Novel signaling pathways : induced by bacterial toxins in eukaryotic cells

Abstract

Inflammation is the process by which the host counteracts microbes that are constantly exposing the human body. These microbes rarely cause disease, due to the effective defense mechanisms of the host immune system. Although inflammation is essential for preventing infections, it can also damage the host. Thus, the host immunity is subjected to a tight control at multiple levels and the responses are results of a complex interplay between microbes and the target cells. It has been shown that interaction between the E. coli toxin Hly and renal epithelial cells induces intracellular signaling resulting in Ca2+ oscillations with the periodicity of 12 min. These Hly-induced Ca2+ oscillations mediate expression and secretion of the pro-inflammatory mediators interleukin (IL)-6 and IL-8.

In this thesis, we provide a model of the molecular mechanisms transmitting the Hly-mediated signaling event into Ca2+ oscillations. This model demonstrates the requirement of interaction between Hly and the trans-membrane protein glycophorin, which acts as a signaling receptor. The binding mechanism, which involves all the functional domains of the Hly-protein, results in activation of the cytoskeleton effector proteins RhoA and ezrin, as well as stimulation of the IP 3 R signaling pathway. The signaling components are recruited to plasma membrane lipid rafts forming a dynamic signalosome, thus mediating efficient transfer of information and stimulation of Ca2+ oscillations with a specific periodicity.

We present moreover a new member ClyA, to the growing family of bacterial pore-forming toxins causing alteration of the intracellular Ca2+ concentration. ClyA is delivered to the target cell as active pore assemblies within outer membrane vesicles (ClyA+.OMV), which upon interaction mediate Ca2+ oscillations with a periodicity of 23 min. Our data suggests that the toxin is being internalized using cholesterol-rich lipid rafts as portals of entry, thus initiating a signaling cascade involving PLC and activation of the 1P3R governed Ca 2+ channels. Molecular investigations show that the ClyA protein is nonimmunogenic, while LPS and OmpW present within the OMVs elicit an IL8 response. The biological outcome of the Ca2+ oscillations induced by ClyA+OMV is currently unknown.

System biology can provide a new facilitating tool to investigating these complex molecular pathways involved in toxin-induced Ca2+ signaling. We present the first theoretical study describing Ca2+ oscillations involving a pore-forming toxin. This minimal generic model describes Ca2+- oscillations in renal epithelial cells stimulated by Hly. This model can improve our understanding of how bacterial toxins can modulates gene expression, which is a mechanism of major importance for the host inflammatory response.

This thesis contributes to our understanding of the interplay between inflammatory responses and the pathogen's ability to regulate and escape the host immune defense system.