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

在我们的世界里，活着的有机体不断地相互作用。这些相互作用的结果是多种多样的，表现为不同的模式，如捕食、共生、共生或寄生。我对了解决定宿主对病原体反应的分子机制特别感兴趣。宿主防御细菌和真菌感染的一个关键因素是将中性粒细胞招募到感染部位。因此，宿主适当地调节中性粒细胞炎症是至关重要的。一旦感觉到病原体，就会发出求救信号，导致中性粒细胞招募。最近，我们在上皮细胞中确定了三条关键的信号通路，这是抵御感染的第一道防线，感染导致中性粒细胞募集反应革兰氏阴性细菌。这三条通路分别是：TAK1-MKK3-P38A、TAK1-IKK？-NFB和TAK1-IKK？-TPL2-MKK1-ERK2。这些途径共同协调炎症介质的合成。尽管我们在确定对细菌感染反应的炎症调控至关重要的关键信号方面取得了良好的进展，但我们仍然缺少许多细节，这些细节有助于更好地了解这一重要的生物反应。目前的大多数方法，包括我们的方法，都使用准时探测来测量信号强度，在获取的信息中留下了重要的空白。这笔赠款旨在开发工具来克服这一限制，以便更全面地了解由斑马鱼上皮细胞或活生物体感染引发的炎症调节。将实现两个目标：目标1：开发实时动态成像炎症的工具。为了动态评估TLR-TAK1介导的MKK3-P38A、ikk？Nf？B和ikk？Tpl2-MKK1-ERK2通路的激活，我们将使用以下技术：双分子荧光互补(BIFC)、生物荧光共振能量转移(BRET)和荧光素酶转录报告。所产生的工具将用于第二个目标，以探索信号转导调节炎症的基本机制。目的2：阐明宿主与病原体相互作用过程中的炎症反应。在第二个目标中，我们想要实时成像在a)培养上皮细胞或b)整个活的有机体--斑马鱼--感染期间发生的炎症。感染后，我们将动态检测细胞内信号通路和相关转录因子的激活情况。同时，使用经典方法，我们将测量细胞因子的产生和中性粒细胞的募集，以评估炎症的程度。综上所述，这些研究应该会为炎症调控的基本分子机制描绘出一幅更加清晰的图景。此外，通过比较鱼类和人类调节宿主防御反应的信号网络的组织，我们将更好地理解炎症反应的进化。最后，我们对斑马鱼感染的调查可以为加拿大养鱼业提供对重大经济影响的感染的新见解，从而使加拿大水产养殖业受益。

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.