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...

In this study, we document that Toxoplasma gondii differentiation and reactivation are mediated by systemic CD8 T-cell dysfunction during chronic infection. We demonstrate that CD8 + T-cell exhaustion occurs despite control of parasitemia during early-chronic toxoplasmosis. During later phases, these cells become exhausted, leading to parasite reactivation and mortality. Concomitant with increased CD8 + T-cell apoptosis and decreased effector response, this dysfunction is characterized by a graded elevation in expression of inhibitory receptor PD-1 on these cells in both lymphoid and nonlymphoid tissue. Blockade of the PD-1–PDL-1 pathway reinvigorates this suboptimal CD8 + T-cell response, resulting in control of parasite reactivation and prevention of mortality in chronically infected animals. To the best of our knowledge, this report is unique in showing that exposure to a persistent pathogen despite initial control of parasitemia can lead to CD8 + T-cell dysfunction and parasite reactivation.

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.

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A single inoculation of mice with the live, attenuated Toxoplasma gondii uracil auxotroph strain cps1-1 induces long-lasting immunity against lethal challenge with hypervirulent strain RH. The mechanism for this robust immunity in the absence of parasite replication has not been addressed. The mechanism of long-lasting immunity, the importance of route of immunization, cellular recruitment to the site of infection, and local and systemic inflammation were evaluated. Our results show that infection with cps1-1 elicits long-lasting CD8+ T cell- mediated immunity. We show that immunization with cps1-1-infected dendritic cells elicits long-lasting immunity. Intraperitoneal infection with cps1-1 induced a rapid influx of GR1+ neutrophils and two stages of GR1+CD68+ inflammatory monocyte infiltration into the site of inoculation. CD19+ B cells and CD3+ T cells steadily increase for 8 days after infection. CD8+ T cells were rapidly recruited to the site of infection and increased faster than CD4+ T cells. Surprisingly, cps1-1 infection induced high systemic levels of bioactive IL-12p70 and a very low level and transient systemic IFN-γ. Furthermore, we show significant levels of these inflammatory cytokines were locally produced at the site of cps1-1 inoculation. These findings offer new insight into immunological mechanisms and local host responses to a non-replicating type I parasite infection associated with development of long-lasting immunity to Toxoplasma gondii.