Abstract

The apicomplexan parasite Cryptosporidium parvum (Cp) is a major global contributor to infectious diarrhea in children and poses a serious threat to immunocompromised adults. This dissertation investigates Cp's unique life cycle, which alternates between sexual and asexual reproduction to achieve three critical objectives: producing oocysts capable of transmission, enhancing genetic variation through recombination, and generating sufficient progeny for survival. Sexual reproduction is essential for forming infectious oocysts that facilitate disease transmission. Yet, the process of meiosis in Cp has been historically understudied due to limitations of traditional in vitro culture methods. The newly developed air-liquid interface (ALI) culture system supports Cp's sexual reproduction in vitro, enabling the exploration of meiosis. Studies using the ALI system and immunocompromised mouse models reveal that Cp chromosomal segregation follows Mendelian inheritance models and displays a high crossover frequency, consistent with observations of Cp's ability to adapt and speciate in nature rapidly. Following excystation, Cp sporozoites invade host cells and rapidly undergo multiple mitotic divisions, which are essential for evading immune responses and establishing infection. Despite the importance of mitosis in Cp pathogenesis, its cellular mechanisms remain unexplored. Using advanced technologies such as CRISPR/Cas9 gene editing, confocal microscopy, and CUT&RUN sequencing, we uncovered features that differentiate Cp from other apicomplexans. We observed a diffuse centromere staining pattern in Cp, distinct from the punctate pattern in Toxoplasma and Plasmodium. Using phospho-histone H3, centrin, and tubulin antibodies, we visualized various stages of Cp mitosis, noting that centromeres remain diffuse even during mitosis. Additionally, Cp telomeres overlap with centromeres at the apical end of the parasite nucleus, rather than at the base as seen in other apicomplexans. We hypothesize that this may be due to the tagged telomere binding protein's occasional occurrence in non-telomeric regions or a tethering of telomeres to the top of the nuclear envelope to be closer to the kinetochore components. This may serve as a mechanism for chromatin organization, epigenetic regulation, etc., and requires additional study. Overall, Cp’s distinctive nuclear architecture may stem from its evolutionary divergence from Toxoplasma and Plasmodium or may be a consequence of its distinct biological adaptations. Cp's unique nuclear configuration may enable the parasite to maintain genome stability while facilitating rapid replication and frequent recombination, overall ensuring the parasite’s adaptability and survival.

Committee Chair

David Sibley

Committee Members

Daniel Goldberg; Douglas Chalker; Megan Baldridge; Tim Schedl

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

7-14-2025

Language

English (en)

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