The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence

Abstract

Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms.

Author Summary

Toxoplasma gondii causes severe encephalitis in immune compromised hosts after reactivation of brain cysts that persist for the life span of the host. The biological mechanisms of bradyzoite persistence within cysts are not fully understood. The glycosylated cyst wall is thought to play a crucial role in survival of bradyzoites during chronic infection as well as successful oral transmission of infection. Here we have identified the gene encoding cyst wall glycoprotein CST1. When we delete the CST1 gene, parasites form dramatically fragile brain cysts. Parasites lacking CST1 develop fewer brain cysts, show dysregulation of bradyzoite-specific gene expression and are less able to grow under stressed conditions. The rescue of these phenotypes requires the heavily glycosylated mucin domain of CST1. These studies demonstrate that the glycosylation of CST1 plays a significant role in the structural integrity and persistence of brain cysts. Agents that perturb CST1 glycosylation have the potential to disrupt formation of latent brain cysts, preventing chronic Toxoplasma infection.