A comprehensive proteome and the first phosphoproteome reveal extensive phosphorylation of carbohydrate metabolism in Cryptosporidium parvum sporozoites

Abstract

Cryptosporidium parvum is an obligate intracellular apicomplexan parasite and a major cause of diarrheal disease in humans and animals worldwide. Despite its public health importance, the molecular regulation of parasite metabolism, particularly at the post-translational level, remains poorly understood. Here, we present a comprehensive proteomic analysis and the first phosphoproteomic profile of excysted C. parvum sporozoites, the invasive stage responsible for host cell entry. Using data-independent acquisition-based mass spectrometry, we identified 2,272 proteins, representing approximately 58% of the predicted parasite proteome, and 8,994 phosphorylation sites across 833 phosphoproteins. Comparative analyses revealed weak correlations between transcript and protein abundance, underscoring extensive post- transcriptional regulation in sporozoites. Functional enrichment analyses showed that proteins involved in carbohydrate metabolism, particularly glycolysis, are highly abundant in the sporozoite proteome. In contrast, phosphoproteomic data revealed that many core glycolytic enzymes exhibit relatively low phosphorylation propensity, suggesting limited reliance on phosphorylation-based regulation for basal energy metabolism at this stage. To integrate proteomic and phosphoproteomic measurements acquired independently, we developed a relative phosphorylation index (RPI) that enables comparative assessment of phosphorylation propensity across proteins. Application of this metric highlighted selective phosphorylation of proteins associated with signaling, cytoskeletal organization, and host-parasite interaction, while key metabolic entry-point enzymes, such as hexokinase, showed high abundance but minimal detectable phosphorylation, in contrast to their mammalian counterparts. Together, these findings provide the most comprehensive molecular resource to date for C. parvum sporozoites and reveal a phosphorylation landscape that emphasizes regulatory and structural processes over core metabolic flux. This work establishes a foundation for understanding parasite-specific regulatory strategies and may inform the identification of novel therapeutic targets against cryptosporidiosis. Author SummaryCryptosporidium parvum is a microscopic parasite that causes diarrheal disease in humans and animals worldwide, yet we still know surprisingly little about how it regulates its biology at the molecular level. In this study, we set out to better understand how the parasite prepares for infection by examining the proteins present in sporozoites, the life stage that actively invades host cells. We generated the most comprehensive protein catalog to date for C. parvum sporozoites and, for the first time, mapped thousands of chemical modifications known as protein phosphorylation. These modifications often act as molecular switches that control how proteins behave. We found that proteins involved in basic energy production are extremely abundant but show relatively little phosphorylation, while proteins linked to cell structure, movement, and interaction with the host are more frequently modified. This suggests that the parasite prioritizes regulation of invasion-related processes over fine-tuning its core metabolism at this stage. By providing a detailed molecular resource and a new way to compare protein phosphorylation patterns, our work lays the groundwork for future studies of parasite biology and may help guide the search for new strategies to combat cryptosporidiosis.