Understanding the molecular mechanisms controlling gametogenesis and virulence in Cryptosporidium parvum

The protozoan parasite, Cryptosporidium parvum, is a leading cause of diarrheal disease and mortality in young children and ruminant animals. This parasite is also a leading cause of waterborne breaks due to its resistance to sanitation, environmental factors, and its easy dissemination through water. There are currently no effective treatments or vaccines available to treat or prevent cryptosporidiosis in humans and animals. There is only one US FDA-approved drug, nitazoxanide, available to treat human cryptosporidiosis, but this drug provides little to no relief to the pediatric and immunocompromised groups. Development of new drugs and vaccines is urgently required to reduce the burden of cryptosporidiosis and thus requires an understanding of parasite biology for the identification of new targets. Cryptosporidium has a highly programmed single-host lifecycle that includes multiple asexual and sexual stages. However, the underlying molecular machinery including signaling mechanisms that control progression of the lifecycle are not known. The Cryptosporidium genome encodes for a family of plant-like calcium-dependent protein kinases (CDPKs). These kinases play important roles in biological processes such as proliferation, invasion, motility, and egress in other related apicomplexan parasites. However, the function of most of these signaling kinases in Cryptosporidium, the parasite life cycle stages at which they are expressed, and the biological processes they regulate are not known. In this study, we aimed at understanding the molecular signaling mechanism controlling sexual development of C. parvum and the role of a transporter in parasite development. Mining the available gene expression data for C. parvum during different timepoints of intracellular development, we identified cdpk5 as a potential sexual stage specific gene based on its late expression at 48 hours. To understand the role of this kinase we employed CRISPR/Cas9 genetic editing and an immunocompromised mouse model of infection to create two transgenic parasite strains, one strain that expressed this kinase along with a hemagglutinin epitope tag (CDPK5-HA) and another strain where this gene was deleted (CDPK5-KO). Localization studies revealed expression of CDPK5 only in the sexual stage, specifically during the development of male gametes. Although genetic ablation of this kinase was not essential for parasite survival, its deletion resulted in significantly reduced virulence and oocyst shedding in immunocompromised mice. Furthermore, we used a phosphoproteomics approach and identified 11 potential substrates of CDPK5. Future characterization of these substrates will reveal their mechanistic role in the male gametogenesis process. Lastly, we wanted to investigate the role of a transporter in the development of parasite stages. Cryptosporidium has a streamlined metabolism and relies on its host for nutrient uptake. We identified a major facilitator superfamily (MFS) domain containing transporter in C. parvum, located upstream of the cdpk5. We found that this protein is expressed throughout the parasite lifecycle and is essential for parasite survival. Overall, this work uncovers molecular mechanisms underlying male gametogenesis, virulence and parasite development and identifies a new target for blocking disease transmission.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

VMS - Pathobiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Nava, Maria G

Contributors dc:contributor

• Vinayak Alam, Sumiti
• Lau, Gee W
• Zhang, Weiping
• Aldridge, Brian M
• Mei, Wenyan

• Genetics
• Kinase
• Gametogenesis
• Egress
• Transporters
• Host-parasite Interactions
• Metabolic Uptake
• Cryptosporidium
• Parasitology
• Apicomplexan Parasite
• Parasite Burden
• Animal Infection
• Virulence
• Diarrhea
• Cell Biology

Statement dc:rights

• Copyright 2024 Maria G Nava

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/127226

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F127226