Molecular basis of gliding motility and cell invasion by sporozoites (Teresa Gil Carvalho, Friedrich Frischknecht, Sabine Thiberge)
Like any invasive stage of the Apicomplexa phylum of protozoa, the Plasmodium sporozoite displays two major phenotypes : it can glide on solid substrates (without changing its shape, which distinguishes gliding from the classical crawling motility) and can invades virtually any adherent cell. These two processes have similar bases and result from capping of interactions between the parasite and the substrate or the cell : posterior translocation of these interactions leads to forward displacement of the parasite (on the substrate or within a parasitophorous vacuole inside the host cell).
Our previous work has shown that 1) sporozoite gliding and cell invasion depend on a transmembrane protein, TRAP; 2) TRAP connects an actin-dependent motor system to receptors on the substrate/cell that are used as anchors for motility and cell invasion by the sporozoite; 3) two adhesive modules present in the ectodomain of TRAP, an I domain of integrin a chains and a thrombospondin type 1 repeat, are necessary and sufficient for sporozoite entry into any host cell (including mosquito salivary glands and rat hepatocytes in vivo). We are currently trying to 1) identify the cell receptor(s) that bind the TRAP I domain, which mediates most of the sporozoite invasive capacities, and 2) characterize the motor system that translocates TRAP and powers sporozoite gliding and cell penetration. This second part is addressed by identifying products that bind the cytoplasmic tail of TRAP and developing conditional mutagenesis techniques, which will be required for analyzing the function of components of the motor system specifically at the sporozoite stage.
Identification of parasite and host cell factors that are necessary for parasite development inside hepatocytes (Hiroshi Sakamoto, Sylviane Pacheco, Alka Agrawal)
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. This is mainly due to the difficulties to obtain pure parasite material and to manipulate the scarce intrahepatocytic stages of the parasite.
To facilitate studies on liver stages, we have constructed a P. berghei clone that expresses GFP at those stages and allows for the first time to isolate by FACS the rare infected cells from noninfected cells. Using this system, we want to characterize the early crosstalk between the parasite and the hepatocyte. We have undertaken the identification of parasite genes expressed after sporozoite entry into host cells, as well as hepatocyte genes whose expression is affected by parasite infection, using two complementary approaches, transcriptome analysis (using shotgun microarrays) and subtractive suppressive hybridization.