo.

The cytoplasmic membrane proteins of bacterial binding protein‐dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA‐‐‐G‐‐‐‐‐‐‐‐‐I‐LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose‐negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane‐bound, as in wild‐type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose‐negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.

The three proteins that comprise anthrax toxin, edema factor (EF), lethal factor (LF), and protective antigen (PA), assemble at the mammalian cell surface into toxic complexes. After binding to its receptor, PA is proteolytically activated, yielding a carboxyl-terminal 63-kDa fragment (PA63) that coordinates assembly of the complexes, promotes their endocytosis, and translocates EF and LF to the cytosol. PA63 spontaneously oligomerizes to form symmetric ring-shaped heptamers that are capable of binding three molecules of EF and͞or LF as competing ligands. To determine whether binding of these ligands depends on oligomerization of PA63, we prepared two oligomerization-deficient forms of this protein, each mutated on a different PA63-PA63 contact face. In solution or when bound to receptors on Chinese hamster ovary K1 cells, neither mutant alone bound ligand, but a mixture of them did. After the two mutants were proteolytically activated and mixed with ligand in solution, a ternary complex was isolated containing one molecule of each protein. Thus EF and LF bind stably only to PA63 dimers or higher order oligomers. These findings are relevant to the kinetics and pathways of assembly of anthrax toxin complexes.

The protective antigen moiety of anthrax toxin translocates the toxin's enzymic moieties to the cytosol of mammalian cells by a mechanism that depends on its ability to heptamerize and insert into membranes. We identified dominantnegative mutants of protective antigen that co-assemble with the wild-type protein and block its ability to translocate the enzymic moieties across membranes. These mutants strongly inhibited toxin action in cell culture and in an animal intoxication model, suggesting that they could be useful in therapy of anthrax.The increase of antibiotic resistance among pathogenic bacteria in recent years has prompted research to identify new approaches to treating bacterial infections. One approach is to develop ways to block the action of virulence factors. Toxic proteins are known to be important factors in many bacterial diseases, in that they are responsible for major symptoms (1), and for some diseases (e.g., diphtheria, tetanus, and pertussis) immunizing against a single toxic protein is known to provide protection against infection. Antibodies against toxins have sometimes been used to treat or prevent toxinrelated diseases, but toxins have generally not been targets for newly developed antibacterial agents. Recently a mutation in a subunit of VacA, a toxin from Helicobacter pylori, was shown to inhibit the action of the toxin in vitro (2). Here we describe mutant forms of a subunit of anthrax toxin that are potent inhibitors of toxin action in vitro and in vivo.Spores of Bacillus anthracis, the etiologic agent of anthrax, infect herbivores most often but can also cause localized or systemic infections in humans (3). The most lethal form of the human disease, inhalational anthrax, is produced when spores in the lungs initiate a systemic infection; death almost inevitably occurs within a few days. Anthrax bacilli produce a set of three proteins, protective antigen (PA; 83 kD), lethal factor (LF; 90 kD), and edema factor (EF; 89 kD) (3), which are known collectively as anthrax toxin (ATx). These proteins are nontoxic individually, but act in binary or ternary combinations to produce shock-like symptoms and death. LF and EF enzymically modify molecular targets within the cytosol, and PA transports them from the mammalian cell surface to that compartment. LF is a Zn 2ϩ -protease that cleaves several mitogen-activated protein kinase kinases, kills macrophages, and causes death of the host (4-6). EF is a calmodulindependent adenylate cyclase that causes edema and impairs neutrophil function (7). After their secretion from B. anthracis as monomeric proteins, PA, LF, and EF undergo self-assembly on the surface of mammalian cells to form toxic cell-bound complexes (Fig. 1). Initially, PA binds its receptor and is activated by furin-related proteases (8, 9). PA 63 (63 kD), the activated form, spontaneously self-associates to form ring-shaped heptamers (10, 11), which bind LF and EF competitively and with high affinity (K d ϳ 1 nM) (12, 13). The resulting cell-associated complexes are endocytosed and ...

Systemic anthrax is caused by unimpeded bacillar replication and toxin secretion. We developed a dually active anthrax vaccine (DAAV) that confers simultaneous protection against both bacilli and toxins. DAAV was constructed by conjugating capsular poly-␥-D-glutamic acid (PGA) to protective antigen (PA), converting the weakly immunogenic PGA to a potent immunogen, and synergistically enhancing the humoral response to PA. PGA-specific antibodies bound to encapsulated bacilli and promoted the killing of bacilli by complement. PA-specific antibodies neutralized toxin activity and protected immunized mice against lethal challenge with anthrax toxin. Thus, DAAV combines both antibacterial and antitoxic components in a single vaccine against anthrax. DAAV introduces a vaccine design that may be widely applicable against infectious diseases and provides additional tools in medicine and biodefense.

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.

Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EF N and LF N , respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LF N had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LF N (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EF N displayed nearly identical defects. Cytotoxicity assays confirmed that the LF N mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.Bacillus anthracis releases three discrete monomeric proteins that assemble at the host cell surface into toxic complexes (1). These proteins are collectively referred to as anthrax toxin (ATx) 1 and include the edema factor (EF) and lethal factor (LF) enzymes and the protective antigen (PA). PA mediates the delivery of EF and LF across the host cell membranes so that they can access their cytosolic substrates. EF is an 89-kDa adenylate cyclase that impairs phagocytosis in macrophages (2). LF is a 90-kDa zinc-dependent protease that cleaves mitogen-activated protein kinase kinases in macrophages (3-5). At high concentrations, the combination of PA and LF can result in death of the host cell macrophages and even the host (6).ATx intoxication involves binding of PA (83 kDa) to a specific mammalian cell-surface receptor and proteolytic activation by furin or a furin-like protease (7). Cleavage results in the release of the N-terminal 20-kDa fragment of PA, and the release in turn allows the receptor-bound 63-kDa domain (PA 63 ) to heptamerize and bind EF and/or LF (8). The complex of heptameric PA 63 ((PA 63 ) 7 ) bound to both receptor(s) and catalytic factor(s) on the cell surface is then internalized by receptor-mediated endocytosis (9). The low pH environment of the endosome is thought to trigger a structural change in (PA 63 ) 7 , allowing it to form a pore and mediate translocation of EF/LF across the endosomal membrane into the cytosol (10). Translocation is also likely to involve unfolding and refolding of the large catalytic moieties before and after traversing the membrane, respectively (11).EF and LF bind to (PA 63 ) 7 competitively (12) and with high affinity (K d ϳ1 nM (13)). These proteins do not share sequence similarity with other proteins in the database but do share significant sequ...