SUMMARY Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.

A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300-to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only ϳ500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of type I strains but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or ␥-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway is present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed postgenome functional analysis and genetic dissection of parasite biology.Toxoplasma gondii is a widespread obligate intracellular protozoan pathogen of virtually all warm-blooded animals and commonly infects humans worldwide. Due to a significant menu of established experimental approaches, T. gondii has become a model for the study of closely related disease-causing parasites (Plasmodium, Theileria, and Cryptosporidium species) belonging to the phylum Apicomplexa (32) and a model for the study of intracellular pathogens (33). Unfortunately, a high frequency of nonhomologous recombination arising from a previously undetermined double-strand break (DSB) DNA repair pathway(s) has hampered gene targeting approaches in this model. DSB repair in most eukaryotes occurs primarily via two different recombination pathways (27). The homologous recombination pathways repair a DSB using mechanisms that recognize highly homologous DNA sequences, while the nonhomologous end-joining (NHEJ) pathway does not rely on DNA sequence homology. Instead, NHEJ involves direct ligation of the ends of broken DNA strands. KU70 and KU80 proteins form a heterodimer that tightly binds the DNA ends at the DSB, an early and essential step of NHEJ (49, 50). In addition to KU70 and KU80 proteins, NHEJ is mediated by the DNA ligase IV-Xrcc4 complex and the DNA-dependent protein kinase catalytic subunit or other functionally equivalent protein complexes (49).Many eukaryotes preferentially use the NHEJ pathway to repair a DSB, and exogenous targeting DNA can be integrated anywhere into the genome independent of DNA sequence homology (27). The NHEJ pathway also appears to be preferentially used by Toxoplasma gondii based on the high rates of nonhomologous recombination a...

The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

Toxoplasma gondii is a ubiquitous protozoan parasite that is responsible for severe congenital birth defects and fatal toxoplasmic encephalitis in immunocompromized people. Fundamental aspects of obligate intracellular replication and pathogenesis are only now beginning to emerge for protozoan parasites. T. gondii has a fragmented pathway for salvaging pyrimidine nucleobases derived from the parasite or host cell, and this limited pyrimidine salvage capacity is funnelled exclusively through uracil phosphoribosyltransferase. Disrupting the function of this enzyme does not affect the growth of T. gondii tachyzoites, which suggests that the de novo pyrimidine biosynthesis pathway may be necessary for growth. We have examined the virulence of T. gondii mutants that lack carbamoyl phosphate synthetase II (uracil auxotrophs) to determine whether de novo pyrimidine biosynthesis is required in vivo. Here we show that T. gondii uracil auxotrophs are completely avirulent not only in immune-competent BALB/c mice but also in mice that lack interferon-gamma. A single injection of the uracil auxotroph into BALB/c mice induces long-term protective immunity to toxoplasmosis. Our findings indicate the significance of the de novo pyrimidine biosynthesis pathway for the virulence of parasitic protozoa, and suggest routes for developing vaccines and chemotherapy.

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.

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