Toxin-deficient mutants of Bacillus anthracis are lethal in a murine model for pulmonary anthrax

Abstract

Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD50) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD50s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.

FIG. 1.

Heat sensitivity of UT500 in the lungs of mice at 3 h postinfection when euthanized using CO₂ or Avertin. The data shown are from one experiment (n = 5). Error bars indicate standard deviations.

FIG. 2.

Dissemination of UT500 and toxin mutants in BALB/c mice. Mice were infected with approximately 5 × 10⁴ spores. CFU were detected in lung and spleen at 24 h (A), 48 h (B), and 72 h (C). Each symbol represents data from one mouse. The numbers of deaths at each time point are indicated for mice infected with each strain. The data shown are from two representative experiments (n = 10).

FIG. 3.

Splenic lesions at 18 h after intravenous inoculation with B. anthracis. (A) Parent. (B) LF⁻ mutant. (C) EF⁻ mutant. Note the presence of bacilli in tissues from mice infected with each strain (arrows in panels A and B). Tissues from mice infected with the LF⁻ mutant (B) display more red and white pulp depletion than tissues from mice infected with the parent (A) and EF⁻ (C) strains. In panel C abundant neutrophils are present in red pulp (arrowhead).

FIG. 4.

Microscopic examination of B. anthracis stained in the lung at 18 h after intravenous inoculation. (A) Parent. (B) LF⁻ mutant. (C) Bacterial counts in the lung. Note the presence of numerous bacilli (arrows) for the LF⁻ mutant (B) compared to the parent strain (A). *, differs significantly from all other strains; +, differs significantly from EF⁻, EF⁻ and LF⁻, and PA⁻ mutants.