Host cell interactions of outer membrane vesicle-associated virulence factors of Enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury

Authors: BIELASZEWSKA, MARTINA - University Of Munster; RUTER, CHRISTIAN - University Of Munster; BAUWENS, ANDREAS - University Of Munster; GREUNE, LILO - University Of Munster; ZHANG, WENLAN - University Of Munster; He, Xiaohua; LLOUBES, ROLAND - Aix-Marseille University; FRUTH, ANGELIKA - Robert Koch Institute; KIM, KWANG - Johns Hopkins University School Of Medicine; SCHMIDT, M. ALEXANDER - University Of Munster; DOBRINDT, ULRICH - University Of Munster; MELLMANN, ALEXANDER - University Of Munster; KARCH, HELGE - University Of Munster

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2016
Publication Date: 2/3/2017
Citation: Bielaszewska, M., Ruter, C., Bauwens, A., Greune, L., Zhang, W., He, X., Lloubes, R., Fruth, A., Kim, K.S., Schmidt, M., Dobrindt, U., Mellmann, A., Karch, H. 2017. Host cell interactions of outer membrane vesicle-associated virulence factors of Enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury. PLoS Pathogens. 13(2):e1006159. doi: 10.1371/journal. ppat. 1006159.

Interpretive Summary: Enterohemorrhagic Escherichia coli (EHEC) O157, the leading EHEC group causing diarrhea and the life-threatening hemolytic uremic syndrome in humans, produce several virulence factors which play distinct roles in the pathogenesis of these diseases. However, the mechanisms of their secretion and host cell injury are poorly understood. We show here that EHEC O157 strains isolated from patients shed nanostructures termed outer membrane vesicles (OMVs) which contain a cocktail of 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 endothelial cells, which are the major targets during EHEC O157 infections, and deliver the virulence factors intracellularly. Inside cells the virulence factors separate from OMVs and are transported via different pathways to their target compartments including the cytosol (Stx2a), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). Cells exposed to EHEC O157 OMVs develop G2 cell cycle arrest induced by CdtV-B-mediated DNA damage. This is followed by apoptotic cell death triggered by CdtV and Stx2a via caspase-9 activation. OMVs thus serve as novel powerful tools of EHEC O157 to injure cells involved in the pathogenesis of EHEC O157-mediated diseases.

Technical 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, 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 target 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 and undergo retrograde transport via the Golgi complex to the endoplasmic reticulum; from there the catalytic Stx2a A1 fragment is translocated to the cytosol to reach ribosomes, and CdtV-B 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 late endosomes/lysosomes and subsequently targets mitochondria. The OMV-delivered CdtV-B subunit causes DNA damage in target cells, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by CdtV and, to a greater extent, by Stx2a, 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 potential vaccine candidates.