A suture-specific oocyst wall protein COWP4 is essential for excystation and infectivity, while COWP6 links wall architecture to host interaction in Cryptosporidium parvum

Abstract The oocyst of Cryptosporidium is a highly resilient transmission stage that protects sporozoites in the environment and mediates infection through a specialized opening known as the suture. While recent studies have identified Cryptosporidium oocyst wall proteins (COWPs) as structural components of the oocyst wall, the functional roles of individual COWPs and the molecular basis of suture biology remain poorly understood. Here, we performed a detailed characterization of two COWP family members, COWP4 and COWP6, combining immunolocalization, ultrastructural analysis, protein interaction assays, and genetic manipulation. We show that COWP4 is strictly localized to the oocyst suture, whereas COWP6 is distributed throughout the oocyst wall inner layer and enriched at the suture. COWP6 is additionally present in sporozoites and is secreted during parasite motility and host cell invasion, where it exhibits high-affinity binding to host cells. Structural analyses indicate that both proteins are cysteine-rich and likely form disulfide-stabilized architectures consistent with roles in wall assembly. Functional analyses reveal that COWP4 is essential for proper suture formation, excystation, and parasite infectivity, establishing the suture as a genetically defined gatekeeper for parasite transmission. In contrast, COWP6 functions as a multifunctional protein linking oocyst wall architecture to host interaction. We further demonstrate that COWP4 and COWP6 interact, suggesting coordinated assembly within the oocyst wall. Together, these findings provide a functional dissection of the Cryptosporidium oocyst suture and reveal distinct, non-redundant roles for individual COWPs. This work advances our understanding of oocyst wall biology and identifies COWP4 as an essential determinant of parasite transmission that may be targeted to disrupt the infectious cycle. Author summary Cryptosporidium is a major cause of diarrheal disease worldwide and is transmitted through environmentally resistant oocysts that protect infectious stages of the parasite. A defining feature of the oocyst is a specialized seam-like structure, called the suture, through which parasites exit to initiate infection. Although oocyst wall proteins (COWPs) have been identified, how individual proteins contribute to the structure and function of the oocyst wall, particularly the suture, has remained unclear. In this study, we investigated two oocyst wall proteins, COWP4 and COWP6, and uncovered distinct roles for each. We found that COWP4 is specifically localized to the suture and is essential for its proper formation, enabling parasites to exit the oocyst and establish infection. In contrast, COWP6 is distributed more broadly in the oocyst wall and also functions beyond it, being secreted during parasite movement and interacting with host cells. These findings reveal that the oocyst wall is composed of specialized proteins with non-redundant functions and identify the suture as a critical control point for parasite transmission. Understanding these mechanisms may help guide the development of new strategies to block infection by disrupting oocyst integrity or parasite release. Highlights COWP4 is a suture-specific protein essential for oocyst excystation and infectivity Genetic disruption of COWP4 produces non-infectious oocysts with impaired viability The oocyst suture is a genetically defined gatekeeper for parasite transmission COWP6 localizes to the wall inner layer and is enriched at the suture COWP6 is secreted by sporozoites and binds host cells with nanomolar affinity · COWP4 and COWP6 directly interact, linking wall architecture to host interaction Competing Interest Statement The authors have declared no competing interest.