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 Kim Chaveroche, Victoria Dominguez and Paul Brey)
The Institut Pasteur is a member of the international consortium that successfully sequenced the genome of Anopheles gambiae, the 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. Our main focus is the genes transcribed in the salivary gland of the mosquito. As a step toward identifying the proteins in the saliva, we are working toward the prediction of all the A. gambiae secreted proteins. 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.
For more information, please visit our unit web site : http://portail.pasteur.fr/recherche/unites/bbmi
Proteomics approach of A. gambiae saliva and salivary glands (Valérie Choumet, Virginie Jan, Annick Carmi-Leroy, Annie Robbe Vincent 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. Different SAGE libraries corresponding to different times after infection have been prepared. Their comparison points out a number of genes which expression is modified during infection. Validation of these results using other methods is underway, in the experimental model A. gambiae/ P. berghei as well as in the natural system A. gambiae/P. falciparum. This work is part of the Anopheles "Strategic Horizontal Program" at the Pasteur Institute.
Differentially expressed genes of the ookinete of Plasmodium (Anna Raibaud and Paul Brey)
The ookinete is responsible for the transmission of Plasmodium to the mosquito. By use of the technique of subtractive hybridization, a cDNA bank had been constructed, which was enriched in ookinete specific sequences from the rodent parasite P. berghei. We carried out a differential screening of the bank, and isolated clones that are specific for the ookinete. The sequence reads of the selected clones were compared with the sequences in the EMBL database. We identified four genes (the Chitinase, CTRP, SOAP and WARP), known to be expressed in the ookinete; the presence of these genes allowed the validation of the bank. However, for 33% of the clones, no homology was found in the EMBL database. The sequences of these clones were searched in the Plasmodium database, PlasmoDB. Strong homology was found with genes from P. yoelii, another rodent Plasmodium. The genome of P. yoelii has been sequenced and the genes are annotated, which allowed us to predict the function of the corresponding proteins. Finally, by expression analysis, we confirmed the differential transcription of the cloned ookinete genes. We also demonstrated the expression of some of these genes in the two other invasive stages of Plasmodium, the sporozoite and the merozoite. This observation indicates that they code for proteins that might play a key role in the general process of invasion.
Evolutionary ecology of Plasmodium and parasite transmission strategies (Richard Paul and Paul Brey)
Transmission of Plasmodium from the vertebrate host to the insect vector is mediated by the sexual stages, the gametocytes. There is growing evidence linking sex allocation to the haematological state of the host suggesting that the parasite is capable of adaptive facultative investment. The parasite seemingly alters its sex allocation to ensure fertilisation. Such an adaptive strategy advocates an important role for mating assurance that may be especially important when gametocyte densities are very low. In areas where the transmission is seasonal, malaria parasites survive in man as low density chronic infections that enable the parasites to survive during the non-transmission season and are the source of gametocytes, infecting the mosquito vectors upon their return. The fragility of the transmission process from low density gametocyte infections and the apparent adaptive fertility assurance responses, would suggest that gametocyte phenotypic evolution is shaped, to a large extent, by the pressure of transmitting from chronic infections. Identifying the key selection pressures is the first step in a constructive approach to disease control. Seasonal adaptive gametocyte production has been suggested to occur in temperate Plasmodium spp. and related haemosporidia. Qualitative analysis of historical data sets leads us to believe that P. falciparum may also respond to seasonal cues (Paul et al. 2004). Our current research focus is to examine the hypothesis that the parasite responds to the sudden seasonal increase in uninfected mosquito bites following the rains by increasing its sex allocation.
Local Expression of Antimicrobial peptide genes in Drosophila epithelia. (P. Brey)
A fundamental question that applies to all organisms is how barrier epithelia efficiently manage continuous contact with microorganisms. In a collaborative study with Won-Jae LEE (Ewha University), we show that in Drosophila an extracellular immune-regulated catalase (IRC) mediates a key host defense system that is needed during host-microbe interactions in the gastrointestinal tract. Strikingly, adult flies with severely reduced IRC expression show high mortality rates even after simple ingestion of microbe-contaminated foods. However, despite the central role that the NF-κ B pathway plays in eliciting antimicrobial responses, NF-κ B pathway mutant flies are totally resistant to such infections. These results imply that homeostasis of redox balance by IRC is one of the most critical factors affecting host survival during continuous host-microbe interactions in the gastrointestinal tract.
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 controlled by a serine protease inhibitor, the Serpin-27A, also involved in the regulation of immune responses and in development. Three genes homologous to the Serpin-27A gene have been cloned in A. gambiae. The differential properties of the three serpins, in particular their potential role in the melanization response, are currently investigated, both in vitro and in vivo.
Keywords: Malaria, Ookinete, Gametocytes, mosquito, catalase, NF-?B, innate immune, genome, Anopheles gambiae, Plasmodium, cDNA « full-length »