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  Director : Paul BREY (pbrey@pasteur.fr)


  abstract

 

Our Unit is concentrating on three inter-related research themes: 1) Anopheles gambiae genomics, 2) Malaria parasite/mosquito interactions especially during sporogonic development and 3) Insect immunity where we are focusing on the enzymatic cascade leading to melanotic encapsulation. Our aim is to better understand how malaria parasites interact with their insect hosts in order to eventually find ways to interrupt or regulate this nteraction.



  report

cale

Gene discovery in Plasmodium- host cell interactions during the sporogonic cycle (R. Paul, A. Raibaud, K. Brahimi and Paul Brey)

Plasmodium infections impose an enormous public health burden on tropical communities resulting in 2 million deaths annually, notably in children. Resistance of Plasmodium to most of the antimalarial drugs available is widespread and no efficient vaccine exists. An increased knowledge of the molecular changes occurring during the life cycle of the parasite and its interactions with host cells should permit the development of alternative strategies to struggle against malaria.

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 the program we propose is to identify and functionally characterise novel genes which are expressed by the two sporogonic invasive stages (the ookinete and the sporozoite) and the hepatocyte. We will apply a recently developed gene discovery approach, suppression subtractive hybridisation (SSH) and use the P. berghei system, whose entire life cycle can be achieved in the laboratory, for which the infection of hepatocytes can go to completion in vitro and which is the model of choice for molecular studies because of the relative ease of targeted mutagenesis. In this way we aim to identify novel therapeutic targets.

 

Genomics of Anopheles gambiae (Charles Roth, Marine Grailles, Inge Holm and Paul Brey)

For the last several years our unit has been working on the genome of the mosquito Anopheles gambiae that is the major vector of malaria in Africa. Understanding the genes that control all aspects of mosquito growth, behavior and environmental response will lead to better control of malaria transmission by this vector. The Institut Pasteur is a partner in an international consortium that is sequencing the entire 270 megabase (millions of bases) genome of this mosquito. This project will identify all the genes in this medically important

mosquito and allow their comparison of those the human and the malaria parasite whose genomes have already been sequenced. This comparison should lead to new ways of understanding the host-vector relationship and to control the disease.

In preparation for this large sequencing effort our laboratory has been working in collaboration with the French National Sequencing Center - "Genoscope" to characterize the genome and its genes. The first project created more than 22,000 sequences from random regions of the genome. This was done by sequencing the ends of large chromosomal fragments cloned in Bacterial Artificial Chromosomes (BACs). The sequences from the ends of these fragments provide information about all the chromosomes, including the genes, the transposons and the repetitive sequences they contain. For example, our analysis of these sequences has identified partial sequences from more than 1000 genes not previously identified in this mosquito. We have also identified numerous new transposable element genes and identified new families of these elements that play a role in rearrangements. We have also identified nearly a thousand microsatellite sequences that could be used as "markers" to identify and follow mosquito population movements in the field.

A second project has been work with Genoscope to sequence a chromosomal section in collaboration with our partners in Germany and the United States to demonstrate the feasibility of sequencing a large genomic region. This sequenced region will provide the standard for the correctness of the full genome sequence. The region was chosen because it contains a gene important for the ability of the malaria parasite to grow and be transmitted by A. gambiae. Genoscope has sequenced a section of 500,000 bases constructed from five overlapping BAC clones and two smaller sections.

The unit is also studying the insect genes coding for transporter proteins associated with "multidrug-resistance" in humans. These transporters function in a network of enzymes that reduce the level of environmental toxins in an animal. We are studying the multidrug-resistance genes and their products to learn how these important transporters work both in humans and in insects. By identifying the parts of these proteins that select the product to be exported, it may be possible to design compounds that can reduce their capacity to transport certain toxic products such as drugs that kill human tumors or insecticides that can control mosquitoes that transmit disease. To this end, we are dissecting the "multidrug-transporter" encoding genes in the fruitfly D. melanogaster and in the malaria vector mosquito A. gambiae. These studies are identifying domains of the transporter protein that these insects can alter rapidly and which suggests that they are important for determining the type of compound the transporter removes from living cells. By comparing these insect genomes and the human genome we are learning how evolution has selected variability and stability in the enzymes that interact with environmental toxins. We believe that studying the structure and function of the genes in parasites such as the malaria parasite P. falciparum and its hosts the mosquito A. gambiae and humans, we will find new ways to manage the and to improve public health.

Studies on a Drosophila serine protease inhibitor (SERPIN) involved in prophenoloxidase pathway (Sung Jun Han and Paul Brey)

The melanization plays several very important roles for their immune response in insects. The melanization process in insects is mediated through phenoloxidase (PO), a key enzyme that exists as pro-form called prophenoloxidase (PPO). The activation of this PPO is regulated by serine protease cascade known as the PPO pathway. Many Prophenoloxidase activating enzyme (PPAE) homologues were found in various kinds of insects. All known PPAEs and PO pathway related proteases are serine proteases, and some serine protease inhibitor (Serpin) molecules which had inhibitory effects against PO pathway were reported in different insects. Few years ago, a serpin molecule was published in the fall webworm, Hyphantria cunea, by using PCR-based differential display and subtractive cloning after bacterial challenge. We found that its C-terminal amino acid sequence was similar to N-terminal sequence of PPOs near the cleavage site cut by PPAE. C-terminal of serpin is corresponding to Reactive Center Loop (RCL) which is exposed on the surface of molecule. After cleavage of RCL by target protease, P1 residue of serpin forms an ester bond with the active site serine residue of the target protease. So amino acid sequence in the RCL is thought to mimic the normal substrate. By screening of BDGP EST data bank with nucleotide sequences of this serpin molecule, we could find one most possible candidate for inhibitor of PPAE in Drosophila. We named it as POPIN (PhenolOxidase Pathway INhibitor). POPIN is a serpin molecule which consists of 447 amino acids and has N-terminal signal peptide, and it's P1 site was determined by amino acid sequences alignment with other known serpins. As expected, POPIN's C-terminal amino acid sequences, which are corresponding to RCL, had high homology with those of PPO's N-terminal amino acid sequences containing cleavage site, even though Arg in the cleavage site of PPO was substituted by Lys in the P1 site of POPIN. We checked POPIN's expression in both transcriptional and translational level. POPIN was expressed in all developmental stages and Drosophila Schneider cell line, even though it was lower expressed in adult stage and higher expressed in pupae stage than in other stages. As expected, POPIN inhibited PO pathway in Drosophila pupae. Furthermore, POPIN could block PO pathway even in Bombyx and Galleria blood as well. The point mutation of reactive site (P1 site) was introduced to check their activity. POPINK406R, which Lys of P1 site is replaced with Arg, showed inhibitory activity against PO pathway even though it is less effective than POPIN wild type But, POPINK406A, which Lys of P1 site is replaced with Ala, had no inhibitory activity. We tested POPIN's inhibitory activity against various kinds of proteases. POPIN could block only trypsin activity. And, the relevancy between PO pathway and cell adhesion was reported recently. So we checked if our POPIN can block hemocyte aggregation in Galleria mellonella. POPIN inhibited both hemocyte aggregation and hemolymph gelation. Further studies are needed to characterize this linkage.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

BLANC, Marie-France, mfblanc@pasteur.fr

BREY, Paul, IP, Head of Laboratory, pbrey@pasteur.fr

ROTH, Charles, CNRS, croth@pasteur.fr

RAIBAUD, Anna, IP, araibaud@pasteur.fr

PAUL, Richard, IP, topotito@pasteur.fr

GRAILLES, Marine, IP Dakar, Postdoc, letocart@pasteur.fr

BRAHIMI, Karima, IP, Postdoc, kbrahimi@pasteur.fr

HAN, Sung-Jun, Yonsei Medical School, Corée, PhD student, sungjun@pasteur.fr

HOLM, Inge, IP, Engineer, holm@pasteur.fr

PERROT, Sylvie, IP, Technician, sperrot@pasteur.fr

CARMI-LEROY, Annick, IP, Technician

GEGAT, Catherine, IP, Laboratory Agent

BLANC, Marie-France, Secretary, mfblanc@pasteur.fr


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