Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.
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.
Institute of Hygiene, University of Münster, Münster, Germany.
Martina
0000-0002-1226-7066
M
Bielaszewska
Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany.
Christian
C
Rüter
Institute of Hygiene, University of Münster, Münster, Germany.
Andreas
A
Bauwens
Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany.
Lilo
L
Greune
Institute of Hygiene, University of Münster, Münster, Germany.
Kevin-André
KA
Jarosch
Institute of Hygiene, University of Münster, Münster, Germany.
Daniel
D
Steil
Institute of Hygiene, University of Münster, Münster, Germany.
Wenlan
W
Zhang
Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture (USDA), Albany, California, United States of America.
Xiaohua
X
He
Laboratoire d'Ingenierie des Systemes Macromoleculaires UMR7255, CNRS-Aix-Marseille Université, Marseille, France.
Roland
R
Lloubes
National Reference Center for Salmonella and Other Enteric Pathogens, Robert Koch Institute, Branch Wernigerode, Wernigerode, Germany.
Angelika
A
Fruth
Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
Kwang Sik
KS
Kim
Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany.
M Alexander
MA
Schmidt
Institute of Hygiene, University of Münster, Münster, Germany.
Ulrich
U
Dobrindt
Institute of Hygiene, University of Münster, Münster, Germany.|Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Münster, Germany.
Alexander
A
Mellmann
Institute of Hygiene, University of Münster, Münster, Germany.|Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Münster, Germany.
Helge
H
Karch
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D004927:Escherichia coli Infections / Q000378:metabolism*; D019453:Escherichia coli O157; D054884:Host-Pathogen Interactions / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D021381:Protein Transport / Q000502:physiology; D022161:Transport Vesicles / Q000502:physiology; D014774:Virulence / Q000502:physiology*; D037521:Virulence Factors / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D037521:Virulence Factors