Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining

Abstract

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...

Phosphorylation of eukaryotic initiation factor-2α promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

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DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

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