Five distinct secretion pathways are used to transport proteins across the Gram-negative bacterial double membrane. This is in addition to three transport routes across the inner membrane. Even so, Escherichia coli, the best-studied bacterium, can still spring a surprise on microbiologists. Reporting in Cell, Wai et al. describe a new mechanism of secretion in which a toxin is exported in vesicles. Vesicle or bleb formation is nothing new — but this is the first time vesicle-mediated secretion has been given clear physiological relevance.

Common laboratory strains of E. coli can produce ClyA, a cytotoxin that is secreted into the bacterial periplasm but acts on mammalian cells. How does the toxin access its target site? Using immunofluorescence, Wai et al. showed that ClyA is surface-exposed. Closer examination with electron microscopy and atomic force microscopy revealed a multitude of small outer-membrane vesicles (OMVs) approximately 50–200 nM in diameter surrounding the bacteria. Analysing the protein content of these vesicles proved that they were derived from the outer membrane. Strains of E. coli that do not produce ClyA still produced OMVs. Reassuringly, pathogenic E. coli and Salmonella enterica serovar Typhi strains that produce ClyA also localized the toxin into OMVs, so this process isn't restricted to laboratory-adapted strains. The OMVs typically had ring-like pores, which were shown to be assemblies of ClyA protein.

Toxicity assays showed that ClyA present in OMVs was at least 8 times more toxic than purified ClyA. This striking result prompted the authors to examine ClyA multimerization, and cross-linking experiments confirmed that periplasmic ClyA is monomeric compared with multimeric ClyA present in OMVs. Further, removing the disulphide-bond oxidation machinery from E.coli dramatically increased the action of ClyA. The authors couldn't detect proteins that participate in disulphide-bond formation in OMVs containing ClyA, raising the possibility that the redox status of ClyA contributes to oligomerization and toxicity. OMVs seem to contain a subset of periplasmic proteins, which means that E.coli could have a crude protein-sorting mechanism that selects which proteins are exported in OMVs.

OMVs are formed by a whole variety of bacterial species, including commensal Bacteroides species and pathogenic Neisseria species. If bacteria can sort periplasmic proteins into vesicles, the challenge will be to find out how sorting is accomplished, how it is regulated and whether this process can be used as a model for eukaryotic vesicle trafficking.