Effects of the Escherichia coli toxin cytolysin A on mucosal immunostimulation via epithelial Ca2+ signalling and Toll-like receptor 4

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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.