Unit: Insect Biochemistry and Molecular Biology
Director: BREY Paul
Our research unit is working on three tightly linked subjects : 1) Anopheles genomics and post-genomics : 2) host-parasite interactions between malaria parasites and mosquito vectors especially during the early phases of the sporogonic cycle, 3) Dipteran innate immune response with special focus on the regulation of the melanization cascade and signal transduction pathways involved in immune response. Our aim is to better understand the natural history and interactions of the malaria parasite within its invertebrate host with the hope of finding ways to interrupt or regulate this interaction.
Genomics of A. gambiae (Charles Roth, Karin Eiglmeier, Shawn Gomez, Inge Holm, Pierre Dehoux and Paul Brey)
The Institut Pasteur is a member of the international consortium that successfully sequenced the genome of this important malaria parasite vector. The sequence of the genome and the preliminary analysis of the genes was completed in 2002 (Science. 2002 Oct 4;298(5591):129-49.). The 278 million base pair sequence covering about 90% of the genome was predicted to contain nearly 15 thousand genes. Our unit is working to increase the accuracy of the gene and protein predictions using full length cDNA sequences. These studies will allow us to predict which mosquito proteins might interact with each other or with the malaria parasite in the mosquito salivary gland and thus pinpoint potential targets for blocking parasite transmission. We are also studying membrane transporters in the mosquito that resemble those used by human cancer cells to evade the killing effects of anit-tumor drugs. These studies could lead to drugs that make human tumor treatment as well as insect control more effective.
Proteomics approach of A. gambiae saliva and salivary glands (Valérie Choumet, Virginie Jan, Annie Robbe-Vincent, Annick Carmi-Leroy and Paul Brey)
Our team carries out a structural and functional proteomics approach of Anopheles gambiae saliva and salivary glands. On one hand, there is now compelling evidence that the pharmacological activity of arthropod saliva has a profound effect on pathogen transmission. The study of its composition could then provide new vaccine targets in the prevention and the treatment of malaria. On the other hand, the saliva of blood-sucking insects is known to contain a large variety of peptides and proteins whose great specificity of action makes them powerful pharmacological tools to dissect certain physiological mechanisms and to propose new drugs or diagnostic tests. The structural proteomics analysis, carried out in collaboration with the platforms "Protéomique" and "Analyse et Microséquençage des Protéines" of the Pasteur Institute. With regard to the functional proteomics approach, our team will more particularly focus her efforts on the characterization of molecules involved in blood feeding.
Alterations of A. gambiae salivary gland transcriptome during infection by Plasmodium (Isabelle Rosinski-Chupin, Sylvie Perrot and Paul Brey).
For the successful completion of the life cycle in mosquitoes, the parasites must migrate through the salivary gland epithelial barrier. Our work hypothesis is that the presence of the parasite inside or in close contact with the salivary gland cells will induce cellular defense mechanism, and a change in gene expression. This change might in turn have some role in the survival of the parasite, and therefore, in the transmission of the parasite. We have recently validated the SAGE (Serial analysis of gene expression) method as a powerful tool for transcriptome approach in Anopheles and are currently using this tool to characterize the changes in gene expression during salivary gland infection by Plasmodium. This work is part of the Anopheles "Strategic Horizontal Program" at the Pasteur Institute.
Gene discovery in Plasmodium- host cell interactions during the sporogonic cycle (Anna Raibaud, Richard Paul and Paul Brey)
Currently, our studies are part of a transversal research program in collaboration with two other laboratories within the Pasteur Institute (Unité Postulante de Biologie et Génétique du Paludisme et Unité Génétique de la Différentiation). The object of our group is to identify and characterise novel genes that are expressed by the ookinete stage of the parasite, being responsible for the invasion of the mosquito. Our model is the rodent malaria parasite P. berghei. In order to isolate ookinete specific sequences we applied a recently developed gene discovery approach; suppression subtractive hybridisation (SSH). We constructed a subtractive ookinete cDNA library, and a differential screening of this library allowed us to isolate clones from this specific parasite stage.
Evolutionary ecology of Plasmodium and virulence-transmission interactions according to infection complexity (Richard Paul and Paul Brey)
The population structure of parasitic organisms determines the degree of parasite inbreeding, which has important epidemiological consequences and is a strong determinant in the evolution of virulence. Virulence is, however, a complex phenotype; parasite sex ratio is predicted to be highly informative for how virulence might evolve in natural populations as both are predicted to respond in the same way to population structure and can thus be modelled similarly. Anaemia is the major form of virulence in malaria and the host haematological state is correlated with parasite sex allocation: reticulocyte density positively correlates with gametocyte density and in animal models of malaria, erythropoietin was implicated in gametocyte sex determination (Paul et al. 2000). Thus anaemia is a physical manifestation of virulence and an indirect trigger of sex allocation. Establishing how infection genetic diversity relates to anaemia has not only considerable importance for disease management but also for influencing parasite transmission phenotypes.
Local Expression of Antimicrobial peptide genes in Drosophila epithelia. (P. Brey)
In Drosophila melanogaster, although the NF-κB transcription factors play a pivotal role in the inducible expression of innate immune genes, such as antimicrobial peptide genes, the exact regulatory mechanism of the tissue-specific constitutive expression of these genes in barrier epithelia has remained largely unknown. In our collaborative study with W-J Lee (Ewha University), we have demonstrated that the Drosophila homeobox gene product Caudal functions as the innate immune transcription modulator that is responsible for the constitutive local expression of antimicrobial peptides cecropin and drosomycin in a tissue-specific manner. These results suggest that certain epithelial tissues have evolved a unique constitutive innate immune strategy by recruiting a developmental "master control" gene.
Characterization of the serpin-27A homologue in A. gambiae (Isabelle Rosinski-Chupin, Sylvie Perrot and P. Brey)
Melanotic encapsulation of Plasmodium in some refractory strains of Anopheles results in a block to parasite transmission. For this reason, we are interested in the regulation of the melanization cascade in A. gambiae, especially in the regulation of phenoloxidase activity, where phenoloxidase is the key enzyme in the melanization response. In Drosophila, the proteolytic cascade leading to phenoloxidase activation might be controled by a serine protease inhibitor, the Serpin-27A, also involved in the regulation of immune responses and in development. We have identified in Anopheles sequence databases three homologues of the Drosophila Serpin 27A and are currently studying the differential properties of these serpins.
Keywords: Mosquito, insect, vector, Drosophila, immune response, parasites, genomics, Anopheles gambiae, Plasmodium