Genetic mechanisms of Host-Salmonella interactions

概述：本研究以沙门氏菌为模式生物，正向遗传学研究细胞内感染的生物学，阐明免疫相关基因的功能。正向遗传学是一种不偏不倚的方法，可以提供有关确定的生物途径所必需的基因功能的重要信息。一旦在体内确定了宿主基因，就可以使用细胞培养模型来表征它们的分子功能。在目前的计划中，我们建议使用协同杂交(CC)小鼠作为一种新的资源来研究宿主与沙门氏菌的相互作用。CC是一组来自8个创始菌株的近亲交配小鼠品系，同时询问多个基因，以揭示意外的基因功能和基因网络关系，使CC成为模拟宿主-沙门氏菌相互作用复杂性的特殊工具。*提出的研究计划有三个具体目标：1-识别新的突变和导致沙门氏菌细胞反应改变的途径。这一目标将通过使用细菌负载作为表型的数量性状基因座(QTL)作图来实现。同时，我们将进行脾组织表达QTL(EQTL)分析，以确定未受到攻击的CC小鼠的基线表型，以确定导致免疫功能变化的可遗传基因组座位。2-CC变异株的细胞表型将通过高通量流式细胞术测定不同组织中存在的免疫细胞的完整性、组成和激活状态，评估脾细胞中TLR信号的完整性，以及利用沙门氏菌感染的细胞培养模型来实现。这些实验对于在细胞水平上更好地表征CC变异株中宿主沙门氏菌相互作用的机制以及在QTL分析中将细胞表型与候选基因联系起来具有重要意义。候选基因的3-功能验证将使用等位基因互补分析、siRNA技术和/或从携带候选基因敲除等位基因的小鼠获得的细胞进行。*意义：该项目将利用尖端的遗传工具来探索复杂的宿主-沙门氏菌相互作用的新参与者。该项目的一项主要资产是，资源既新颖，确保将产生的结果的原创性，又随时可用，这保证了拟议工作的可行性。拟议的计划将促进我们对宿主与沙门氏菌复杂相互作用的了解，并将导致识别与先天免疫最相关的其他新机制。从长远来看，这项研究计划将提供涉及免疫细胞功能的新基因的详细特征。

Overview : My research program is using Salmonella as model organism and forward genetics for studying the biology of intracellular infection and to elucidate the function of immune related genes. Forward genetics is an unbiased approach for providing important information on gene function essential to defined biological pathways. Once the host genes are identified in vivo, their molecular function is characterized using cell culture models. In the current program we are proposing to use mice of the collaborative cross (CC) as a new resource to study the interaction between the host and Salmonella. The CC is a large panel of inbred mouse strains derived from eight founder strains and interrogates multiple genes at the same time to reveal unsuspected gene functions and gene network relationships, making the CC an exceptional tool to model the complexity of host-Salmonella interaction.******Specific Objectives and Methodology : There are three specific objectives to the proposed research program : 1 – Identification of novel mutations and pathways responsible for altered cellular response to Salmonella. This objective will be approached using Quantitative Trait Loci (QTL) mapping using bacterial load as phenotype. In parallel, we will perform expression QTL (eQTL) analysis in spleen to determine baseline phenotype in unchallenged CC mice to identify heritable genomic loci that contribute to the variation in immune function. 2 – Cellular phenotyping of CC deviant strains will be achieved by determining the integrity, composition and activation status of immune cells present in different tissues using high throughput flow cytometry, evaluation of the integrity of TLR signalling in splenocytes and by using cell culture model of Salmonella infection. These experiments are important to better characterize at the cellular level the mechanisms involved in host Salmonella interaction in CC deviant strains and to relate the cellular phenotype to candidate genes in QTLs analysis. 3 – Functional validation of candidate genes will be done using allelic complementation assays, siRNA technologies and/or cells derived from mice carrying a knock out allele at the candidate gene. ******Significance : This project will take advantage of cutting-edge genetic tools to explore new players of the complex host-Salmonella interaction. A major asset of this project is that the resources are both novel, ensuring the originality of the results that will be produced, and are readily available, which warranties the feasibility of the proposed work. The proposed program will advance our knowledge of the complex interaction of the host with Salmonella, and will lead to the identification of additional novel mechanisms most relevant to innate immunity. In long-term, this research program will provide detailed characterization of novel genes involved in immune cell function.