An electron microscopy study of Bacillus anthracis capsule reveals early expression and novel strand-like structures

Capsule is a key virulence factor for Bacillus anthracis. In this study, we examined the onset of capsule expression and the ultrastructure of capsule expressed in vitro and in infected hosts. We determined by immunofluorescence and immunoelectron microscopy that capsule was produced as early as 15 min after onset of germination. Further analysis revealed bacilli were often fully encapsulated when they emerged from the exosporium. Using conventional fixative to prepare samples for electron microscopy resulted in loss of capsular material, but using a fixative containing ruthenium red and lysine acetate preserved it. Transmission electron microscopy using enhanced fixation revealed that B. anthracis grown in vitro were surrounded by a thick capsule layer comprised of globular structures and a slime layer. When examined using the enhanced fixation, extracellular bacilli in tissues from moribund animals were surrounded by a thick capsule layer, and had long branching strands like those observed on live but not fixed bacilli by fluorescence microscopy. Scanning electron microscopy using enhanced fixation similarly revealed B. anthracis within spleen sinusoids were covered with long branching strands that appeared to tether them to host cells. This is the first report describing the presence of long capsule strands during infection in vivo.