Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Boris Striepen

Abstract

Cryptosporidium has emerged as one of the leading causes of diarrhea induced-mortality in children and immunocompromised HIV+ individuals. Other than the acute infection, chronic and asymptomatic cryptosporidiosis results in stunted physical and mental development in children. Drugs and vaccines are needed to combat cryptosporidiosis, and a better understanding of the biology of the parasite will help in developing therapeutics against the parasite. Cryptosporidium has a single host lifecycle. Ingested meiotic spores called oocysts release invasive sporozoites in gut. Sporozoites infect intestinal enterocytes where parasites multiply asexually followed by sexual differentiation. Parasites have sex and then undergo sporulation in the host to produce mature oocysts. Oocysts re-infect the host or are transmitted via feces. Cryptosporidium infection in cancerous cell lines (HCT-8 and Caco-2) lasts for only three days but mice stay infected for a month We engineered a strain that allows to discern different stages and used it to study the developmental kinetics in HCT-8 cells and mice. Parasites replicated asexually in culture followed by sexual differentiation of the 80% of the total population after 48 hours. However, parasites failed to fertilize in culture. Contrastingly, parasites undergo mating, post-fertilization development and sporulation in mice. These studies suggested that sex and renewed production of oocyst is necessary to maintain infection in a host. As a result, disruption of sexual development or mating should break the cycle of infection in mice. This requires an in-depth understanding of sexual stage processes. We identified sexual stage-specific markers and engineered male- and female-specific reporter strains to isolate sexual stages from infected mice and culture for RNA sequencing. Sexual stages were enriched for genes required for meiosis, oocyst development, gamete recognition and fusion. Transcriptional analyses further confirmed four sex specific ApiAP2 genes, and ApiAP2s in Plasmodium are involved in stage-specific development. We intend to disrupt sexual development by targeting one of these ApiAP2s, and cgd4_1110 was confirmed as an essential, female specific ApiAP2. We engineered rapamycin inducible DiCre gene KO system to conditionally disrupt AP2-F. Our next step is to conditionally disrupt AP2-F in infected mice to test essentiality of sex to maintain infection.

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