Characterization of Dominant-Negative Forms of Anthrax Protective Antigen

Ming Yan,R John Collier

doi:10.1007/bf03402106

Abstract

Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped heptameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2beta2-2beta3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of testing in vivo as a new type of antitoxic agent for treatment of anthrax.

Highlights

The major symptoms of anthrax are believed to result from the effects of a tripartite toxin secreted by the anthrax bacillus, Bacillus anthracis
Demonstration that certain mutations within the protective antigen (PA) moiety of anthrax toxin convert the protein into a potent inhibitor of toxin action is relevant both to the mechanism of toxin action and to development of new types of therapeutics for anthrax. The existence of such mutations strongly supports the notion that translocation of edema factor (EF) and lethal factor (LF) is mediated by a multisubunit form of PA that forms a transmembrane pathway for EF and LF to cross to the cytosol
Introduction of a translocation-defective subunit into the heptamer—perhaps even in single copy—is able to interfere with the membrane-penetration and translocation processes, thereby blocking toxin action

Summary

INTRODUCTION

The major symptoms of anthrax are believed to result from the effects of a tripartite toxin secreted by the anthrax bacillus, Bacillus anthracis. It was proposed that for the transmembrane β-barrel to form, the 2 β strands flanking the loop must be stripped out from the body of the domain, allowing the loop to relocate to the base of the structure This implies a major conformational change in the domain. We believe the various dominant-negative mutations in some way block the conformational change in domain 2 required for conversion of the prepore to the pore. These findings raised the possibility that DN-PAs might represent a novel approach to therapy of anthrax. The double, K397D + D425K, mutant was used as the primary template to analyze the effects of additional mutations

MATERIALS AND METHODS

RESULTS

Findings

DISCUSSION