Abstract 2478 Defining the essential host genome for Cryptosporidium replication and development

Cryptosporidium is an obligate intracellular parasite that invades and replicates within a single cell type – epithelium, that lines the intestine of an infected host. Within these epithelial cells, the parasite remodels the cytosketeton, siphoning nutrients, and modulating host signalling pathways to hijack cellular processes and evade immune detection. To better understand how the parasite interacts with the host cell, we developed a high-throughput imaging screen to look for genetic dependencies. Our screen employed an arrayed CRISPR library, targeting all protein coding genes in the human genome, with a microscopy-based readout of parasite and host cellular markers. This approach allowed us to systematically measure the impact of every human protein coding gene on the growth and development of the parasite. Although several host metabolic pathways were identified to influence parasite growth, cholesterol biosynthesis attracted our attention due to the appearance of contrasting effects. Loss of genes early in the cholesterol synthesis pathway decreased parasite growth, while loss of genes late in the pathway led to increased parasite growth and development. Using a variety of genetic and chemical approaches, we then determined that the basis of these opposing results is the level of the intermediate metabolite squalene. Further, chemical modulation of squalene levels in vivo shows promise as an effective treatment option for Cryptosporidium infection. This unique data set furthers our understanding of intestinal epithelial cell biology and has the potential to establish new lines of host-pathogen research. This work was supported by the Francis Crick Institute-which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.