Unit: Insect Biochemistry and Molecular Biology
Director: Charles ROTH par intérim
Our Unit works on three general research topics associated with the salivary gland. 1) Annotation of the Anopheles gambiae genome. 2) The transcriptome and proteome of the salivary gland and its parasite induced alteration. 3) The effects of saliva on the transmission of Plasmodium between the mosquito and the animal host. Our goal is to better understand how the malaria parasite interacts with its insect host and to find methods to interupt or regulate these interactions.
Annotation of the Anopheles gambiae genome (Charles Roth, Karin Eiglmeier, Shawn Gomez, Inge Holm, Pierre Dehoux, Kim Chaveroche)
Our unit participated in international consortium that recently sequenced the genome of Anophleles gambiae the principle vector of malaria (Science, 2002, 4 octobre, 298 (5591) : 129-49) and we are working with the consortium that is currently sequencing the genome of Aedes aegypti, the vector of dengue fever. The A. gambiae genome contians 278 million base pairs and encodes about 15,000 proteins. We have used the "full-length" or "5'-enriched" cDNA procedure to identify new genes and to improve the definition of previously identified genes. We are currently directing our studies toward the genes expressed in the mosquito salivary gland and the identification of those proteins that are excreted. The sequence of the A. aegypti genome, about 5X longer than that of A. gambiae. and its cDNAs will allow comparative analyses of the two mosquito genomes and better identify genes, including salivary gland genes, in both mosquitoes. These studies should help identify salivary proteins and understand their role in parasite maturation and transmission. Additionally, the accurate identification of mosquito proteins will help us predict the interactions between various parasite and mosquito salivary gland proteins in order to identify targets for reducing parasite transmission.
Alterations of Anopheles gambiae salivary gland transcriptome during infection by Plasmodium (Isabelle Rosinski-Chupin, Sylvie Perrot and Paul Brey)
For the successful completion of their life cycle in mosquitoes, the malaria parasites must migrate through the salivary gland epithelial barrier. Our working hypothesis is that the presence of the parasite inside or in close contact with the salivary gland cells will induce cellular defense mechanisms and a change in gene expression. This change might in turn have a role in the survival of the parasite and ; therefore, in its transmission. We have recently validated the SAGE (Serial Analysis of Gene Expression) method as a powerful tool for transcriptome studies 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. The function of these genes in the Anopheles/Plasmodium interactions are under study. This work is part of the Anopheles "Strategic Horizontal Program" at the Pasteur Institute.
Contributions of Anopheles/Plasmodium interactions to sporozoite infectivity (Thomas Chertemps, Sylvie Perrot, Anna raibaud and Isabelle Rosinski-Chupin)
During the time-course of their storage in the gland, sporozoites undergo a genetic program that equips them with the proteins required for hepatocyte invasion and subsequent liver stage development in their vertebrate host. The transcriptome analysis of infected salivary glands allowed us to characterize some of these Plasmodium genes whose expression varies during the salivary phase. Our aim is now to use these genes as markers of sporozoite maturation and to determine the contribution of Plasmodium/ salivary epithelium interactions to the modulation of gene expression in sporozoite.
Proteomics approach of A. gambiae saliva and salivary glands (Valérie Choumet, Virginie Jan, Annick Carmi, Annie Robbe-Vincent)
Our group carries out a proteomics approach toward 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 display proteomics analysis, carried out in collaboration with the platforms "Protéomique" and "Analyse et Microséquençage des Protéines" of the Pasteur Institute. We thus could identify 86 components from salivary gland extract of young blood-fed females, 46% of them being proteins of saliva (vasodilators, allergens, enzymes of digestion of sugars and proteins of unknown functions). With regard to the functional proteomics approach, our team focusses its efforts on the characterization of molecules involved in blood feeding. The effects of the components of salivary glands on proteins of the cascade of coagulation were characterized. The kinetics and the nature of the pro-inflammatory effects were studied on a murine model in collaboration with the Unit of Histotechnology et Pathology. The screening of the pharmacological activities made it possible to characterize several activities never yet described in the Anopheles mosquitoes. Generally, the Anopheles gambiae salivary glands contain inhibitory compounds for active factors such as: kallikrein, FXa, thrombin and protein Ca, urokinase and plasmin. The composition of salivary glands was shown to vary according to the age of the mosquito, to whether it took a blood meal or not and to whether the mosquito is infected by Plasmodium or not. The identification as well as the characterization of the mechanism of action of the proteins implied in these pharmacological effects could allow their use as tools to dissect certain physiological mechanisms and to propose new drugs or diagnostic tests. In addition, certain components could provide new targets within the framework of studies of transgenesis for a better understanding of host/vector/parasite interactions.
Differential gene expression in the ookinete stage of the malaria parasite Plasmodium berghei (A. Raibaud)
The ookinete is the Plasmodium stage, which invades the mosquito midgut. We constructed an ookinete Suppression Subtractive Hybridization (SSH) cDNA library, subtracted by zygote cDNA, the zygote being the preceeding developmental stage. Ookinete specific clones were selected by a differential screening of the library. Sequence analysis revealed that a majority of the clones carried sequences for four known abundant ookinete secreted proteins, which allowed for validation of the library. The remaining clones were further analysed. Their sequences showed a strong homology with Plasmodium yoelii, whose genome had been sequenced and annotated, thus a probable funcion could be assigned to the P. berghei orthologs..
Analysis of the ookinete specific genes' transcription in ookinete and zygote stages demonstrated that all the genes are differentially transcribed in the ookinete. For 14 of the 15 genes, our data present the first evidence of transcription in ookinetes. Four genes are also expressed in other Plasmodium invasive stages : the sporozoite and/or the merozoite, suggesting that they code for proteins with a common function.
10 of the 15 genes encode proteins with a putative function. The identified proteins include invasion proteins such as the MACPF (membrane-attack complex/perforin)and regulatory proteins such as P-ATPase, an ion transporting protein,and Ca-dependent protein kinase (CDPK). Additional genes include defense proteins, such as thioredoxin, which could have a protective effect for the ookinete, which has to resist the mosquito immune defences.
Evolutionary ecology of Plasmodium and parasite transmission strategies (Richard Paul and Ramatoulaye Lawaly)
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 hematological state of the host suggesting that the parasite is capable of adaptive facultative investment. The parasite seemingly alters its sex allocation to ensure fertilization. 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, notably the sudden seasonal increase in mosquito bites per se (Paul et al. 2004). Indeed, preliminary results show a strong correlation between the tendency for an infection to produce gametocytes and the biting density. Our current research focus is to examine the potential role that allergy may play in this response.
Keywords: insect vectors, malaria, paludism, salivary gland, transcriptome, SAGE, proteome, parasite transmission