Regulation of Cryptosporidium Development

The parasite Cryptosporidium is the leading cause of waterborne illness from treated recreational water in the United States and causes severe diarrheal disease worldwide. There is no vaccine, and the only drug with FDA approval, called nitazoxanide, is ineffective at treating immunocompromised individuals. Therefore, a dire need exists for new and effective therapeutics. Cryptosporidium can survive harsh environments, including chlorinated water, due to its oocyst form which is the product of sexual reproduction. Transmission occurs through ingestion of the oocyst, and the entire parasite life cycle takes place in a single host. Cryptosporidium propagates asexually in the epithelial cells of the small intestine for three cycles before transitioning to a sexual fate, producing male and female gametes. Sexual reproduction takes place and produces oocysts which either exit the host or reinfect intestinal cells. This indicates that sexual reproduction is necessary for both continuous infection and transmission. Previous work showed that transcription factor Myb-M acts as a master regulator of sex determination and that its expression initiates male development. The work proposed here will define the molecular events that direct Cryptosporidium male gametogenesis by accomplishing the following aims. First, which signaling pathways are controlled by Myb-M? Aim 1 will utilize chromatin profiling to define the target genes of Myb-M and immunoprecipitation assays to determine the protein complex that coordinates male differentiation. This will uncover the binding partners of Myb-M. Second, how do male-specific RNA-binding proteins (RBPs) direct the transcriptional switch to male fate? Aim 2 will focus on elucidating the role of a putative zinc finger CCCH-type RBP that is expressed in the earliest males. It is hypothesized that this gene, RBP-M1, translationally controls early male genes, including Myb-M, by binding their transcripts. It is also predicted that RBP-M1 is necessary but not sufficient to drive male fate. Immunoprecipitation of RBP-M1 and sequencing of its targets will reveal the abundance of Myb-M and other transcripts. The generation of an RBP-M1 loss-of-function mutant will show which step of male development this gene controls and if it regulates Myb-M protein levels. The creation of an RBP-M1 overexpression strain will test whether RBPM1 is sufficient to drive male fate. Overall, these experiments will uncover the regulatory mechanisms that direct male gametogenesis in Cryptosporidium and will provide new drug and vaccine targets to block sexual reproduction, thereby halting infection and transmission. Project Number: 5P20GM146584-04 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Katelyn Walzer | Institution: CLEMSON UNIVERSITY, CLEMSON, SC | Award Amount: $251,625 | Activity Code: P20 | Study Section: ZGM1-RCB-3(C2) View on NIH RePORTER: https://reporter.nih.gov/project-details/11566081