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  Director : Robert Ménard (rmenard@pasteur.fr)


  abstract

 

We study Plasmodium, the causing agent of malaria, and particularly the parasite stages that infect the mosquito vector and the liver of the mammalian host. Our studies have three main axes: 1) the functional analysis of the mosquito genes that are differentially expressed during infection by the ookinete, the parasite stage that traverses the digestive epithelium in the mosquito, 2) the biology of the sporozoite, the stage formed in the mosquito intestine and transmitted to the mammalian host, and particularly its capacity to glide on susbtrates and invade host cells, and 3) the crosstalk between the sporozoite and the hepatocyte, and the products that are involved in the early stages of parasite differentiation inside hepatocytes.



  report

cale

1. Characterization of Anopheles gambiae genes expressed during Anopheles gambiae-Plasmodium falciparum interaction (C. Lavazec, I. Thiery, C. Bourgouin)

During its early sporogonic development in the mosquito, the parasite crosses two barriers successively: the peritrophic membrane and the intestinal epithelium. In natural conditions of P.falciparum transmission, a sharp decrease in parasite loads occurs in the intestine lumen so that only a few oocysts will become mature. We have previously identified genes from An. gambiae, the main P. falciparum vector in Africa, whose expression is regulated in the mosquito midgut by the presence of P. falciparum (Bonnet et al, 2001). The fine regulation of expression of these genes during mosquito development and parasite development inside mosquitoes was monitored by quantitative RT-PCR. In addition, two of these genes were characterized. Their function will be investigated by RNA interference.

2. Analysis of An gambiae immune response during Plasmodium sporogonic development (R. Tahar, I. Thiery, C. Bourgouin)

P. falciparum development inside An. gambiae is likely the result of a fine adaptation between the parasite and its mosquito host. We have shown by quantitative RT-PCR analysis that the early sporogonic stages of P. falciparum and P. berghei, a parasite species that infect rodents and that is not naturally transmitted by An gambiae, had different impacts on the mosquito immune response. The P. falciparum invasive stages do not induce a stage-specific response in the mosquito digestive track, which suggests that this parasite species specifically manipulates the An gambiae immune response (Tahar et al, 2002).

3. Sporozoite gliding mobility (F. Frischknecht, P. Baldacci, B. Martin)

Like any other invasive stage of the Apicomplexan phylum of protozoans, the Plasmodium sporozoite is capable of gliding (moving without changing its shape) on a solid substrate and to invade any adherent cell. These two processes have a common molecular basis and result from the posterior translocation of parasite-susbtrate/cell interactions, which lead to the forward movement of the parasite. Presently, we are developing methodologies to track the infectious process in vivo, using GFP-expressing sporozoites and in collaboration with ‘Unité d'Analyse d'Images Quantitative' and the ‘Centre d'Imagerie Dynamique'. In particular, we want to determine whether sporozoite gliding motility is necessary for host cell invasion only, or whether it is also responsible for parasite locomotion in vivo.

4. Host cell invasion by sporozoites (P. Baldacci, F. Frischknecht)

Plasmodium TRAP is a transmembrane protein whose cytoplasmic domain binds a motor system located underneath the parasite plasma membrane. Our recent results have shown that sporozoite entry into cultured cells or into target organs in vivo (mosquito salivary glands and mammalian liver) depends on two TRAP extracellular domains, a thrombospondin-type I motif and an integrin A-like domain (Matuschewski et al., 2002). The results also suggest that these two binding domains are the parasite ligands of the parasite-cell junction on which the parasite motor system exerts force to propel the parasite inside the host cell. We are in the process of identifying the host cell molecules that are present in the junction, as well as the receptor(s) of the TRAP A domain that mediates most the sporozoite invasive capacities.

5. Developing gene modification tools in P. berghei (T. Gil Carvalho, S. Thiberge. H. Sakamoto)

Reverse genetics is straightforward in Plasmodium, because of the high rate of DNA integration via homologous recombination and the haploid nature of its genome. P. berghei offers the double advantage of allowing in vivo analysis of mammalian liver infection as well as more reproducible and rapid gene targeting procedures than with P. falciparum. We are currently trying to develop a conditional mutagenesis system in P. berghei (using Cre or FLP) that would allow to analyse the function of essential products, particularly those necessary for red blood cell infection. In the context of a PTR, we are also trying to develop negative selection procedures, which would allow by ‘Hit and Run' to recycle selection markers and introduce mutations in the absence of these markers.

6. Genomic analysis of the parasite-hépatocyte interaction (H. Sakamoto, A. Agrawal)

Once the sporozoite is within a vacuole inside the hepatocyte, the parasite dedifferentiates and generates tens of thousands of merozoites, the parasite stage that infects erythrocytes and causes all symptoms of the disease. Most studies on parasite liver stages have attempted to characterize new vaccine candidates, as well as the immunological basis of the solid protection induced by injection of irradiated sporozoites (whose development inside hepatocytes is blocked). On the other hand, our understanding of parasite maturation in the liver is limited to a few descriptive studies, and only a handful of parasite molecules expressed inside hepatocytes have been identified.

In collaboration with the Genopole, we have constructed macroarrays (membranes) containing 13,000 GSTs, swhich are currently being hybridized with RNA from parasites collected at various time points of their development inside primary hepatocytes. In addition, our collaborators in PTR 59 are identifying genes of primary hepatocytes that are differentially expressed in the presence of the parasite.

Keywords: Plasmodium, Anopheles, cell invasion, gliding motility, intrahepatic development, mosquito immune response



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Delacourtie, Claudine,cdelac@pasteur.fr Baldacci, Patricia, IP, Chargée de Recherches,baldacci@pasteur.fr

Bourgouin, Catherine, IP, Chargée de Recherches,cabourg@pasteur.fr

Ménard, Robert, IP, Chef de Laboratoire,rmenard@pasteur.fr

Agrawal, Alka, post-doc,alka@pasteur.fr

Frischknecht, Friedrich, post-doc,freddy@pasteur.fr

Gil Carvalho, Teresa, étudiante,carvalho@pasteur.fr

Lavazec, Catherine, étudiante,calavazec@pasteur.fr

Tahar, Rachida, post-doc,tahar@pasteur.fr

Martin, Béatrice, Technicienne (GPH),

Thiberge, Sabine, Technicienne,thiberge@pasteur.fr

Thiery, Isabelle, Ingénieur,ithiery@pasteur.fr

Sakamoto, Hiroshi, Ingénieur,hiroshi@pasteur.fr


Activity Reports 2002 - Institut Pasteur
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