A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes required for Cryptosporidium parvum infection and/or host cell survival. The gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFNλ reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFNλ production critical to early protection against this infection. This work includes two indepedent RNAseq experiments. The first is a 48 hour infection with C. parvum using either wild type parasites or transgenics propagated by our lab. The 48 hour experiment includes 3 biological replicates of uninfected, wild type infected, and transgenic infected. The second experiment was a 10 hour infection with a C. parvum strain expressing an mNeon green fluorescent protein. In this experiment cells were sorted to assess infection status with GFP negative as bystanders and GFP positive as infected cells. Three biological replicates were double sorted for these data.