Unit: Malaria Biology and Genetics
Director: Robert Ménard
Our laboratory works on Plasmodium, the agent of malaria, and particularly on two phases of the parasite life cycle that are targets of vaccination strategies. The first is the transmigration of the parasite (or ookinete) through the mosquito intestinal barrier. This phase is the target of altruistic vaccines, which do not protect the vaccinated individuals but block transmission of the parasite. The second phase is the so-called pre-erythrocytic phase. It consists of the sporozoite's journey from the site of the mosquito bite to a hepatocyte, as well as the parasite intra-hepatocytic differentiation into the form that will infect erythrocytes and cause the symptoms of the disease. Pre-erythrocytic vaccines aim at preventing sporozoite entry into the hepatocyte and/or its intra-hepatocytic development. We want to characterize the host-parasite interactions that are vital to the parasite. For this, we study P. falciparum, the most deadly species to humans, and P. berghei, a species that infects rodents.
We are following five main directions in the laboratory
1. Caracterization of Anopheles gambiae genes whose expression is modified by mosquito interaction with Plasmodium falciparum (C. Lavazec, M. Gendrin, C. Bourgouin)
During its early sporogonic development in the mosquito, the parasite crosses two barriers: the peritrophic matrix and the intestinal epithelium. In conditions of natural transmission of P.falciparum, the deadliest species to humans, an important reduction in the parasite load occurs in the intestinal lumen so that only a few oocyst reach maturity. We have characterized two genes encoding carboxypeptidases, and demonstrated that they are involved in the development of P. falciparum in the mosquito. Using P. berghei, our results suggest that these carboxypeptidases could constitute components of a vaccine that would block Plasmodium transmission. We are also analyzing cDNA libraries constructed by suppressive and subtractive hybridization to identify A. gambiae genes whose expression is modified during interactions between intestinal cells and ookinetes of P. falciparum.
2. Functional genomics by RNA interference in An. gambiae (B. Boisson, J.-C. Jacques, C. Bourgouin, Program GPH Anophèle)
For assessing the function of mosquito proteins during Anopheles-Plasmodium interactions, we are trying to develop techniques for inactivating gene expression using RNA interference (RNAi) based on inoculation of double stranded RNA in the insect hemolymph. Our current results suggest that the efficiency of the technique depends on the target cells/tissue; whereas it can silence genes that are expressed in the digestive tract or in cells that are in direct contact with the hemolymph, it has so far failed to silence genes expressed in salivary glands (where sporozoites are stored before transmission to the mammalian host). We are now trying to develop transgenesis in A. gambiae, using the piggyBac transposon, as an alternative strategy to silence genes that are expressed in the mosquito salivary glands.
3. In vivo imaging of the Plasmodium sporozoite (R. Amino, F. Frischknecht, B. Martin, R. Ménard, Program GPH Anophèle)
The sporozoite is formed inside an oocyst in the mosquito intestinal epithelium, and can only complete its development inside a mammalian hepatocyte. Using sporozoites expressing various fluorescent proteins, and in collaboration with the Unité d'Analyse d'Images Quantitative' and the Centre d'Imagerie Dynamique' in the campus, we have analyzed each phase of the in vivo journey of the sporozoite in the P. berghei rodent system. These studies have shown the importance of in vivo mobility of these parasites, not only for invading host cells but also for locomoting in host tissues (both in the mosquito and the mammal). They have also revealed unexpected host-parasite interactions, including invasion of both blood and lymph capillaries in the mouse skin, infection of the proximal lymph node, intra-vascular adherence and gliding motility, as well as complex leukocyte-parasite interactions in the liver sinusoids. We are now attempting to characterize the interactions between sporozoites and the draining lymph node, and their consequences on the parasitic process.
4. Development and use of conditional mutagenesis in P. berghei (T. Gil Carvalho, S. Thiberge, A. Combe, D. Giovannini, R. Ménard)
Reverse genetics in Plasmodium is facilitated by the high efficiency of homologous recombination and the haploid nature of the parasite genome. The P. berghei system has several attractive features: the entire parasite life cycle can be maintained in the laboratory, liver infection can be analyzed in vivo, and mutants can be created more easily than with P. falciparum. However, manipulation of the Plasmodium genome has only been constitutive so far, based on transfection of erythrocytic stages of the parasite, and did not allow the studies of numerous essential parasite proteins. We have developed two complementary strategies for conditional mutagenesis in P. berghei, using the Flp-FRT site-specific recombination system of yeast. We are currently using these techniques to assess the function, at pre-erythrocytic stages of the parasite, of two proteins that are essential for the erythrocytic stages of the parasite.
5. Functional genomics of parasite-hepatocyte interactions (P. Baldacci, H. Sakamoto, S. Thiberge, S. Akerman, R. Ménard)
Once the sporozoite is within a vacuole inside the hepatocyte, the parasite transforms 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.
Our goal is to identify parasite proteins that are essential for parasite development in the hepatocyte, and particularly those that act at the hepatocyte-parasite interface. For this, we have developed a shuttle mutagenesis technique: a gene of interest is mutagenized by transposon insertion (mini-Tn5) in E. coli, and the resulting fragment is then reintroduced in the parasite where it replaces the wild-type copy of the gene by double cross-over homologous recombination. This technique allows for a systematic mutagenesis of selected P. berghei genes by the simultaneous construction of multiple mutants in a single transfection experiment. We are now using this approach to assess the function of several tens of genes that are specifically expressed by the parasite inside the hepatocyte, which have been selected by a bio-informatic screen.
Keywords: Plasmodium, Anopheles, malaria, molecular genetics, génomics, in vivo imaging