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

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.

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1‐independent manner

“…Cholesterol-rich lipid rafts have been commonly reported to be involved in the uptake of OMVs produced by a number of species, including enterotoxigenic Escherichia coli (ETEC), Haemophilus influenzae , Campylobacter jejuni , and Pseudomonas aeruginosa , among others (Bomberger et al, 2009; Elmi et al, 2012; Kaparakis et al, 2010; Kesty, Mason, Reedy, Miller, & Kuehn, 2004; Mondal et al, 2016; Sharpe, Kuehn, & Mason, 2011), while OMVs produced by other organisms, including Helicobacter pylori and enterohemorrhagic E. coli (EHEC), have been reported to enter cells in cholesterol-independent endocytic processes (Bielaszewska et al, 2017; Bielaszewska et al, 2013; Canas et al, 2016; Kunsmann et al, 2015; Parker, Chitcholtan, Hampton, & Keenan, 2010). …”

Intrahost milieu modulates production of outer membrane vesicles, vesicle‐associated Shiga toxin 2a and cytotoxicity in Escherichia coli O157:H7 and O104:H4

“…However, the most devastating outbreak with nearly 4000 infected persons, more than 900 cases of HUS and 54 deaths was caused in 2011 by a rare E. coli serotype O104:H4 (Frank et al, 2011;Karch et al, 2012), which is a hybrid of EHEC and enteroaggregative E. coli (Bielaszewska et al, 2011a;Mellmann et al, 2011). Both these pathogens secrete cocktails of their virulence factors via outer membrane vesicles (OMVs), nanostructures released during growth (Kunsmann et al, 2015;Bielaszewska et al, 2017). Shiga toxin 2a (Stx2a), the major virulence factor of EHEC implicated in the pathogenesis of HUS (Karch et al, 2005;Tarr et al, 2005), is an important component of both O157 and O104 OMVs (Kunsmann et al, 2015;Bielaszewska et al, 2017).…”

“…Both these pathogens secrete cocktails of their virulence factors via outer membrane vesicles (OMVs), nanostructures released during growth (Kunsmann et al, 2015;Bielaszewska et al, 2017). Shiga toxin 2a (Stx2a), the major virulence factor of EHEC implicated in the pathogenesis of HUS (Karch et al, 2005;Tarr et al, 2005), is an important component of both O157 and O104 OMVs (Kunsmann et al, 2015;Bielaszewska et al, 2017).…”

Estudo dinâmico da expressão gênica global durante a interação STEC-enterócito utilizando séries temporais

“…É curioso que três hubs -CHAF1A, RAD51 e UBE2T -estão envolvidos no reparo de DNA !Bhattacharya et al, 2017;Kelsall et al, 2012). Bielaszewska et al (2017) mostraram que algumas toxinas, como a toxina citoletal CdtV-B de EHEC O157, provoca danos no DNA celular ativando respostas para o reparo de DNA.…”

“…Outro hub interessante desta janela é SURF4, que codifica para uma proteína de membrana envolvida no transporte de proteínas do complexo de Golgi para o retículo endoplasmático. Esse tipo de transporte é utilizado para a internalização de Stx (Bielaszewska et al, 2017).…”