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Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System

Zheng, Xiao Nan; Wang, Jin Lei; Elsheikha, Hany M.; Wang, Meng; Zhang, Zhi Wei; Sun, Li Xiu; Wang, Xin Cheng; Zhu, Xing Quan; Li, Ting Ting


Xiao Nan Zheng

Jin Lei Wang

Meng Wang

Zhi Wei Zhang

Li Xiu Sun

Xin Cheng Wang

Xing Quan Zhu

Ting Ting Li


The analysis of the subcellular localization and function of dense granule proteins (GRAs) is of central importance for the understanding of host-parasite interaction and pathogenesis of Toxoplasma gondii infection. Here, we identified 15 novel GRAs and used C-terminal endogenous gene tagging to determine their localization at the intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in the tachyzoites and at the periphery of the bradyzoites-containing cysts. The functions of the 15 gra genes were examined in type I RH strain and 5 of these gra genes were also evaluated in the cyst-forming type II Pru strain. The 15 novel gra genes were successfully disrupted by using CRISPR-Cas9 mediated homologous recombination and the results showed that 13 gra genes were not individually essential for T. gondii replication in vitro or virulence in mice during acute and chronic infection. Intriguingly, deletion of TGME49_266410 and TGME49_315910 in both RH and Pru strains decreased the parasite replication in vitro and attenuated its virulence, and also reduced the cyst-forming ability of the Pru strain in mice during chronic infection. Comparison of the transcriptomic profiles of the 15 gra genes suggests that they may play roles in other life cycle stages and genotypes of T. gondii. Taken together, our findings improve the understanding of T. gondii pathogenesis and demonstrate the involvement of two novel GRAs, TGME49_266410 and TGME49_315910, in the parasite replication and virulence.


Zheng, X. N., Wang, J. L., Elsheikha, H. M., Wang, M., Zhang, Z. W., Sun, L. X., …Li, T. T. (2022). Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System. Microbiology Spectrum, 11(1),

Journal Article Type Article
Acceptance Date Nov 16, 2022
Online Publication Date Dec 14, 2022
Publication Date Dec 14, 2022
Deposit Date Dec 18, 2022
Publicly Available Date Dec 20, 2022
Journal Microbiology Spectrum
Electronic ISSN 2165-0497
Publisher American Society for Microbiology
Peer Reviewed Peer Reviewed
Volume 11
Issue 1
Public URL
Publisher URL


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