Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Christopher D. Huston

Abstract

Cryptosporidium species are eukaryotic intracellular parasites belonging to the phylum Apicomplexa. C. hominis and C. parvum cause diarrhea in humans which is self-limiting in immunocompetent adults but can have severe consequences in immunocompromised individuals and malnourished children. In developing countries, Cryptosporidium is one of the leading causes of moderate to severe diarrhea in children under five years of age. There is an urgent need for novel therapeutics against this parasite as the current treatment option is inadequate to treat the most vulnerable population to Cryptosporidium infection. Better understandings of the biology of Cryptosporidium would greatly enhance our capability to design effective control measures.

The parasite initially multiplies through several rounds of asexual replication within the intestinal epithelial cells before differentiating into sexual forms. Here we have tested the ReFRAME library, a set of ~12,000 biologically active compounds, for activities against the asexual and sexual forms of C. parvum separately. We identified and validated compounds that inhibit and/or induce the growth of either or both life cycle stages. Compounds that inhibit both stages are potentially good drug candidates. Other compounds have promising tool-like properties that can be utilized to probe different aspects of C. parvum sexual differentiation. Several inhibitors of host cell oxidative phosphorylation and purine nucleotide biosynthesis disproportionately inhibited the sexual differentiation of C. parvum, highlighting the necessity of these processes in facilitating C. parvum sexual differentiation.

All the apicomplexan parasites differentiate into distinct forms in the course of their life cycle and each of the differentiation steps is associated with large scale changes in gene expression. To identify such stage specific genes and pathways associated with C. parvum sexual differentiation, we performed mRNA-seq of host cells infected with C. parvum in the presence of nine differentiation inhibitors. Ribosomal proteins were the most significantly enriched group of genes that were upregulated with multiple differentiation inhibitors, which suggested that the downregulation of these genes is associated with C. parvum sexual differentiation. Comparison of our results with a publicly available stage-specific mRNA-seq dataset of C. parvum validated this conclusion and analysis of transcriptomic datasets from other apicomplexan parasites revealed that this is a common mechanism of regulating life cycle stage differentiation in these parasites. In addition, identification of significantly enriched DNA motifs at promoters of dysregulated genes coupled with the expression pattern of several apicomplexan AP2 transcription factors strongly suggested that they play a critical role in regulating C. parvum sexual differentiation.

Language

en

Number of Pages

222 p.

Available for download on Saturday, July 31, 2021

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