Protozoan parasites of the genus Plasmodiumare the vector-borne causative agents of malaria, responsible for more than 350 million clinical cases and one million deaths annually. Studies using rodent malaria models have shown that during the bite of an infected Anopheles mosquito, Plasmodiumsporozoites are transmitted and subsequently traverse a variety of cell types, wounding their membranes on the way1. This allows sporozoites to travel through the dermis into the bloodstream and then pass through the cell layer that lines blood vessels in the liver as well as several hepatocytes before taking up residence in host hepatocytes for further development as exoerythrocytic forms (EEFs) 2. Although cell traversal provides a means to cross tissue barriers, it is unclear whether this constitutes its only biological role. A novel function for parasite host cell traversal was proposed by Carrolo et al. 3, who showed that cell traversal by sporozoites of the rodent malaria parasite Plasmodium berghei induces secretion of hepatocyte growth factor (HGF). HGF is a soluble factor that activates the receptor tyrosine kinase MET, which is capable of initiating a cascade of signaling events that result in hepatocyte proliferation and survival. Carrolo et al. 3 further showed that this signaling cascade is crucial to promote early development of EEFs and thus substantially contributes to transmission success. More recently, the same group demonstrated, again using P. berghei, that MET signaling events are important for the parasite to protect itself from host hepatocyte apoptosis4 and subsequently showed that a dietary supplement, genistein, which inhibits MET activation, can treat liver-stage malaria infection5. A fundamental question, then, is whether this unique function for cell traversal is broadly conserved among Plasmodium species and whether it is found in human malaria parasites. To test the ability of another rodent malaria parasite species, Plasmodium yoelii, and the human malaria parasite, Plasmodium falciparum, to activate MET by cell traversal, we incubated sporozoites of each species with HepG2-CD81 hepatoma cells for 1 h to allow traversal and then collected cellular lysates. Using an antibody specific to the phosphorylated activation loop of MET, we probed the lysates for MET activation. As a negative control, we used lysates from cells that had been incubated with salivary gland extracts from uninfected mosquitoes (Supplementary Methods). We found that cell traversal by P. berghei sporozoites but not P. yoelii or P. falciparum sporozoites led to the activation of MET (Fig. 1a). To ensure that P. falciparumdoes not fail to activate MET because it is being assayed in a cell type that does not support its EEF development, we allowed P. falciparumsporozoites to traverse HC04 cells, a hepatocyte-derived cell line that does support P. falciparum EEF development6, but, again, we did not detect MET activation (Fig. 1b). To ensure that differences in MET activation were not due strictly to differences in traversal rates, we incubated sporozoites with HepG2-CD81 cells in the presence of labeled dextran. Given that cells that are wounded by traversal take up the labeled dextran, we used FACS to identify traversed cells (Supplementary Methods).

We observed comparable rates of traversal for each of the three species. Modest differences in traversal rates between species did not correlate with MET activation. Instead, MET activation was only observed for P. berghei, which had slightly lower traversal rates than P. yoelii, and similar traversal rates when compared to P. falciparum