@article {69397, title = {Expansion of in vitro Toxoplasma gondii cysts using enzymatically enhanced ultrastructure expansion microscopy}, journal = {mSphere}, volume = {e0032224}, year = {2024}, month = {2024 Aug 27}, abstract = {

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked the detail of super-resolution images for a broader scientific circle, lowering the cost and entry skill requirements for the field. One of its branches, ultrastructure expansion microscopy (U-ExM), has become popular among research groups studying apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. Here, we show that the chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localization. We then solve the in vitro cyst{\textquoteright}s resistance to denaturation required for successful U-ExM. As the cyst{\textquoteright}s main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without disrupting immunofluorescence analysis of parasite proteins. Using StcE-enhanced U-ExM, we clarified the localization of the GRA2 protein, which is important for establishing a normal cyst, observing GRA2 granules spanning across the CST1 cyst wall. The StcE-U-ExM protocol allows accurate pinpointing of proteins in the bradyzoite cyst, which will greatly facilitate investigation of the underlying biology of cyst formation and its vulnerabilities.

}, doi = {10.1128/msphere.00322-24}, url = {https://www.doi.org/10.1128/msphere.00322-24}, author = {Bondarenko, Kseniia and Limoge, Floriane and Pedram, Kayvon and Gissot, Mathieu and Young, Joanna C.} }