Increased efficiency of homologous recombination in Toxoplasma gondii dense granule protein 3 demonstrates that GRA3 is not necessary in cell culture but does contribute to virulence

Toxoplasma gondii possesses unique secretory organelles, which synchronously release proteins during and after invasion. One of these organelles, the dense granules, secrete proteins after invasion which are thought to be important in development of the parasite throughout all stages of its life cycle. Dense granule protein 3 (GRA3) is a 30 kDa protein localized to the intravacuolar network and parasitophorous vacuole membrane (PVM). Like many dense granule proteins, GRA3 has no homology to proteins with described functions. However, it has been hypothesized to be involved in nutrient acquisition for the parasite due to its localization on the PVM. To begin to investigate the importance of GRA3, the locus was disrupted by homologous replacement with a chloramphenicol resistance gene in a type II strain. Two ΔGRA3 strains were obtained after two independent electroporations with efficiency greater than 80%. No differences between wild-type and ΔGRA3 were detected in cell culture growth rate or bradyzoite formation. Location of other parasite dense granule proteins and association with host cell organelles were also not affected in ΔGRA3. Interestingly, at an infectious dose approximately four-fold above the lethal dose 50% for wild-type parasites, all mice infected with ΔGRA3-2 infected mice survived acute infection. Complementation of GRA3 expression in the ΔGRA3-2 strain restored virulence to wild-type levels, and increased the virulence of the ΔGRA3-1, confirming that the GRA3 protein plays a role during acute infection in a type II strain.