Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins

Johanna Rivera, Radames J.B. Cordero, Antonio S. Nakouzi, Susana Frases, André Nicola, Arturo Casadevall

Research output: Contribution to journalArticlepeer-review

279 Scopus citations


Extracellular vesicle production is a ubiquitous process in Gramnegative bacteria, but little is known about such process in Grampositive 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 doublemembrane spheres and ranged from 50 to150 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 analysisof vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb toprotective antigen protected macrophages against vesicles from an anthrolysin-deficientstrain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistentwith 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 componentstotarget host cells, a mechanism that may increase toxin potency. Our observations may haveimportant implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.

Original languageEnglish (US)
Pages (from-to)19002-19007
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number44
StatePublished - Nov 2 2010


  • Immunizations
  • Monoclonal antibody
  • Passive immunity

ASJC Scopus subject areas

  • General


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