Functional consequences of dynamic MAPK signalling on the host defence response of vertebrates to bacterial infections

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In our world, living organisms constantly interact with each other. The outcome of these interactions is diverse and represented by different modes like predation, symbiosis, co-habitation or parasitism. I am particularly interested in understanding the molecular mechanisms determining the host response to pathogens. A key element of host defense against bacterial and fungal infections is the recruitment of neutrophils to site of infections. Therefore it is vitally important for the host to properly regulate neutrophilic inflammation. Upon sensing of pathogens, distress signals are emitted leading to the recruitment of neutrophils. Recently we have identified three key signaling pathways in epithelial cells, the first line of defense against infections that lead to neutrophil recruitment in response to gram-negative bacteria. The three pathways are: TAK1-MKK3-p38a, TAK1-IKKß-NF?B and TAK1-IKKß-TPL2-MKK1-ERK2. Together, these pathways coordinate the synthesis of inflammatory mediators. Although we have made good progress in identifying key signals important for the regulation of inflammation in response to bacterial infection, we are still missing a lot of the details that would contribute to a much better understanding of this important biological response. The majority of current approaches, including ours, use punctual probing to measure signal intensities, leaving important gaps in the information acquired. This grant aims to develop tools to overcome this limitation in order to get a more complete understanding of the regulation of inflammation triggered by infection of epithelial cells or a living organism, the zebrafish. Two objectives will be undertaken: Objective 1: Developing tools to dynamically image inflammation in real-time. To dynamically assess TLR-TAK1-mediated activation of the MKK3-p38a, IKKß-NF?B and IKKß-TPL2-MKK1-ERK2 pathways, we will be using the following technologies: Bimolecular fluorescence complementation (BiFC), Bioluminescence Resonance Energy Transfer (BRET) and luciferase transcriptional reporters. The tools generated will be used in the second objective to probe fundamental mechanisms of signal transduction regulating inflammation. Objective 2: Shedding light on the inflammatory response during host-pathogen interactions. In this second objective, we want to image inflammation in real-time occurring during infection of a) epithelial cells in culture or b) a whole living organism, the zebrafish. Following infection, we will measure dynamically the activation of intracellular signaling pathways and associated transcription factors. In parallel, using classical approaches we will measure the production of cytokines and neutrophil recruitment to evaluate the magnitude of inflammation. Taken together, these studies should paint a much clearer picture of the fundamental molecular mechanisms underlying the regulation of inflammation. Moreover, by comparing the organization of signaling networks in fish and humans regulating host defense responses, we will have a better understanding of the evolution of inflammatory response. Finally, our investigation in zebrafish infection can benefit the Canadian fish farming industry by providing novel insights into infections that have a significant economic impact.