Role of toxin functional domains in anthrax pathogenesis

Abstract

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live B. anthracis was used to follow the in vivo interaction of LF and PA. We found that the binding of LF to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.

FIG. 1

Schematic representation of the mutagenesis of PA. PA (residues 1 to 735) is shown without its leader peptide. The four folding domains, according to the crystal structure, are represented by rectangles and are numbered (24). The positions of short in-frame deletions (Δ) and of the stop codon (∗) are indicated.

FIG. 2

Schematic representation of the production of the B. anthracis RPL686 mutant. (A) The integrative plasmid, pBF686, carrying an erythromycin resistance cassette (erm) and the lef686 toxin gene, was integrated into the pXO1 of the RPL strain. This strain has a lef deletion and a spectinomycin resistance cassette (spc) insertion (Table 1). (B) The resulting heterodiploid clone was grown in the absence of antibiotic selection pressure. We screened for a second crossover resulting in either the expected allelic exchange (B to C) or a reversion event (B to A). The black square indicates the point mutation (H686→A) in lef686.

FIG. 3

In vitro characterization of B. anthracis recombinant strains. The B. anthracis recombinants were grown in R medium for toxin production. Supernatants from 500 μl of culture were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with PA-, LF-, and EF-specific monoclonal antibodies. MW, molecular weight in thousands.

FIG. 4

Edema formation induced by B. anthracis mutants in mice footpads. Groups of five mice were injected in the right footpad with 5 × 10⁸ spores from various B. anthracis strains. Strains: 7702 (×), RPL200 (○), RPL686 (▴), RPE (□), RPE346 (●), RPLC2 (■), and RPA705 (▵). Swelling was monitored at intervals by measuring, three times for each mouse, the thickness of the infected footpad relative to the contralateral footpad.

FIG. 5

Cleavage of wild-type and mutant PA proteins in serum. PA proteins (100 ng per sample) were incubated for 60 min at 37°C with (+) or without (−) mouse serum. The samples were then subjected to electrophoresis, and the PA proteins were detected by Western blotting by using polyclonal antibodies specific for PA.