Malaria Liver Infection: molecular mechanisms of host cell invasion by Plasmodium sporozoites. – MaLI

In this project, we propose to develop a proteomic-based screen to identify Plasmodium molecules that could play a role during infection of hepatocytes, and to exploit genetically modified parasites to characterize the host-parasite interactions taking place during sporozoite invasion. Sporozoite invasion of host cells is associated with proteolytic processing and shedding of proteins derived from intracellular organelles, the secretion of which is triggered by interactions with the host cell. Using mass spectrometry (MS/MS), we will identify secreted/excreted proteins released in the supernatant of hepatocytic cell cultures infected with sporozoites. To assess the functional importance of these factors, the corresponding genes will be deleted, using a novel gene targeting approach allowing sensitive and reliable assessment of the dynamics of sporozoite invasion in mutant knockout parasites. In addition, we will generate genetically modified sporozoites expressing fluorescent markers of the moving junction, in order to establish a model for imaging the process of sporozoite entry. In parallel, we will characterize the role of host molecules during sporozoite invasion, focussing on the tetraspanin CD81, which remains the only host molecule reported as essential for Plasmodium sporozoite invasion. The combination of proteomic analysis of sporozoite secretome/excretome with reverse genetics should provide new insights in the molecular mechanisms of Plasmodium sporozoite infection.

The MaLI project should provide new insights into the molecular events associated with infection of liver cells by Plasmodium sporozoites. In particular, the combination of proteomic screening with reverse genetics should allow the identification of parasite proteins involved in interactions with the host cell, which until now remain unknown. Also, this project may help deciphering the precise molecular role of host molecules involved during sporozoite invasion, notably the tetraspanin CD81, which plays an essential role through a still unknown mechanism. Finally, the use of reporter parasites expressing fluorescent markers of the junction will constitute novel invaluable tools for live imaging of the moving junction during sporozoite invasion, which has been virtually impossible so far.

Altogether, this project may lead to a better understanding of the process of sporozoite invasion, and to the discovery of potential new targets for novel vaccine formulations aimed at blocking the first steps of malaria infection, before parasite development in the liver. An efficient vaccine against Plasmodium parasites would have tremendous effects in terms of public health, as malaria still causes more than one million deaths each year in the world.

Presentations at international conferences

Risco-Castillo V, Manzoni G, Briquet S, Topçu S, Silvie O. “Targeted gene deletion of perforin-like protein 1/SPECT2 in Plasmodium yoelii.” 9th Annual BioMalPar/EVIMalaR Conference, Heildelberg, Germany, 2013 (poster).

Manzoni G, Briquet S, Topçu S, Risco-Castillo V, Boggetto N, Silvie O. “Positive/negative selection combined with flow cytometry for rapid generation of knockout parasites in Plasmodium berghei and Plasmodium yoelii.” 9th Annual BioMalPar/EVIMalaR Conference, Heildelberg, Germany, 2013 (poster).

Manzoni G, Briquet S, Topçu S, Risco-Castillo V, Boggetto N, Silvie O. “Positive/negative selection combined with flow cytometry for rapid generation of knockout parasites in Plasmodium berghei and Plasmodium yoelii.” Young Researchers in Life Sciences Meeting, Paris, France, 2013 (poster).

Topçu S, Manzoni G, Risco-Castillo V, Briquet S, Silvie O. “Host and parasite factors involved in Plasmodium sporozoite entry into hepatocytes.” Young Researchers in Life Sciences Meeting, Paris, France, 2013 (poster).

Manzoni G, Briquet S, Topçu S, Risco-Castillo V, Boggetto N, Silvie O. “Positive/negative selection combined with flow cytometry for rapid generation of knockout parasites in Plasmodium berghei and Plasmodium yoelii.” Research Advances in Malaria Meeting, Baltimore, USA, 2013 (poster).

Risco-Castillo V, Son O, Silvie O. “Novel Transgenic Plasmodium yoelii parasites to study sporozoite invasion” 8th Annual BioMalPar/EVIMalaR Conference, Heildelberg, Germany, 2012 (poster).

The malaria parasite Plasmodium is transmitted by Anopheles mosquitoes, which inject infective forms called sporozoites into the host skin. Motile sporozoites rapidly travel to the liver and invade hepatocytes by forming a parasitophorous vacuole, where they transform into liver stages. After a few days of intense multiplication, liver stages release merozoites that invade erythrocytes, and are responsible for symtoms and complications of malaria. Sporozoite molecules interacting with hepatocytes during invasion represent ideal vaccine targets, simply because blocking liver infection prevents formation of merozoites and thus the pathogenicity associated with erythrocytic multiplication of the parasite. In this context, a better understanding of the molecular mecanisms of sporozoite invasion is essential. The nature of parasite and host molecules that interact during sporozoite invasion remains elusive. In this project, I propose to develop a targeted proteomic approach to specifically identify molecules that could play a role in the invasion process. To assess the functional importance of these candidate factors, I will perform targeted gene deletions. Using this strategy, I expect to identify novel sporozoite proteins that participate in parasite entry into hepatocytes. In parallel, I will develop an approach to visualize interaction of invading sporozoites with their target host cells, using transgenic parasites expressing fluorescent markers. This will be particularly helpful to decipher the role of parasite and host cell molecules during the process of invasion. Altogether, these approaches may lead to a better understanding of the process of sporozoite invasion, and to the discovery of potential new targets for novel intervention strategies aimed at blocking the first steps of malaria infection, before parasite development in the liver.