BACKGROUND: E. coli O83 (Colinfant Newborn) is a Gram-negative (G-) probiotic bacterium used in the clinic. When administered orally, it reduces allergic sensitisation but not allergic asthma. Intranasal administration offers a non-invasive and convenient delivery method. This route bypasses the gastrointestinal tract and provides direct access to the airways, which are the target of asthma prevention. G- bacteria such as E. coli O83 release outer membrane vesicles (OMVs) to communicate with the environment. Here we investigate whether intranasally administered E. coli O83 OMVs (EcO83-OMVs) can reduce allergic airway inflammation in mice. METHODS: EcO83-OMVs were isolated by ultracentrifugation and characterised their number, morphology (shape and size), composition (proteins and lipopolysaccharide; LPS), recognition by innate receptors (using transfected HEK293 cells) and immunomodulatory potential (in naïve splenocytes and bone marrow-derived dendritic cells; BMDCs). Their allergy-preventive effect was investigated in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: EcO83-OMVs are spherical nanoparticles with a size of about 110 nm. They contain LPS and protein cargo. We identified a total of 1120 proteins, 136 of which were enriched in OMVs compared to parent bacteria. Proteins from the flagellum dominated. OMVs activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. EcO83-OMVs induced the production of pro- and anti-inflammatory cytokines in splenocytes and BMDCs. Intranasal administration of EcO83-OMVs inhibited airway hyperresponsiveness, and decreased airway eosinophilia, Th2 cytokine production and mucus secretion. CONCLUSIONS: We demonstrate for the first time that intranasally administered OMVs from probiotic G- bacteria have an anti-allergic effect. Our study highlights the advantages of OMVs as a safe platform for the prophylactic treatment of allergy. Video Abstract.

• Keywords
• Allergic airway inflammation, Extracellular vesicles, Microbiota and innate immunity, Nasal route of administration, Outer membrane vesicles,
• MeSH
• Hypersensitivity * prevention & control   metabolism   MeSH
• Asthma * metabolism   MeSH
• Escherichia coli MeSH
• Extracellular Vesicles * metabolism   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Lipopolysaccharides MeSH
• Mice MeSH
• Immunity, Innate MeSH
• Probiotics * pharmacology   MeSH
• Inflammation metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Names of Substances
• Lipopolysaccharides MeSH

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.

• Keywords
• Caco-2 cells, enterohemorrhagic Escherichia coli, hemolytic uremic syndrome, human colonoids, intestinal epithelial barrier, outer membrane vesicles, translocation,
• Publication type
• Journal Article 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.

• Keywords
• 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
• Bacterial Toxins metabolism   MeSH
• Endothelial Cells metabolism   microbiology   pathology   MeSH
• Escherichia coli O157 * metabolism   pathogenicity   MeSH
• Virulence Factors metabolism   MeSH
• Humans MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Shiga Toxin 2 metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Toxins MeSH
• cytolethal distending toxin MeSH Browser
• Virulence Factors 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.

• Keywords
• EHEC, ETEC, intestinal pathogenic Escherichia coli, outer membrane vesicles, toxins, virulence factors,
• MeSH
• Bacterial Secretion Systems metabolism   MeSH
• Bacterial Toxins metabolism   MeSH
• Enterohemorrhagic Escherichia coli metabolism   pathogenicity   MeSH
• Enterotoxigenic Escherichia coli metabolism   pathogenicity   MeSH
• Enterotoxins metabolism   MeSH
• Virulence Factors metabolism   MeSH
• Stress, Physiological MeSH
• Escherichia coli Infections microbiology   MeSH
• Humans MeSH
• Proteolipids metabolism   ultrastructure   MeSH
• Intestines microbiology   MeSH
• Protein Transport MeSH
• Virulence MeSH
• Bacterial Outer Membrane metabolism   ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Bacterial Secretion Systems MeSH
• Bacterial Toxins MeSH
• Enterotoxins MeSH
• Virulence Factors MeSH
• Proteolipids MeSH
• proteoliposomes MeSH Browser