Genomic analysis of NF-kappaB signalling in Anopheles gambiae

先天免疫系统是抵御高等生物体感染的第一道防线。对于缺乏抗体的昆虫来说，先天免疫是唯一的防御系统。它依赖于识别微生物之间共享的特定分子结构的受体，然后将危险信号传递到对抗感染的效应器机制。在许多情况下，这些效应机制需要从头产生，这是通过表达受REL/核因子/kappaB(NF-kappaB)家族转录因子控制的基因来实现的。该项目旨在剖析非洲冈比亚按蚊中受核因子-kappaB信号通路控制的基因表达机制。这种蚊子是动物和人类疾病的媒介，包括由寄生虫疟原虫引起的疟疾。近年来，由于冈比亚按蚊基因组序列的公布和便捷的基因功能分析的发展，冈比亚按蚊已经成为研究微生物，特别是寄生虫如何与其免疫系统相互作用以及如何逃脱其免疫系统的模式系统。我们最近发现，蚊子的NF-kappaB免疫途径，即IMD或REL2，在感染期间负责杀死大量的疟原虫，从而限制蚊子的感染，从而限制其感染能力。同样的途径也与对抗蚊子感染细菌有关。此外，我们未发表的数据显示，另一个NF-kappaB途径，Toll或REL1的人工激活，可以导致疟原虫感染的完全阻断；有趣的是，该途径在寄生虫感染期间保持不活跃。在过去的五年里，我们开发了基于冈比亚按蚊基因组序列信息的高通量新技术。到目前为止，我们已经使用这些技术来了解蚊子生物学的各个方面，包括对细菌和寄生虫感染的反应。在这里，我们将利用并进一步开发这些技术来识别组成蚊子NF-kappaB信号通路的基因和基因网络。我们的第一个目标是确定核因子-kappaB转录网络。为此，我们将监测蚊子整个基因组的表达，以检测每个途径调控的基因，并直接研究核因子-kappaB因子与基因组之间的相互作用。下一个目标是确定在每条途径的激活过程中发挥作用的基因。这些基因及其相互关系的集合将定义核因子-kappaB基因网络。这是一项要求非常严格的研究，需要对蚊子基因组的几乎所有基因进行特定的沉默，并随后监测沉默效果。为此，我们将开发一种简单而强大的技术-dsRNA芯片，通过它，可以在玻璃片上进行一次测试来分析整个基因组。这项技术将使蚊子生物学取得重大突破，并将广泛适用于其他研究和生物体。根据我们目前的知识和上述研究的结果，我们最终将深入分析NF-kappaB通路在蚊子感染各种微生物过程中的作用。将特别强调这些途径在感染疟疾寄生虫期间的作用，因为这种寄生虫似乎正在操纵这些途径或逃避其激活。免疫逃避是免疫的一个重要但鲜为人知的方面。

The innate immune system is the first line of defence against infections in higher organisms. In insects, which lack antibodies, innate immunity is the only defence system. It relies on receptors that recognize specific molecular structures shared between microbes and then convey danger signals to effector mechanisms that counteract the infection. In many cases, these effector mechanisms require de novo production, which is achieved through expression of genes controlled by transcription factors of the Rel/Nuclear Factor/kappaB (NF-kappaB) family. This project aims to dissect the mechanisms of gene expression that are under the control of NF-kappaB signalling pathways in the African mosquito Anopheles gambiae. This mosquito is a vector of animal and human diseases including malaria which is caused by the parasite Plasmodium. In recent years, thanks to the availability of its genome sequence and development of convenient assays of gene function, A. gambiae has become a model system for studies of how microbes, especially parasites, interact with and, in the case of Plasmodium, escape its immune system. We have recently shown that a mosquito NF-kappaB immunity pathway, the Imd or REL2, is responsible for killing substantial numbers of Plasmodium parasites during an infection, thus limiting the mosquito infection and thereby its infectious capacity. The same pathway is also implicated in confronting mosquito infections with bacteria. In addition, our unpublished data reveal that artificial activation of another NF-kappaB pathway, the Toll or REL1, can lead to total blockade of Plasmodium parasite infection; interestingly, the pathway remains inactive during a parasitic infection. During the last five years, we have developed new high throughput technologies based on the sequence information of the A. gambiae genome. To date we have used these technologies to understand various aspects of the mosquito biology, including the reactions to bacterial and parasite infections. Here, we will exploit and further develop these technologies to identify the genes and gene networks that make up the mosquito NF-kappaB signalling pathways. Our first goal is to determine the NF-kappaB transcription networks. For this, we will monitor the expression of the entire mosquito genome to detect genes regulated by each pathway, and directly investigate the interactions between the NF-kappaB factors and the genome. The next goal is to identify genes playing a role in the activation of each pathway. Collections of such genes and their relationships will define the NF-kappaB genetic networks. This is a very demanding research requiring specific silencing of practically all the genes of the mosquito genome and subsequent monitoring of the silencing effect. For this reason, we will develop a simple and powerful technology, the dsRNA chip, by which the entire genome can be analyzed with a single test performed on a glass slide. This technique will permit a major breakthrough for mosquito biology and will be widely applicable to other studies and organisms. Based on our current knowledge and the results from the research described above, we will finally analyze in depth the function of NF-kappaB pathways during mosquito infections with various microbes. Special emphasis will be placed on the role of the pathways during infections with the malaria parasite, as it appears that the parasite is manipulating the pathways or evading their activation. Immune evasion is an important but little understood aspect of immunity.