Eukaryot Cell. 2011 Apr 29. [Epub ahead of print]
Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection
Fox BA, Falla A, Rommereim LM, Tomita T, Gigley JP, Mercier C, Cesbron-Delauw MF, Weiss LM, Bzik DJ.
SourceDepartment of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756 USA.
Type II Toxoplasma gondii KU80 knockouts (Δku80) 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 Δku80Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80Δhxgprt 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, 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. By contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development as brain tissue cyst burdens were drastically reduced specifically in mutant strains deleted for GRA4 and/or GRA6. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under 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 the type II Δku80Δhxgprt genetic background enables a higher throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
PMID:21531875[PubMed - as supplied by publisher]