The cyst wall, acting as the interface between dormant cysts and the host cell, is thought to offer Toxoplasma gondii protection from the host environment. However, the specifics of cyst wall formation and the functional roles of CST1 are not well understood. Our study seeks to elucidate the role of CST1, a major cyst wall protein, in constructing the cyst wall layer through the formation of a filament network mediated by its distinct domains. To this end, we engineered several CST1 truncation mutants and assessed their impact on cyst wall integrity and the localization of other cyst wall proteins such as MCP4. Our findings reveal that the cysteine-rich domain of CST1 is pivotal for polymerization, facilitating the formation of inter-molecular disulfide bonds. The mucin and SRS domains are crucial for maintaining the integrity of the cyst wall and retaining various cyst wall proteins, including MAG1. Utilizing a crosslinking mass spectrometry approach (XL-MS) we are constructing an interactome of the cyst wall, delineating the interactions between proteins, and investigation the structure of CST1. Our findings led to the development of a model for the cyst wall filament network, in which the CST1 homopolymers form the spongy inner layer, acting as a scaffold for other cyst wall proteins to establish the structure. These insights into the function of CST1 provide a deeper understanding of cyst wall architecture and its critical role in parasite survival.

[Funding: NIHAI134753]