Unit: Malaria Biology and Genetics
Director: Ménard Robert
We study Plasmodium, the causative agent of malaria, and particularly the parasite stages that infect the mosquito vector and the liver of the mammalian host. We are addressing: 1) the functional analysis of 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 gut 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 proteins that are involved in the early stages of parasite differentiation inside hepatocytes.
1. Characterization of Anopheles gambiae genes involved in Anopheles gambiae-Plasmodium falciparum interaction (C. Lavazec, R. Tahar, I. Thiery, C. Bourgouin)
During its early sporogonic development in the mosquito, the parasite crosses two barriers: the peritrophic membrane and the intestinal epithelium. During natural transmission of P.falciparum, which infects humans, a sharp decrease in parasite loads occurs in the intestine lumen so that only a few oocysts 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. Of these genes, two encoding carboxypeptidases have been analyzed and shown to be important to P. falciparum development in An. gambiae. Also, a subtractive and suppressive hybridization approach has been initiated for identifying mosquito genes whose expression is modified during the interaction between An. gambiae midgut epithelial cells and P. falciparum ookinetes.
2. Functional Genomics in Anopheles gambiae (B. Boisson, JC Jacques, C. Bourgouin)
Towards addressing the role of mosquito products during Plasmodium infection, we are developing a systematic approach of gene silencing by RNA interference based on injection of double stranded RNA in the mosquito hemolymph. We are also establishing transgenesis in An. gambiae, using the piggyBac transposon. The goal is to create mosquito lines stably expressing double-stranded RNA or overexpressing genes of interest, in a tissue-specific fashion or inducible by a blood meal.
3. Sporozoite gliding mobility and in vivo biology (F. Frischknecht, R. Amino, B. Martin)
Like any other invasive stage of the Apicomplexan phylum of protozoans, the Plasmodium sporozoite is capable of gliding 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 leads to the forward movement of the parasite. We have shown, using GFP-expressing sporozoites and in collaboration with Unité d'Analyse d'Images Quantitative' and the Centre d'Imagerie Dynamique', that sporozoite gliding motility is not only necessary for its cell invasive capacity but is also responsible for its locomotion in vivo, particularly inside the mosquito salivary ducts. This active motility of the Plasmodium sporozoite, which is independent of the saliva flow, is the first direct proof of active motility of an apicomplexan parasite in vivo. Currently, we are analyzing the sporozoite infectious process in the mammalian host, in the skin and the liver.
4. Host cell invasion by sporozoites (P. Baldacci, S. Thiberge)
Plasmodium TRAP is a transmembrane protein whose cytoplasmic domain binds the motor system located underneath the parasite plasma membrane. We have previously 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. These two adhesive modules appear to constitute the parasite ligands of the parasite-cell junction on which the parasite motor system exerts force to propel the parasite inside the host cell. Our recent results suggest that two types of ubiquitous host cell receptors may play an important role in sporozoite internalization.
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 liver infection as well as more reproducible and rapid gene targeting procedures than with P. falciparum. We have developed conditional mutagenesis in P. berghei, using the Flp/FRT system of yeast. This system will allow the analysis of the function of any gene in this parasite, at any time of its life cycle.
6. Genomic analysis of the parasite-hepatocyte interaction (H. Sakamoto, P. Baldacci)
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 Platform, we have constructed macroarrays on membranes containing 13,000 GSTs, which are currently being hybridized with RNA from parasites collected at various developmental time points, ie sporozoites, liver stages, and red blood cell stages. In addition, we are attempting to develop a systematic/random mutagenesis technique for identifying genes that are essential to our stages of interest. This system, based on Plasmodium DNA mutagenesis by Tn5 in E. coli followed by reintroduction of the mutagenized DNA in the parasite by homologous recombination, should permit to select genes of interest on the basis of their function.
Keywords: Plasmodium, Anopheles, malaria, pre-erythrocytic stages, molecular genetics, in vivo imaging, genomics