Our laboratory focuses on the pre-erythrocytic phase of malaria. This phase extends from injection of the parasite in the skin by the mosquito to the onset of red blood cell infection, which causes the clinical symptoms of the disease. Our major aim is a functional understanding of the important host-parasite interactions that occur during the parasite journey to the liver, as well as inside the hepatocyte. For this, we use a combination of molecular genetics, cell biology and in vivo imaging approaches. We are also interested in understanding how the host can mount a protective immune response against this phase of Plasmodium infection. We use rodent Plasmodium models of infection for our studies.

Imaging the Plasmodiumpre-erythrocytic phase

The clinically silent pre-erythrocytic phase of malaria amounts to the journey of the sporozoites, the parasite form injected by the mosquito, from the skin to the liver. Inside hepatocytes, sporozoites transform into the red blood cell-infecting forms of the parasite. In recent years, our intra-vital imaging studies in rodents have revealed a more complex picture of the pre-erythrocytic phase. We have shown that (i) 20% of the inoculated sporozoites, which are motile, actively reach the lymph node draining the site of mosquito bite, (ii) while about half of the sporozoites stay at the site of bite. The latter can either fully develop into red blood cell-infecting forms in the dermis (inside fibroblasts) or the epidermis (inside keratinocytes), or persist as growth-arrested/slowly-growing parasites in association with hair follicles, an immuno-privileged site of the mammalian body. We have also shown that the cell traversal activity of the sporozoite, initially thought to be important for activating its capacity to enter hepatocytes, instead (i) allows sporozoite progression through host cells and cellular barriers and (ii) confers sporozoite resistance to destruction by phagocytic leukocytes, both in the skin and in the liver sinusoids. These data thus depict a new pre-erythrocytic phase of malaria, at least in rodents, which will now need to be addressed using primate/human-infecting species of Plasmodium.

Dissecting the basis of protection against the pre-erythrocytic phase

It has been known for over 40 years that injection of sporozoites attenuated by irradiation induces sterile protection, ie, prevents blood infection after subsequent injection of WT sporozoites. So far, this protection model has been analyzed in rodents after intravenous injection of attenuated sporozoites. We have shown that sterile protection can also be induced by injection of irradiated sporozoites in the skin, and we are currently trying to pinpoint the parasite stages (sporozoites, early-, mid- or late- intracellular stages) that are crucial for inducing protection, using parasite mutants defective in specific steps of the infective/developmental process.

Characterizing the important molecular players of parasite infection

We have recently established new molecular genetics tools and approaches for studying gene/protein function in pre-erythrocytic stages of the parasite, including a new conditional, Flp/FRT-based mutagenesis procedure that allows for modifying any gene of interest specifically at the sporozoite stage (only erythrocytic stages of the parasite can be transfected). Our primary goal is to better understand the function of parasite proteins, from both the extracellular sporozoite and the intracellular liver stage, which act at the interface with the host.

In particular, we have recently characterized the role of two proteins highly conserved in the apicomplexan phylum of parasites, called AMA1 and RON4. Initially thought to be components of the conserved junction that forms between the parasite and the host cell, we have gathered conclusive evidence that AMA1 does not act at the junction but as an adhesin prior to junction formation. We have also characterized a transporter, named NPT1, which appears to be crucial for gametocytogenesis as well as for liver stage development, ie, at both parasite transmission steps. Finally, we have also found that sporozoite latency is an active process controlled by an initiation factor-2a kinase and a phosphatase, the activity of the former being dominant in sporozoites, leading to translation inhibition and preventing the premature transformation of the sporozoite into a liver stage.

Keywords: Plasmodium, pre-erythrocytic phase, sporozoite, skin, liver, intravital imaging, molecular genetics

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Gonzalez V, Combe A, David V, Malmquist N, Delorme V, Leroy C, Blazquez S, Ménard R, Tardieux I. 2009. Host cell entry by apicomplexa parasites requires actin polymerization in the host cell. Cell Host and Microbes19, 259-272.

Ishino T, Boisson B, Orito Y, Lacroix C, Bischoff E, Loussert C, Janse C, Ménard R, Yuda M, Baldacci P. 2009. LISP1 is important for the egress of Plasmodium berghei parasites from liver cells. Cellular Microbiology 11, 1329-1339.

Combe A, Giovannini D, Gil Carvalho T, Späth S, Boisson B, Loussert C, Lacroix C, Gueirard P, Ménard R. 2009. Clonal conditional mutagenesis in malaria parasites. Cell Host and Microbes5, 386-96.

Gueirard P, Tavares J, Thiberge S, Bernex F, Ishino T, Milon G, Franke-Fayard B, Janse C, Ménard R, Amino R. Development of the malaria parasite in the skin of the mammalian host. 2010. Proc. Natl Acad Sci USA107, 18640-18645.