Enrichment and Proteomic Characterization of the Cyst Wall from In Vitro Toxoplasma gondii Cysts

Abstract

The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface. IMPORTANCE Toxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.

Novel roles of dense granule protein 12 (GRA12) inToxoplasma gondiiinfection

“…Consistent with previously reported data, we found that GRA12 is secreted into the PV, co‐localized with GRA2 detected using polyclonal rabbit anti‐GRA12 or anti‐HA antibody, and is associated with the IVN. GRA12 was also localized to the cyst wall and matrix, confirming previous proteomic finding that GRA12 exists in the cyst wall fractions …”

“…GRA12 was also localized to the cyst wall and matrix, confirming previous proteomic finding that GRA12 exists in the cyst wall fractions. 47 The IVN serves diverse functions, such as nutrient acquisition through trafficking of host-derived vesicles, 48 uptake | 3175 WANG et Al.…”

Identification of a master regulator of differentiation inToxoplasma

“…Endogenous tagging of two additional strong, early markers of differentiation (TGME49_312330, TGME49_208740) identified both as cyst-wall proteins, confirming the localization recently shown for TGME49_208740 ( Fig. S8C) (Tu et al, 2019). While BFD1 transcription is not exclusive to bradyzoites, BFD1 is identified as modestly upregulated at 72 h in stressed wild-type parasites by our scRNA-Seq data, and is significantly enriched in bradyzoite-containing clusters ( Fig.…”

Global profiling of myristoylation inToxoplasma gondiireveals key roles for lipidation in CDPK1 and MIC7 function

“…The log2 fold changes in protein abundance for MIC7 and the bradyzoite-specific marker, MAG1 ( (Tu et al, 2019); Figure 5A, Table S4) revealed that in contrast to MAG1, MIC7 is expressed in tachyzoites, supporting the MS and transcriptional evidence in ToxoDB. We next aimed to validate the protein's myristoylation as our MS screen did not identify a myristoylated peptide for MIC7.…”