A synergistic approach identifies key host factors required for apicomplexan parasites

Abstract text

Apicomplexan parasites are obligate intracellular organisms responsible for major human and bovine infectious diseases, including malaria, toxoplasmosis and theileriosis. In this project we used a synergistic approach to identify host cell pathways that are essential for the parasite but dispensable for the host cell. By performing a genome-wide CRISPR/Cas9 drop out screen in Theileria-infected and non-infected bovine macrophages we obtained a list of 111 genes specifically essential for infected cells, with one third of these involved in metabolic pathways. We then combined genetic screens with chemoinformatic modelling, and ran gene knock-out simulations of these candidates in a human hepatocyte-Plasmodium falciparum metabolic model. Hydroxymethylbilane synthase (HMBS), an enzyme involved in heme biosynthesis, was predicted to be dispensable for uninfected hepatocytes but necessary for the survival of Plasmodium in the host cell. In order to confirm this prediction, we generated HMBS knockout clones in the human haploid cell line HAP1. Upon infection with the widely used rodent model organism Plasmodium berghei, we observed a significant decrease in parasite numbers, accompanied by a striking reduction in parasite liberation from infected cells. To exclude Cas9 off-target effects, we complemented HMBS and obtained a complete reversion of the phenotype. In conclusion, our results indicate that both Plasmodium and Theileria parasites depend on host HMBS for their survival. This suggests that Plasmodium, which expresses all enzymes of the heme pathway, not only relies on endogenous heme biosynthesis but also requires an intact host pathway for liver stage development.