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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE185247 | GEO | 2021/10/06

REPOSITORIES: GEO

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Project description:Purpose: Transcriptome profiling of Crytosporidium parvum infected lung and small intestinal organoids was performed to access the response of epithelial cells upon parasitic infection and to do a temporal analysis of the transcriptome of the parasite inside the organoid lumen. We isolated RNA from infected human lung and small intestinal organoids at 24 and 72 hour post infection. Methods: Organoids were grown in expansion or differentiation media and microinjected with equal amounts of Cryptosporidium oocysts. Media-injected organoids were used as a control .Expanding SI organoids were microinjected at 5-6 days after seeding, differentiated SI organoids were injected at 5 days after inducing differentiation. Lung organoids were incubated for 2 weeks after seeding for microinjection. RNA was extracted from 1-2 matrigel drops containing organoids. RNA was converted to cDNA and libraries were prepared using the CelSeq2 method and sequenced. Samples were sequenced on Illumina NextSeq500 by using 75-bp paired-end sequencing. Methods: Paired-end reads from Illumina sequencing were aligned to the human transcriptome genome and C. parvum transcriptome genome (Iowa strain) by BWA. DeSeq (v1.18.0) was used for read normalization and differential expression analysis (p-value adjustment 0.05 by method Benjamini-Hochberg). Gene set enrichment analysis (GSEA) was performed using gene lists for type I interferon response and regulation against normalized RNA-seq reads of injected SI and lung organoids using GSEA software v3.0 beta2. Results: At 24 hr post-infection,GO (gene ontology)-term analysis revealed that a substantial number of genes related to ‘cytoskeleton’ and ‘cell mobility’ were up-regulated in lung organoids. This suggests that infection by the parasites and subsequent formation of the intracellular stages within 24 hrs might affects cytoskeleton structures of host cells. After 72 hrs, many genes associated with the type I interferon pathway increased dramatically in lung and intestinal organoids. Results: After 72 hrs, many genes associated with the type I interferon pathway increased dramatically in lung and intestinal organoids. Multiple C. parvum genes were differentially expressed with a large fold change between 24 and 72 hr post-injection.At 24 hr post-infection, most of the enriched genes represented ribosomal proteins and ribosomal RNA subunits in both intestinal organoids and lung organoids. By contrast, at 72 hr post-infection, multiple oocyst-wall protein genes were up-regulated, confirming that the parasites formed new oocysts within the organoids. Conclusions: RNA sequencing of injected organoids revealed host epithelial responses upon parasite infection in differentiated SI organoids as well as in lung organoids.Upregulation of genes associated with type I interferon immunity in both SI and lung organoids.

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

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