Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins

Abstract

Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

Fig. 1.

(A) Immunogold localization of toxin components and anthrolysin in isolated vesicles. Immunoelectron microscopy with mAbs 7.5G (PA), BD3 (EF), 14FA (LF), and 64F8 (ALO) revealed gold particles in isolated vesicles. Arrow shows a vesicle with double membrane. (B) Immunogold double-labeling with mAbs 10F4 (PA), BD3 (EF), and 14FA (LF). Solid arrows, gold particles (10 nm) depicting EF binding; dashed arrows, gold particles (6 nm) depicting PA or LF binding. (C) Immunogold labeling with irrelevant IgG did not reveal gold particles in isolated vesicles. (Scale bars, 200 nm.)

Fig. 2.

Immunogold localization of PA, LF, and EF in B. anthracis Sterne 34F2. IEM with mAbs (A) FF7 (EF), (B) 10F4 (PA), and (C) 14FA (LF) revealed gold particles (solid arrows) in structures resembling vesicles. (D) Immunogold labeling with irrelevant IgG did not reveal gold particles in bacterial cells. (Scale bars, 200 nm.) (E and F) High-resolution IEM with mAb 7.5G (PA) of bacterial cells revealed concentrations of gold particles (solid arrows) at or near the bacterial surface. (Scale bars, 100 nm.)

Fig. 3.

Immunogold localization of macrophages incubated with B. anthracis with mAb 10F4. (A) Gold particles (10 nm) depicting PA in vesicle-like structure were detected bound to the surface of macrophages (arrow). (Scale bar, 100 nm.) (B) B. anthracis bacterial cells (Ba) are located within phagosomes (P) of macrophages (MØ). We note a disruption in phagosome double membrane (dashed arrow) and a vesicle containing PA (solid arrow) in the cytoplasm of the host cell in close proximity to an ingested bacterial cell. (Scale bar, 500 nm.)

Fig. 4.

Immunogenicity of isolated vesicles from B. anthracis Sterne 34F2 in mice. (A) Ab titer of BALB/c mice immunized with isolated vesicles as measured by ELISA. Mice (M1, M2, and M3) were immunized with vesicles. Inset, immunoblot of Tox⁺ and Tox⁻ vesicles incubated with sera from vesicle-immunized mice. (B) Isotype analysis of immune sera from vesicle-immunized mice reveals a robust IgM response. (Inset) ELISA schematic which applies to A and B. (C) Survival of BALB/c mice immunized with isolated vesicles, mice immunized with adjuvant alone (control) and challenged with B. anthracis Sterne (Tox+) (10⁶ cells i.v.). n = 3 each group.