Fully virulent Bacillus anthracis does not require the immunodominant protein BclA for pathogenesis

Abstract

The BclA protein is the immunodominant epitope on the surface of Bacillus anthracis spores; however, its roles in pathogenesis are unclear. We constructed a BclA deletion mutant (bclA) of the fully virulent Ames strain. This derivative retained full virulence in several small-animal models of infection despite the bclA deletion.

FIG. 1.

Effect of the BclA protein on the germination rates of B. anthracis spores in vitro. (A) Microtiter spectrofluorometric assay of germination of spores in the presence of a germination medium with alanine, adenosine, and Casamino Acids (AAC), as described previously (30). The germination of spores of the wild-type Ames strain (○) and bclA strain (Δ) was monitored every minute for 1 h. The data (percent increase in relative fluorescence units [RFU]) are the relative increase in RFU at a given time point compared to the RFU at time zero, multiplied by 100. Representative data are presented, and similar results were obtained in at least two additional experiments. (B) Germination was also measured by absorbance readings as previously described (19). The A₆₀₀ of each sample was measured at various times and is plotted as the percentage of the initial A₆₀₀ [A₆₀₀ (init)] at time zero that is represented by the A₆₀₀ at a given time point [A₆₀₀ (t)]. Absorbance data are from three independent experiments, and standard errors of the means are depicted.

FIG. 2.

Effect of the BclA protein on virulence in the guinea pig infection model. (A) Female Hartley guinea pigs were challenged i.m. with spores of the Ames wild-type strain (○) or spores of the bclA strain (Δ). (B) The effect of the bclA mutation on in vivo fitness in a competitive guinea pig infection model. Guinea pigs were coinfected i.m. with spores of both the Ames wild-type strain and the bclA strain. The error bars represent standard errors of the means. Wt, wild type.

FIG. 3.

Effect of the BclA protein on virulence in the mouse infection model. (A) BALB/c mice were challenged intranasally with spores of the Ames wild-type strain (○) or the bclA strain (Δ). (B) The effect of the bclA mutation on the rate of spore clearance from mouse lungs. Mice were exposed to an aerosolized inoculum of spores of both wild-type Ames and bclA mutant. The number of heat-resistant (65°C for 30 min) spores retained within the lungs at designated time points was determined by bacterial counts on selective and nonselective media. The error bars represent standard errors of the means. *, P = 0.03; **, P = 0.0005.