Genomic analysis of NF-kappaB signalling in Anopheles gambiae

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 In addition, our unpublished data reveal that artificial activation of another NF-kappaB pathway, the Toll or REL1, can lead to total blockade of Plasmodium parasite infection;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.

Insect immune responses that are based upon activation of antimicrobial peptides and other defence proteins usually result from signalling through NF-kappaB transcription factors. We are studying NF-kappaB signalling in the mosquito Anopheles gambiae. Genome sequencing and subsequent technological breakthroughs have made A. gambiae a model system to study immunity, as it is a natural host of parasites and viruses that cause human and animal diseases. Its genome encodes two NF-kappaB factors, REL1 and REL2, orthologous to the Drosophila Dorsal and Relish, respectively. Our work has shown that signalling through REL2 is important for defence against infections with Gram-positive and negative bacteria, revealing a significant difference in the immune systems of Anopheles and Drosophila. The pathway also reduces significantly the prevalence of mosquito infection with the protozoan parasite Plasmodium. Preliminary data show that signalling through REL1 is not normally important in immunity against bacteria and parasites; however, constitutive activation of REL1 leads to complete blockade of parasite infection. This project will employ existing and develop new functional genomic technologies towards better understanding of NF-kappaB signalling in A. gambiae. Genome-wide RNAi screens are expected to illuminate NF-kappaB genetic networks and reveal essential players of the signalling pathways. This approach will be drastically enhanced by development of dsRNA chips allowing simultaneous assaying of the entire genome. Dam methylase identity assays in conjunction with gene silencing and DNA microarray analysis as well as chIP-chip assays will detect targets of the NF-kappaB factors in the mosquito genome. Finally, the role of NF-kappaB networks and their components during infections will be investigated in adult mosquitoes. Putative evasion of the mosquito immune responses by Plasmodium parasites through suppression of NF-kappaB signalling will be specifically addressed.