Type II Toxoplasma gondii KU80 knockouts (⌬ku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II ⌬ku80 ⌬hxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II ⌬ku80 ⌬hxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8 ؉ T cell epitopes that elicit corresponding antigen-specific CD8 ؉ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8 ؉ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the ⌬gra4 and ⌬gra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II ⌬ku80 ⌬hxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

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.

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.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii. Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals.

The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism.