Dominant-Negative Mutants of a Toxin Subunit: An Approach to Therapy of Anthrax

Abstract

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 ...

Immunogenicity of Bacillus anthracis Protective Antigen Domains and Efficacy of Elicited Antibody Responses Depend on Host Genetic Background

“…This peptide segment includes residues in and near a solvent-exposed loop of domain 4 (aa 679 to 693) that play a critical role in receptor binding (46) and that are recognized by several neutralizing MAbs to PA (22). Furthermore, previous studies have shown that mutations in PA, including a deletion of the 2␤2-2␤3 loop (27) and point mutations in any of the three residues K397, D425, and F427, which confer a dominant negative phenotype (38), resulted in impaired pore formation and impaired the translocation of EF and LF across the endosomal membrane.…”

Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

“…A polyvalent peptide inhibitor that binds to the heptameric PA 63 prepore and prevents the interaction between cell-binding and enzymatic moieties has been reported (14). Dominant-negative mutants of protective antigen that co-assemble with the wildtype PA 63 protein and block its ability to translocate the LF and EF across membranes have also been developed (15). Here we propose another way to prevent LF and EF translocation inside the cell that involves blocking the PA 63 channel with specially designed low molecular weight compounds.…”

Efficient synthetic inhibitors of anthrax lethal factor

“…Inside the cell compartment, LF cleaves mitogen-activated protein kinase kinases (MAPKK) (7)(8)(9), disrupts signal transduction, and finally leads to macrophage lysis through a mechanism that is not completely understood to date (10). Accordingly, inhibition of LF is the most promising means for treating postexposure anthrax (11,12).…”