Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection

Barbara A. Fox, Alejandra Falla, Leah M. Rommereim, Tadakimi Tomita, Jason P. Gigley, Corinne Mercier, Marie France Cesbron-Delauw, Louis M. Weiss, David J. Bzik

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Abstract

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 Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt 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 Δgra4 and Δgra6 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 Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

Original languageEnglish (US)
Pages (from-to)1193-1206
Number of pages14
JournalEukaryotic Cell
Volume10
Issue number9
DOIs
StatePublished - Sep 2011

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Toxoplasma
Cysts
Infection
Genes
CD8 Antigens
Parasites
uracil phosphoribosyltransferase
T-Lymphocytes
T-Lymphocyte Epitopes
Gene Targeting
Brain
Infection Control
Gene Frequency
Virulence
Proteins
Plasmids
Alleles
Genome
Antigens
Population

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Fox, B. A., Falla, A., Rommereim, L. M., Tomita, T., Gigley, J. P., Mercier, C., ... Bzik, D. J. (2011). Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection. Eukaryotic Cell, 10(9), 1193-1206. https://doi.org/10.1128/EC.00297-10

Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection. / Fox, Barbara A.; Falla, Alejandra; Rommereim, Leah M.; Tomita, Tadakimi; Gigley, Jason P.; Mercier, Corinne; Cesbron-Delauw, Marie France; Weiss, Louis M.; Bzik, David J.

In: Eukaryotic Cell, Vol. 10, No. 9, 09.2011, p. 1193-1206.

Research output: Contribution to journalArticle

Fox, BA, Falla, A, Rommereim, LM, Tomita, T, Gigley, JP, Mercier, C, Cesbron-Delauw, MF, Weiss, LM & Bzik, DJ 2011, 'Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection', Eukaryotic Cell, vol. 10, no. 9, pp. 1193-1206. https://doi.org/10.1128/EC.00297-10
Fox, Barbara A. ; Falla, Alejandra ; Rommereim, Leah M. ; Tomita, Tadakimi ; Gigley, Jason P. ; Mercier, Corinne ; Cesbron-Delauw, Marie France ; Weiss, Louis M. ; Bzik, David J. / Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection. In: Eukaryotic Cell. 2011 ; Vol. 10, No. 9. pp. 1193-1206.
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