Three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF; a calmodulin-dependent adenylate cyclase), compose the lethal (PA+LF) and edema (PA+EF) toxins secreted by Bacillus anthracis. Mutant strains, each deficient in the production of one toxin component, were constructed, and their virulence was then studied. A kanamycin resistance cassette was inserted in each cya (encoding EF) and lef(encoding LF) gene, and the constructs were separately introduced into B. anthracis Sterne on a mobilizable shuttle plasmid. An EF-strain and an LF-strain were then isolated after homologous recombination with the resident toxin-encoding plasmid, pXO1. Spores from these mutants and from a previously constructed PA-mutant were used to inoculate mice, and the lethality and local edema formation were monitored. LF-or PA-mutants were not lethal even at high inocula, whereas the EF-mutant induced lethal infections. This indicates that LF in combination with PA is a key virulence factor required for lethality. Skin edema formation was observed with the LF-mutant, which produces only the combination of PA and EF. However, EF-and LFmutants were significantly less efficient at inducing, respectively, lethality and edema than was the parental Sterne strain. These results suggest that the three toxin components might act synergistically in vivo to cause lethality and edema formation.

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The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components : protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitvo and in vivo.