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

动态 MAPK 信号传导对脊椎动物细菌感染宿主防御反应的功能影响

• 批准号: RGPIN-2014-05963
• 负责人: Rousseau, Simon
• 金额: $ 2.55万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=567498
• 关键词: Functional; consequences; dynamic; MAPK; signalling

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.

在我们的世界里，生物体不断地相互作用。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，同居或寄生。我对理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募集到感染部位。因此，宿主适当调节嗜酸性炎症是至关重要的。在感测到病原体时，发出求救信号，导致中性粒细胞的募集。最近，我们已经确定了上皮细胞中的三个关键信号通路，这是对抗感染的第一道防线，导致革兰氏阴性细菌引起中性粒细胞募集。这三条通路分别是：TAK 1-MKK 3-p38 a，TAK 1-IKK 3-NF？B和TAK1-IKK1-TPL 2-MKK 1-ERK 2。这些通路共同协调炎症介质的合成。尽管我们在识别对细菌感染引起的炎症反应具有重要调节作用的关键信号方面取得了很大进展，但我们仍然缺少许多有助于更好地理解这一重要生物反应的细节。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。这项资助旨在开发克服这一限制的工具，以便更全面地了解上皮细胞或活生物体（斑马鱼）感染引发的炎症调节。目标1：开发实时动态成像炎症的工具。动态评估TLR-TAK 1介导的MKK 3-p38 a、IKK 3-NF？B和IKK 1-TPL 2-MKK 1-ERK 2通路，我们将使用以下技术：双分子荧光互补（BiFC）、生物发光共振能量转移（BRET）和荧光素酶转录报告基因。所产生的工具将用于第二个目标，以探索调节炎症的信号转导的基本机制。目的2：阐明宿主-病原体相互作用过程中的炎症反应。在第二个目标中，我们希望对a）培养中的上皮细胞或B）整个活生物体（斑马鱼）感染期间发生的炎症进行实时成像。感染后，我们将动态测量细胞内信号通路和相关转录因子的激活。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募集，以评估炎症的程度。总之，这些研究应该描绘了一个更清晰的图片的基本分子机制的调节炎症。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，我们将更好地了解炎症反应的演变。最后，我们对斑马鱼感染的调查可以通过提供对具有重大经济影响的感染的新见解而使加拿大鱼类养殖业受益。