Unit: Malaria Biology and Genetics
Director: Robert Ménard
Our laboratory focuses on Plasmodium, the agent of malaria, and particularly on two distinct phases of the parasite life cycle that are targets of vaccine strategies. The first is the migration of the parasite (called ookinete at this stage) through the intestinal barrier of the mosquito. This phase of infection is the target of so-called transmission blocking vaccines', which do not protect the vaccinated individual but its community by blocking parasite transmission. The second is the pre-erythrocytic phase of the parasite life cycle. This phase includes the journey of the sporozoite (the stage of the parasite inoculated by the mosquito) from the mosquito site of bite to a hepatocyte, as well as the phase of parasite differentiation in the liver into the parasite form that infect erythrocytes and causes the symtoms of the disease. We want to gain functional understanding of these steps of the parasite life cycle. In our studies, we use P. falciparum, the species the mots deadly to humans, and P. berghei, a species that infects rodents.
Several lines of research are developed in the laboratory
1. Characterization of Anopheles gambiae genes whose expression is modified by mosquito interaction with Plasmodium falciparum (C. Lavazec, M. Gendrin, R. Lacroix, C. Bourgouin)
In conditions of natural transmission of P. falciparum, the Plasmodium species deadliest to humans, an important reduction in the parasite numbers occurs in the mosquito intestinal lumen so that only a few oocysts become mature. We have characterized two genes encoding midgut carboxypeptidases and demonstrated that these genes are involved in P. falciparum development in the mosquito. Antibodies directed against one carboxypeptidase strongly reduce the sporogonic development of both P. falciparum and P. berghei and also reduce mosquito fecundity. Therefore, An. gambiae midgut carboxypeptidases could constitute components of a vaccine that would block Plasmodium transmission, by reducing Plasmodium sporogonic development and mosquito populations. Elucidating the role of these carboxypeptidases in Plasmodium development is under way using both biochemical and RNAi approaches
2. Functional genomics by RNA interference in An. gambiae (B. Boisson, J.-C. Jacques, C. Bourgouin, Program GPH Anopheles)
Before being transmitted to the mammalian host, sporozoites reside in the salivary glands of the mosquito. We have established the conditions for efficiently silencing gene expression in the mosquito salivary glands by RNA interference. These conditions are different from those used for silencing gene expression in the mosquito midgut, fat body and hemocytes. Using this approach, we are now assessing the role of saliva components in the transmission of Plasmodium sporozoites to the mammalian host. In particular, we will examine the role of salivary gland genes whose expression is modified by the presence of sporozoites, which have been identified by SAGE analysis by the I. Chupin's group in the GPH program.
3. In vivo imaging of the Plasmodium sporozoite (R. Amino, S. Thiberge, D. Giovannini, R. Ménard, Program GPH Anopheles)
The parasite is inoculated in the dermis of the mammalian host by the mosquito vector. The parasite journey from the site of bite to the target hepatocyte, however, remains elusive. To better understand the natural history of the sporozoite in the mammalian host, we are using a rodent system, sporozoites that express fluorescent proteins, and various in vivo imaging approaches (in collaboration with the " Centre d'Imagerie Dynamique "). In vivo studies have shown the importance of sporozoite motility not only for invading target cells, but also as a means of locomotion in the mosquito as well as in the mammalian hosts. They have also revealed numerous unexpected host-parasite interactions, including invasion of lymphatic vessels in the mammalian dermis, infection of the draining lymph node, intra-vascular gliding and complex leucocyte-parasite interactions in the liver sinusoids. We are now trying to characterize the host-parasite interactions in the lymph nodes, their consequences on the infection process, as well as the role of traversal of host cells on sporozoite infectivity.
4. Molecular basis of the sporozoite cell invasion capacity (A. Combe, D. Giovannini, S. Thiberge, R. Ménard)
The final destination of the sporozoite is the hepatocyte, which the sporozoite invades in a parasitophorous vacuole and inside which it develops in the parasite stage that invades erythrocytes. Invasion of the host cell by the sporozoite is a rapid and active process, and the force that drives the parasite inside the cell depends on the actin cytoskeleton in the parasite, and not in the host cell. The parasite ligands and the host cell receptors involved in the process remain elusive; in particular, the parasite ligands, which are common to other invasive stages of the parasite, cannot be genetically inactivated using the available transfection technology. To study the function of the sporozoite surface proteins that are potentially involved in host cell invasion, we have developed conditional mutagenesis in P. berghei, which now allows us to mutagenize specifically at the sporozoite stage any gene of interest. We are currently focusing our attention on the function of MSP-1, AMA-1, RON-4, and TLP.
5. Mutagenesis of Plasmodium genes expressed in pre-erythrocytic stages (P Baldacci, B Boisson, S Akerman, C Lacroix, S Thiberge, H Sakamoto, R Ménard).
The liver is the first organ of the mammalian host targeted by Plasmodium parasites. Although no pathology is associated with the liver phase of infection, it has been demonstrated that this phase can induce an immune response that protects the host against a challenge with infectious sporozoites. Little is known about the molecular biology of Plasmodium in liver stages (LS) due to the difficuty in obtaining parasite material from this stage of development. Our main objective is to identify and characterize the parasite-hepatocyte interactions which are important for the intra-hepatocytic development of P. berghei. To this end, we are identifying parasite genes expressed specifically in liver stages, and will be creating the corresponding P. berghei mutants. An in silico analysis of published transcription profiles and proteomics databases has allowed us to identify about 50 P. berghei genes that are potentially expressed exclusively in liver stages. The candidate genes have been analyzed by qPCR and are undergoing systematic mutagenesis via a technique of shuttle mutagenesis recently published by our laboratory. The mutant parasites will be characterized for their capacity to invade/develop in the liver, and their ability to protect the host against a subsequent challenge with infectious sporozoites will be analyzed..
Plasmodium berghei sporozoites gliding in the dermis of a mouse. The parasite is shown in yellow at t0. During the next 10 minutes, the movement of the parasite is represented by a maximal projection in red. The blood vessel is represented in blue.
Keywords: Plasmodium, malaria, Anopheles, transmission blocking vaccine, invasion, differentiation