Cryptosporidium parvum is a major cause of diarrheal disease worldwide, particularly affecting children and immunocompromised individuals. Sexual reproduction is essential for parasite transmission, and disrupting fertilization has emerged as a promising strategy to block infection. Understanding the molecular mechanisms underlying this process could reveal new targets for therapeutic intervention.Recent studies have revealed that male gametes of C. parvum express a complex and specialized fertilization machinery. While components such as the fusogen HAP2 have been identified (Tandel et al., 2019), the overall structure and function of this machinery remain poorly understood. To address this knowledge gap, we aim to use cryo-electron tomography (cryo-ET) to directly visualize the architecture of the male gamete and its fertilization apparatus at molecular resolution. A critical bottleneck for structural studies is the ability to generate and isolate enough male gametes. To address this, we engineered a new C. parvum strain that integrates three key components: inducible maleness (previously validated in Walzer et al., 2024), a fluorescent male-specific reporter, and a high-production genetic background. Our results demonstrate that this system allows robust induction and enrichment of male gametes suitable for cryo-ET and downstream analyses.This system paves the way for high-resolution structural studies and functional dissection of the fertilization machinery. Insights gained from this work will advance our understanding of parasite reproduction and may inform the development of transmission-blocking strategies against cryptosporidiosis.
Fenglin Li (Sun,) studied this question.