Abstract
Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) O157, the leading EHEC serogroup causing human diseases including life-threatening hemolytic uremic syndrome (HUS) [1], consist of classical non-sorbitol-fermenting (NSF) O157:H7 and sorbitol-fermenting (SF) O157:H(non-motile) strains [2]
We show here that EHEC O157 strains isolated from patients shed nanostructures termed outer membrane vesicles (OMVs) which contain major EHEC O157 virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin
The OMVs are taken up by human intestinal epithelial and renal and brain microvascular endothelial cells, which are the major targets during EHEC O157 infections, and deliver the virulence factors intracellularly
Summary
Enterohemorrhagic Escherichia coli (EHEC) O157, the leading EHEC serogroup causing human diseases including life-threatening hemolytic uremic syndrome (HUS) [1], consist of classical non-sorbitol-fermenting (NSF) O157:H7 and sorbitol-fermenting (SF) O157:H(non-motile) strains [2]. Soluble EHEC-Hly lyses these cells [9], whereas EHEC-Hly bound to bacterial membrane vesicles causes apoptosis [10] Besides their endothelial cytotoxicity, Stxs and EHEC-Hly induce, alone or together with H7 flagellin and/or O157 lipopolysaccharide (LPS), secretion of proinflammatory cytokines [15,16,17], which play multiple roles in HUS development [1, 5]. Stxs and EHEC-Hly induce, alone or together with H7 flagellin and/or O157 lipopolysaccharide (LPS), secretion of proinflammatory cytokines [15,16,17], which play multiple roles in HUS development [1, 5] Another virulence factor, the serine protease EspPα produced by NSF O157 strains [18], may contribute to the pathogenesis of HUS by interacting with the coagulation cascade by cleaving factor V [19] and with the complement system by degrading C3 and C5 [20]
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