Bacillus anthracis spores of the bclA mutant exhibit increased adherence to epithelial cells, fibroblasts, and endothelial cells but not to macrophages

Abstract

Bacillus anthracis is the causative agent of anthrax, and the spore form of the bacterium represents the infectious particle introduced into a host. The spore is surrounded by an exosporium, a loose-fitting membrane composed of proteins and carbohydrates from which hair-like projections extend. These projections are composed mainly of BclA (Bacillus-collagen-like protein of B. anthracis). To date, exact roles of the exosporium structure and BclA protein remain undetermined. We examined differences in spore binding of wild-type Ames and a bclA mutant of B. anthracis to bronchial epithelial cells as well as to the following other epithelial cells: A549, CHO, and Caco-2 cells; the IMR-90 fibroblast line; and human umbilical vein vascular endothelium cells. The binding of wild-type Ames spores to bronchial epithelial cells appeared to be a dose-dependent, receptor-ligand-mediated event. There were similar findings for the bclA mutant, with an additional nonspecific binding component likely leading to significantly more adherence to all nonprofessional phagocytic cell types. In contrast, we detected no difference in adherence and uptake of spores by macrophages for either the wild-type Ames or the bclA mutant strain. These results suggest that one potential role of the BclA fibers may be to inhibit nonspecific interactions between B. anthracis spores with nonprofessional phagocytic cells and thus direct the spores towards uptake by macrophages during initiation of infection in mammals.

FIG. 1.

Association of spores to BEC. (A) BEC were incubated with either Ames wild-type (MOI of 32 to 50 spores for one BEC) or bclA mutant spores (MOI of 21 to 47 spores for one BEC) for 1 h. Following this incubation, the cells were washed and lysed and the bacteria were plated upon LB agar plates. The percent recovery of spores was determined by dividing the number of CFU counts from the lysed cells by the number of spores added. The difference between spore types associated with the BEC was significant (P = 0.0005). Error bars represent the standard errors of the means from three independent experiments. (B and C) Micrographs of spores associated with BEC. Samples were stained with spore stain (malachite green) and counterstained with a Wright-Giemsa stain. (B) Ames spores (indicated by white arrows) with BEC. (C) bclA mutant spores with BEC.

FIG. 2.

SEM and TEM micrographs of the interaction between B. anthracis spores and BEC. (A) Adherence of Ames spores to BEC 2 h postinfection. (B) Adherence of bclA mutant spores to BEC 2 h postinfection. (C) bclA mutant spores incubated with BEC. (D) Close-up view from boxed area of panel C.

FIG. 3.

Interaction of BEC with Ames spores treated with trypsin. Cytochalasin D-treated BEC were incubated with either untreated Ames spores or trypsin-treated spores for 1 h at an MOI of 23 to 26 spores for one BEC. Following this incubation, the cells were washed and lysed and the bacteria were plated upon LB agar plates. The percent recovery of spores was determined, and the difference in the number of spores (with or without trypsin treatment) associated with the BEC was significant (P = 0.0003). Error bars represent the standard errors of the means from triplicate independent experiments.

FIG. 4.

Saturation of the BEC binding sites for B. anthracis spores. Cytochalasin D-treated BEC were incubated with increasing MOI concentrations of either Ames wild-type or bclA mutant spores, and the numbers of adherent spores were determined by CFU counts. Error bars represent standard errors of the means from the CFU counts. Shown are representative results from an experiment performed in triplicate.

FIG. 5.

B. anthracis spores within BEC were recovered by gentamicin protection assays. BEC were incubated with either Ames wild-type or bclA mutant spores for 1 h at an MOI of 33 to 39 spores for one BEC. Following this incubation, the medium was replaced with that containing 10% FBS, 5 μg/ml of cytochalasin D, and 2.5 μg/ml of gentamicin. Cells were then lysed, the bacteria were plated, and the percent recovery of the intracellular spores was determined. A difference of spore numbers within the BEC was significant between strains (P = 0.0004). Error bars represent standard errors of the means from three independent experiments.

FIG. 6.

Effect of the bclA mutation on uptake of spores by macrophages. RAW264.7 cells were infected with spores at an MOI of 1 to 3 for one cell. (A) Data depict viable counts. The error bars represent standard errors of the means. (B) Microscopic counts of fluorescently labeled samples. (C) Microscopic counts of samples stained with the malachite green spore stain. The phagocytic index is the mean number of spores phagocytosed per macrophage multiplied by 100. Similar results were obtained in at least two additional experiments.