Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.

• Klíčová slova
• Caco-2 cells, enterohemorrhagic Escherichia coli, hemolytic uremic syndrome, human colonoids, intestinal epithelial barrier, outer membrane vesicles, translocation,
• Publikační typ
• časopisecké články MeSH

Outer membrane vesicles (OMVs), nanoparticles released by Shiga toxin-producing Escherichia coli (STEC), have been identified as novel efficient virulence tools of these pathogens. STEC O157 OMVs carry a cocktail of virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, flagellin, and lipopolysaccharide. OMVs are taken up by human intestinal epithelial and microvascular endothelial cells, the major targets during STEC infection, and deliver the virulence factors into host cells. There the toxins separate from OMVs and are trafficked via different pathways to their target compartments, i.e., the cytosol (Stx2a-A subunit), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). This leads to a toxin-specific host cell injury and ultimately apoptotic cell death. Besides their cytotoxic effects, STEC OMVs trigger an inflammatory response via their lipopolysaccharide and flagellin components. In this chapter, we describe methods for the isolation and purification of STEC OMVs, for the detection of OMV-associated virulence factors, and for the analysis of OMV interactions with host cells including OMV cellular uptake and intracellular trafficking of OMVs and OMV-delivered toxins.

• Klíčová slova
• Confocal laser scanning microscopy, Flow cytometry, Host cell interactions, Immunogold labeling, OMVs, Outer membrane vesicles, STEC, Shiga toxin-producing Escherichia coli, Transmission electron microscopy, Virulence cargo, Western blot,
• MeSH
• bakteriální toxiny metabolismus   MeSH
• endoteliální buňky metabolismus   mikrobiologie   patologie   MeSH
• Escherichia coli O157 * metabolismus   patogenita   MeSH
• faktory virulence metabolismus   MeSH
• lidé MeSH
• mikropartikule metabolismus   MeSH
• shiga toxin 2 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální toxiny MeSH
• cytolethal distending toxin MeSH Prohlížeč
• faktory virulence MeSH
• shiga toxin 2 MeSH

Outer membrane vesicles (OMVs) are nanoscale proteoliposomes secreted from the cell envelope of all Gram-negative bacteria. Originally considered as an artifact of the cell wall, OMVs are now recognized as a general secretion system, which serves to improve the fitness of bacteria and facilitate bacterial interactions in polymicrobial communities as well as interactions between the microbe and the host. In general, OMVs are released in increased amounts from pathogenic bacteria and have been found to harbor much of the contents of the parental bacterium. They mainly encompass components of the outer membrane and the periplasm including various virulence factors such as toxins, adhesins, and immunomodulatory molecules. Numerous studies have clearly shown that the delivery of toxins and other virulence factors via OMVs essentially influences their interactions with host cells. Here, we review the OMV-mediated intracellular deployment of toxins and other virulence factors with a special focus on intestinal pathogenic Escherichia coli. Especially, OMVs ubiquitously produced and secreted by enterohemorrhagic E. coli (EHEC) appear as a highly advanced mechanism for secretion and simultaneous, coordinated and direct delivery of bacterial virulence factors into host cells. OMV-associated virulence factors are not only stabilized by the association with OMVs, but can also often target previously unknown target structures and perform novel activities. The toxins are released by OMVs in their active forms and are transported via cell sorting processes to their specific cell compartments, where they can develop their detrimental effects. OMVs can be considered as bacterial "long distance weapons" that attack host tissues and help bacterial pathogens to establish the colonization of their biological niche(s), impair host cell function, and modulate the defense of the host. Thus, OMVs contribute significantly to the virulence of the pathogenic bacteria.

• Klíčová slova
• EHEC, ETEC, intestinal pathogenic Escherichia coli, outer membrane vesicles, toxins, virulence factors,
• MeSH
• bakteriální sekreční systémy metabolismus   MeSH
• bakteriální toxiny metabolismus   MeSH
• enterohemoragická Escherichia coli metabolismus   patogenita   MeSH
• enterotoxigenní Escherichia coli metabolismus   patogenita   MeSH
• enterotoxiny metabolismus   MeSH
• faktory virulence metabolismus   MeSH
• fyziologický stres MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• lidé MeSH
• proteolipidy metabolismus   ultrastruktura   MeSH
• střeva mikrobiologie   MeSH
• transport proteinů MeSH
• virulence MeSH
• vnější bakteriální membrána metabolismus   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální sekreční systémy MeSH
• bakteriální toxiny MeSH
• enterotoxiny MeSH
• faktory virulence MeSH
• proteolipidy MeSH
• proteoliposomes MeSH Prohlížeč

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H- strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H- strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H- strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H- strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology.IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H- patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

• Klíčová slova
• EHEC O157, Sfp fimbriae, enterohemorrhagic E. coli, hemolytic-uremic syndrome, outbreaks, plasmid analysis,
• MeSH
• Escherichia coli O157 klasifikace   izolace a purifikace   metabolismus   MeSH
• faktory virulence genetika   metabolismus   MeSH
• fermentace MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• lidé MeSH
• plazmidy genetika   metabolismus   MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• sorbitol metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Německo MeSH
• Názvy látek
• faktory virulence MeSH
• proteiny z Escherichia coli MeSH
• sorbitol MeSH