Structure and Function of Drosophila NF-kappaB Signaling Pathways

 描述(申请人提供)：Toll和Toll样受体(TLRs)在从昆虫到人类的各种动物的先天性免疫反应中发挥核心作用。虽然我们对启动Toll信号的病原体识别和信号机制本身知道很多，但我们对介导先天性免疫防御的效应器知之甚少。为了满足这一需求，我们对基因表达进行了跨物种比较分析，以确定果蝇中Toll调节的免疫谱系的保守特征。将我们的注意力集中在以这种方式确定的一个新的免疫效应器家族上，我们使用了最先进的基因组工程方法来消除12个家族成员中的10个成员的功能。值得注意的是，失活这10个基因降低了微生物感染后的存活率，程度和特异性与完全阻断Toll信号的程度相同。这些发现确立了我们所命名的Bom基因家族的基本免疫功能，并为原创性和创新性的效应肽功能分析提供了基础。通过对果蝇的研究，我们可以很容易地产生扰乱途径活动的突变，监测和操纵基因活动，并通过分子、生化和生物信息学方法绘制出基因功能的网络。利用转基因和诱变的组合，我们将把Bom介导的防御与特定的病原体相匹配，并描述Bom多肽之间的结构-功能关系。然后，我们将把这些发现应用于多肽功能的体外研究的设计和解释，重点放在抗菌活性上。接下来，我们将探索Bom基因在体内的充分性，并描述Bom与先前描述的抗菌肽的功能相交或重叠的程度。最后，我们将开始通过对其他新的效应器基因簇的遗传和表型分析，绘制出更广泛的Toll介导的防御网络。

 DESCRIPTION (provided by applicant): Toll and Toll-like receptors (TLRs) have a central role in innate immune responses in animals ranging from insects to humans. Whereas we know a great deal about the pathogen recognition that initiates Toll signaling and the signaling mechanism itself, we know considerably less about the effectors that mediate innate immune defenses. To address this need, we carried out a cross- species comparative analysis of gene expression to identify conserved features of the Toll- regulated immune repertoire in Drosophila. Focusing our attention on a novel family of immune effectors identified in this manner, we used a state-of-the-art genomic engineering approach to eliminate function in ten of twelve family members. Remarkably, inactivating these ten genes decreased survival upon microbial infection to the same extent and with the same specificity as a complete block in Toll signaling. These findings establish the essential immune function of what we have named the Bom gene family and provide the basis for an original and innovative analysis of effector peptide function. By working in Drosophila, we can readily generate mutations that disrupt pathway activity, monitor and manipulate gene activity, and map out networks of gene function via molecular, biochemical, and bioinformatic approaches. Using a combination of transgenesis and mutagenesis, we will match Bom-mediated defenses to particular pathogens and delineate the structure-function relationships among the Bom peptides. We will then apply these findings to the design and interpretation of in vitro studies of peptide function, focusing on antimicrobial activity. Next, we will explore Bom gene sufficiency in vivo and delineate the extent to which the function of Bom and previously described antimicrobial peptides intersect or overlap. Finally, we will begin mapping out the broader network of Toll mediated defenses by genetic and phenotypic analysis of clusters of other novel effector genes.