Genomic analysis of NF-kappaB signalling in Anopheles gambiae

先天免疫系统是高等生物体抵御感染的第一道防线。在缺乏抗体的昆虫中，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给抵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，这是通过表达由Rel/核因子/kappaB（NF-κ B）家族的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NF-κ B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，包括由疟原虫寄生虫引起的疟疾。近年来，由于其基因组序列的获得和方便的基因功能检测方法的发展，A。冈比亚疟原虫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，以及疟原虫如何逃脱其免疫系统的模型系统。我们最近已经表明，蚊子NF-κ B免疫途径，Imd或IMD 2，负责在感染期间杀死大量的疟原虫寄生虫，从而限制蚊子感染，从而限制其感染能力。同样的途径也与蚊子感染细菌有关。此外，我们未发表的数据显示，人工激活另一个NF-κ B通路，Toll或Toll 1，可以导致疟原虫感染的完全阻断;有趣的是，该通路在寄生虫感染期间保持不活动。在过去的五年中，我们开发了新的高通量技术的基础上的序列信息的A。冈比亚基因组。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构成蚊子NF-κ B信号通路的基因和基因网络。我们的第一个目标是确定NF-κ B转录网络。为此，我们将监测整个蚊子基因组的表达，以检测由每个途径调节的基因，并直接研究NF-κ B因子与基因组之间的相互作用。下一个目标是确定在每个通路的激活中起作用的基因。这些基因的集合及其关系将定义NF-κ B遗传网络。这是一项非常苛刻的研究，需要特异性沉默蚊子基因组的几乎所有基因，并随后监测沉默效应。因此，我们将开发一种简单而强大的技术，即dsRNA芯片，通过该芯片，可以在载玻片上进行单一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。基于我们目前的知识和上述研究结果，我们最终将深入分析NF-κ B通路在蚊子感染各种微生物期间的功能。特别强调的是，在疟疾寄生虫感染过程中的作用的途径，因为它似乎是寄生虫操纵的途径或逃避其激活。免疫逃避是免疫的一个重要方面，但了解甚少。

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.