Detailed analysis of Cryptosporidium non-coding gene expression

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ABSTRACT Cryptosporidium is a zoonotic apicomplexan protist parasite recently identified as the second most prevalent diarrheal pathogen of infants globally (1-6). It is spread via an oral-fecal route and it can be lethal in the immunocompromised since there are no therapeutics approved for use in this population. The molecular parasitology of Cryptosporidium has been lacking but this situation is changing with the recent advent of in vitro culture systems, better parasite enrichment protocols and a nascent genetic system (8-11). This project proposal focuses on an analysis of Cryptosporidium transcription and the host-pathogen interaction. Specifically, we propose to generate candidate molecules for experimental testing of the recent hypothesis that Cryptosporidium alters host-cell gene expression in trans, via the export of long non-coding RNA molecules (lncRNA) that alter host cell gene expression thereby affecting host cell response and pathogenesis. Recent work by our group and others (7, 9, 12) has revealed that the C. parvum transcriptome is complex and laden with a variety of non-coding RNAs, both long and short. Recent work by Dr. Xian-Ming Chen has detected C. parvum transcripts in host-cell nuclei and demonstrated that several C. parvum lncRNAs, when introduced affect host-cell gene expression (13- 18). Sadly, due to the historical limitations of working with C. parvum, transcriptional data are sorely lacking for this important pathogen. Building on our proven track record of generating and sharing C. parvum transcriptional data, this project proposes to systematically characterize C. parvum coding and non-coding RNAs including small RNAs from several developmental stages of the parasite, pre- and post-infection (in vitro) using PacBio Iso-Seq (19) to identify complete transcripts and Illumina technologies to study small RNAs as Aim 1. Aim 2 focuses on examining the host-pathogen interaction. Bioinformatics will be used to identify select lncRNA molecules that may warrant experimental testing in the laboratory of Dr. Chen for host cell effects or knockout via CRISPR in the laboratory of Dr. Boris Striepen. Additionally, NovaSeq, which generates billions of reads (20) will be used for deep RNA sequencing of in vitro heavily-infected (0-48 hr) and uninfected host cells to characterize the gene transcriptional changes occurring in both the host and pathogen at three post-infection time-points. It is imperative to characterize transcriptional responses that may play a role in the host-pathogen interaction. They will expose new insights into parasite survival mechanisms, the development of pathogenesis and reveal much needed new targets for future therapeutic interventions (21-24).