Abstract
The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.
Highlights
The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown
We investigated the interactions of the outer membrane vesicles (OMVs) with intestinal epithelial cells (IECs), which are the first cellular targets for E. coli O104:H4 during human disease, and determined biological consequences of such interactions
Electron microscopy of Luria-Bertani (LB) agar culture of E. coli O104:H4 outbreak strain LB226692 demonstrated blebbing of OMVs from the bacterial surface (Fig. 1a–c) as well as free OMVs that had already been released from bacteria (Fig. 1b)
Summary
The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. Clinical observations and studies in animal models and tissue cultures indicate that Stx2a, the SPATEs Pic and SigA, as well as the pAA-encoded virulence factors, in particular AAF/I, contributed to the high pathogenicity of the outbreak strain[5,6,7,8]. The key role of OMVs in bacterial virulence is supported by their ability to mimic in animal models diseases caused by the parental pathogens[13] It is presently unknown in which forms the outbreak strain secretes its virulence factors, in particular whether or not it releases OMVs and which role(s) they may play in its virulence. We investigated the interactions of the OMVs with intestinal epithelial cells (IECs), which are the first cellular targets for E. coli O104:H4 during human disease, and determined biological consequences of such interactions
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