title: Effects of the Escherichia coli toxin cytolysin A on mucosal immunostimulation via epithelial Ca2+ signalling and Toll-like receptor 4.
abstract: Epithelial cells are vital to sense the presence of bacteria, thereby initiating a proper innate immune response. This occurs via different mechanisms, e.g. recognition by pattern recognition receptors (TLR), or alteration of the cellular Ca2+ homeostasis. The Escherichia coli toxin cytolysin A (ClyA) is naturally delivered to target cells as active pore assemblies within outer membrane vesicles (OMVs), and we here investigate a possible role of ClyA-containing OMVs (ClyA+ (OMV)) for induction of proinflammatory responses via the above-mentioned mechanisms. We report that low, sublytic concentrations of ClyA+ (OMV) affect the Ca2+ homeostasis in epithelial cells by induction of slow, intracellular Ca2+ oscillations, while increased concentrations act cytolytically. Thus, ClyA belongs to the novel group of pore-forming toxins shown to elicit such biphasic responses. Ca2+ waves in the minute range have been shown to regulate gene transcription of, e.g. interleukin (IL)-6 and -8. While the periodicity of ClyA+ (OMV)-induced Ca2+ waves (22.9 +/- 0.9 min) fail to induce an IL-8 response, our data fit to the general concept of frequency-specific gene expression. Molecular investigations of the signal transduction pathway reveals that ClyA+ (OMV) utilize a different one as compared with those previously reported for other toxins causing Ca2+ waves. The ClyA protein per se and ClyA pore assemblies are non-immunogenic, while lipopolysaccharide present on the OMVs induces a TLR4-dependent proinflammatory response as expected. Additional membrane components of the OMV, e.g. OmpW, was also found to elicit proinflammatory responses that was independent of TLR4 and Ca2+ signalling.
authors.affiliation: Microbiology and Tumor Biology Centre, Karolinska Institutet, S-171 77, Stockholm, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Söderblom
authors.forename: Camilla
authors.initials: C
authors.lastname: Oxhamre
authors.forename: Sun Nyunt
authors.initials: SN
authors.lastname: Wai
authors.forename: Per
authors.initials: P
authors.lastname: Uhlén
authors.forename: Anita
authors.initials: A
authors.lastname: Aperia
authors.forename: Bernt Eric
authors.initials: BE
authors.lastname: Uhlin
authors.forename: Agneta
authors.initials: A
authors.lastname: Richter-Dahlfors
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000276:immunology* / Q000382:microbiology; D004926:Escherichia coli / Q000276:immunology; D029968:Escherichia coli Proteins / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D007113:Immunity, Innate; D018928:Immunity, Mucosal; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D008562:Membrane Glycoproteins / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000502:physiology*; D015290:Second Messenger Systems; D015398:Signal Transduction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; C119944:hlyE protein, E coli; C064035:ompW protein, E coli; D002118:Calcium