Contribution of ExsFA and ExsFB Proteins to the Localization of BclA on the Spore Surface and to the Stability of the Bacillus anthracis Exosporium

Abstract

Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the ⌬exsFA and ⌬exsFA ⌬exsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The ⌬exsFA and ⌬exsFA ⌬exsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.

Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization

“…2c, d), although other spore antigens were also recognized ( Fig. 2f) (Steichen et al, 2003(Steichen et al, , 2005Sylvestre et al, 2002Sylvestre et al, , 2005. We observed that bclA : : Kan spores were also not significantly opsonized by anti-spore IgG when compared to wild-type Ames strain spores (Fig.…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…B. anthracis was grown in BHI broth (Becton Dickson, MD) supplemented with 0.5% glycerol at 37°C unless otherwise noted. Spores were produced by plating on NBY media with antibiotics when necessary and incubated at 30°C for 10 days, then harvested and purified on an Omnipaq gradient (350 milligrams of iodine/mL) (GE Healthcare, NJ), similar to previously described methods [81,261,289]. Escherichia coli strains were either α-select (Bioline, MA) or dcm, dam-deficient GM119 (dcm-6, dam-3) [290] and grown in LB broth with appropriate antibiotics when necessary (Becton Dickson, MD).…”

“…Omnipaq gradient (350 milligrams of iodine/mL), similar to previously described methods [81,261,289]. The E.coli strains used were either α-select (Bioline, MA) or dcm, dam-deficient GM119…”

“…Spores were then harvested and separated from vegetative bacilli on an Omnipaq gradient (350 milligrams of iodine/mL) (GE Healthcare, NJ), similar to previously described methods [81,261,289]. MDCK and Caco2 cell lines were grown in DMEM + 10% FBS and incubated at 37°C with 10% CO2.…”

“…The spores were then collected from plates and purified on an Omnipaq gradient (350 mg of iodine/mL, GE Healthcare, NJ) as described previously [81,264,289].…”