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.

Bacterial Virulence Factors: Secreted for Survival

“…Bacillus anthracis is among the few Grampositive bacteria, which produces membrane vesicles. Membrane vesicles derived from Bacillus anthracis is reported to contain the anthrolysin O apart from lethal toxin/edema toxin, indicating the physiological role of anthrolysin O during anthrax infections [62]. Bishop and colleague have studied the effect of recombinant purified anthrolysin O on the integrity of gut epithelial barrier in human C2BBE and ex vivo mouse intestinal segments.…”

Origin of life: LUCA and extracellular membrane vesicles (EMVs)

“…EMVs produced by these bacteria are derived from the cytoplasmic membrane and are 20-250 nm in diameter (Lee et al 2009;Schrempf et al 2011). It has been suggested that the vesicles produced by monoderm bacteria are like their diderm counterparts, involved in pathogenesis (Rivera et al 2010;Prados-Rosales et al 2011). Enzymes involved in peptidoglycan degradation, antibiotic degradation, virulence factors (anthrolysin, anthrax toxin components, coagulases, hemolysins and lipases) and immunologically-active compounds have been identified in these vesicles (Marsollier et al 2007;Lee et al 2009;Rivera et al 2010;Gurung et al 2011;Prados-Rosales et al 2011;Thay et al 2013;Brown et al 2014).…”

Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

“…They are naturally produced by both pathogenic and nonpathogenic Gram negative bacteria like -Neisseria, Helicobacter pylori, Vibrio cholerae, Haemophilus influenzae (Schooling et al, 2006). However Gram positive bacteria like Bacillus anthracis (Rivera et al, 2010) and Mycobacteria spp (Prados-Rosales et al, 2011) have also been reported to produce outer membrane vesicles. OMV allows bacteria to secrete insoluble and complex molecules with the soluble material so that it reach the target in a concentrated and protected form and helps in bacterial stress response, nutrient acquisition, biofilm development (Mashburn-Warren et al, 2006).…”