Discriminating Virulence Mechanisms amongBacillus anthracisStrains by Using a Murine Subcutaneous Infection Model

Abstract

Bacillus anthracis strains harboring virulence plasmid pXO1 that encodes the toxin protein protective antigen (PA), lethal factor, and edema factor and virulence plasmid pXO2 that encodes capsule biosynthetic enzymes exhibit different levels of virulence in certain animal models. In the murine model of pulmonary infection, B. anthracis virulence was capsule dependent but toxin independent. We examined the role of toxins in subcutaneous (s.c.) infections using two different genetically complete (pXO1 ؉ pXO2 ؉ ) strains of B. anthracis, strains Ames and UT500. Similar to findings for the pulmonary model, toxin was not required for infection by the Ames strain, because the 50% lethal dose (LD 50 ) of a PA-deficient (PA ؊ ) Ames mutant was identical to that of the parent Ames strain. However, PA was required for efficient s.c. infection by the UT500 strain, because the s.c. LD 50 of a UT500 PA ؊ mutant was 10,000-fold higher than the LD 50 of the parent UT500 strain. This difference between the Ames strain and the UT500 strain could not be attributed to differences in spore coat properties or the rate of germination, because s.c. inoculation with the capsulated bacillus forms also required toxin synthesis by the UT500 strain to cause lethal infection. The toxin-dependent phenotype of the UT500 strain was host phagocyte dependent, because eliminating Gr-1 ؉ phagocytes restored virulence to the UT500 PA ؊ mutant. These experiments demonstrate that the dominant virulence factors used to establish infection by B. anthracis depend on the route of inoculation and the bacterial strain.

Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax

“…The Ames-derived cap-null strain (UTA8 [⌬capBCADE]) was similarly constructed using an omega-spectinomycin resistance cassette, as previously described (36). Spore stocks were prepared in phage assay broth as previously described (8,24), and kanamycin (Sigma; 50 g/ml) or spectinomycin (100 g/ml) was added to phage assay media for preparation of UTA mutant spore stocks. The extent of sporulation was assessed by phase-contrast microscopy.…”

“…Inoculums were prepared by thawing a stock vial to room temperature and diluting it in DPBS to the desired concentration. Vegetative cultures were prepared as previously described (8,24), and the parent and mutant strains were shown to have similar growth rates under these growth conditions. Briefly, a B. anthracis spore stock was used to streak a nutrient broth yeast agar plate supplemented with 0.8% sodium bicarbonate (NBY-NaHCO 3 ) plus kanamycin (50 g/ml) when appropriate and incubated at 5% CO 2 , 37°C for 24 h. Next, two to three colonies were inoculated into 15 ml of LuriaBertani (LB) broth containing 0.5% glycerol (and antibiotics as appropriate) and shaken at 200 rpm overnight, and then an aliquot of the overnight culture was added to NBY-NaHCO 3 broth at an initial optical density at 600 nm (OD 600 ) of 0.1 and shaken at 200 rpm in 10% CO 2 in a 37°C incubator to a final OD 600 of 1.2 to give a known concentration.…”

Anthrax Edema Toxin Impairs Clearance in Mice

“…B. anthracis mutants defective in EF production are attenuated for virulence in mice in some infection models (22,40) but not in others (4), and EF has been reported to influence bacterial dissemination (8). ET injection into mice causes symptoms typical of anthrax disease, including massive edema, hemorrhages in organs, hypotension, bradycardia, and death (12).…”

Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores

“…Spore stocks were prepared in phage assay medium as previously described (7,16), and inocula were prepared from frozen aliquots. The actual number of spores delivered was determined by culturing the inoculum on sheep blood agar plates directly from the bronchoscope.…”