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

Abstract

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.

Citation

Zheng, X. N., Wang, J. L., Elsheikha, H. M., Wang, M., Zhang, Z. W., Sun, L. X., …Li, T. T. (2023). Functional Characterization of 15 Novel Dense Granule Proteins in Toxoplasma gondii Using the CRISPR-Cas9 System. Microbiology Spectrum, 11(1), https://doi.org/10.1128/spectrum.03078-22

• Journal Article Type: Article
• Acceptance Date: Nov 16, 2022
• Online Publication Date: Dec 14, 2022
• Publication Date: 2023-02
• Deposit Date: Dec 18, 2022
• Publicly Available Date: Dec 20, 2022
• Journal: Microbiology Spectrum
• Electronic ISSN: 2165-0497
• Peer Reviewed: Peer Reviewed
• Volume: 11
• Issue: 1
• DOI: https://doi.org/10.1128/spectrum.03078-22
• Public URL: https://nottingham-repository.worktribe.com/output/14596502
• Publisher URL: https://journals.asm.org/doi/10.1128/spectrum.03078-22