Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Christopher D. Huston
Abstract
Cryptosporidium, an intestinal protozoan parasite, is a major cause of diarrheal disease worldwide for which there is no effective vaccine and only one FDA approved drug for treatment which is ineffective for those most prone to developing severe disease. A better understanding of sexual reproduction in Cryptosporidium could inform new treatment options.Cryptosporidium’s life cycle begins with the excystation of the oocyst in the small intestine which releases sporozoites that infect intestinal epithelial cells where they develop into trophozoites in an epicellular niche, which is intracellular but extracytoplasmic. Trophozoites transition to merogony which produces 8 merozoites which escape and infect new host cells. This process of merogony is repeated 3 times after which parasites develop into the sexual stages (microgamonts (male) or macrogamonts (female)) upon infecting new host cells. Microgametes egress and find and fertilize macrogamonts to form zygotes, the only diploid stage of the parasite’s life cycle. Meiosis produces 4 haploid sporozoites within an oocyst which is excreted into the environment to infect new hosts or excysts within the intestine causing autoinfection. Sex is essential for transmission and important for determining how long an infection can last. In typical cell culture, the life cycle stalls at the point of fertilization for an unknown reason. Recent approaches have reported to overcome this barrier but have not been widely adopted due to excessive complexity or inability to reproduce the results. Here, 2D and 3D organoid-based approaches were developed to facilitate sexual reproduction in vitro. Both models utilize differentiated mouse intestinal epithelial cells that represent each of the major cell types found in the small intestinal epithelium. The 3D intestinal organoid model employs an inverted organoid with the apical surface facing the outside of the organoid which enables infection without the need for microinjection. Inverted organoids supported C. parvum infection and subculture of the parasites. In addition, 2D mouse organoid-derived monolayers grown on permeable supports facilitated polarization of the cells and supported C. parvum infection, fertilization, and oocyst production for at least 3 weeks. Fertilization was confirmed by production of new oocysts and a mating assay in which mating of fluorescent reporter strains of C. parvum produced double positive zygotes. We developed two fertilization reporters to investigate factors involved in C. parvum fertilization. An offspring reporter utilizes the inducible di-Cre system for an inducible switch after fertilization that is spatially and temporally controlled resulting in red parasites in the first generation and green parasites in the second generation. A dual sexual differentiation reporter utilizes mCherry and mNeonGreen sequences under the control of microgamete and macrogamont specific promoters, which allows investigation of differential expression in sexual stages and detection of zygotes. These reporters were validated in infected mice and/or in organoid-based cultures. Using these organoid-based cell cultures and fertilization reporters could help to gain better understanding of sexual reproduction in Cryptosporidium that could inform the development of treatments to inhibit sexual reproduction that could significantly shorten the duration of disease and prevent transmission.
Language
en
Number of Pages
211 p.
Recommended Citation
Mihavics, Bethany R., "Development Of A Toolbox For The Study Of Cryptosporidium Sexual Reproduction" (2024). Graduate College Dissertations and Theses. 1954.
https://scholarworks.uvm.edu/graddis/1954