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Bacterial protein secretion updated

A novel vesicle-mediated pathogenic protein secretion pathway in gram-negative bacteria

By | October 6, 2003

Bacterial interaction with plants and animals, symbiotic or pathogenic, involves the transfer to host cells and tissues of a range of bacterial proteins whose biochemical activities are key to establishing both commensalism and infection. Export of protein molecules across the bacterial membranes takes place via a variety of mechanisms, from simple one-component systems to complex multicomponent pathways, with five types of nonhomologous protein secretion system characterized in pathogenic bacteria so far. In the October 3 Cell, Sun Nyunt Wai and colleagues at Umeå University describe an additional protein transfer strategy in gram-negative bacteria that furthers our understanding of host–bacteria interactions (Cell, 115:25-35, October 3, 2003).

Wai et al. dissected the secretion pathway of cytolysin A (ClyA), a pore-forming toxin with hemolytic and cytolytic properties produced by the gram-negative bacterium Escherichia coli and closely related species. Gram-negative bacteria have two distinct membrane bilayers separated by a periplasmic space that complicates the secretion of proteins. While the inner membrane has a normal phospholipid protein composition, the outer membrane has a more complex structure, with the outer leaflet comprising the glycolipid lipopolysaccharide (or endotoxin) that is toxic to animals. ClyA accumulates in the periplasmic space in overproducing strains, but how it is transported to the membrane surface and released from bacterial cells had been unclear.

Using the E. coli K12 strain, the authors observed that ClyA was exported from the bacterial cells in outer-membrane vesicles that can fuse with mammalian target cells and subsequently discharge their toxic cargo. These outer-membrane vesicles are constantly discharged from the surface of the cell during bacterial growth, but their role in protein transport had been poorly documented. In the case of ClyA, electron microscopy showed that the protein formed pore-like structures on the vesicles, and immunoblot analysis of the vesicle proteic components confirmed that the toxin was the most abundant protein, although other periplasmic proteins were also present. Vesicle ClyA was more than eight-fold more active that the protein purified from the periplasm.

Periplasmic ClyA is kept in monomeric form by a disulphide bond, whereas the relevant cysteine residues are reduced in the protein from the vesicles, which oligomerize to form the large complexes suggested to be the active form of pore assemblies. Since the redox status of periplasmic proteins depends on the activity of specific membrane-bound and periplasmic disulphide bond isomerases/oxidases, that ClyA can bypass this thiol-redox pathway suggests that bacteria can sort proteins when forming vesicles, excluding certain periplasmic proteins such as those catalyzing the formation of disulphide bonds.

"The results define a vesicle-mediated transport mechanism in bacteria that is responsible for the activation and delivery of pathogenic effector proteins," conclude the authors.

"The discovery that bacterial vesicles are not simply a phenomenon of in vitro growth should result in studies that will increase our knowledge of the interactions of bacteria with eukaryotic organisms," highlight Samuel Miller and colleagues at the University of Washington in an accompanying preview article.

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