Functional Characterization of Eight Zinc Finger Motif-Containing Proteins in Toxoplasma gondii Type I RH Strain Using the CRISPR-Cas9 System

Author:

Gao Jin12,Wu Xiao-Jing12,Zheng Xiao-Nan1,Li Ting-Ting2,Kou Yong-Jie12,Wang Xin-Cheng2,Wang Meng2,Zhu Xing-Quan13ORCID

Affiliation:

1. Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China

2. State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

3. Key Laboratory of Veterinary Public Health of Higher Education of Yunnan Province, College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China

Abstract

The Zinc finger protein (ZFP) family is widely distributed in eukaryotes and interacts with DNA, RNA, and various proteins to participate in many molecular processes. In the present study, the biological functions of eight ZFP genes in the lytic cycle and the pathogenicity of Toxoplasma gondii were examined using the CRISPR-Cas9 system. Immunofluorescence showed that four ZFPs (RH248270-HA, RH255310-HA, RH309200-HA, and RH236640-HA) were localized in the cytoplasm, and one ZFP (RH273150-HA) was located in the nucleus, while the expression level of RH285190-HA, RH260870-HA, and RH248450-HA was undetectable. No significant differences were detected between seven RHΔzfp strains (RHΔ285190, RHΔ248270, RHΔ260870, RHΔ255310, RHΔ309200, RHΔ248450, and RHΔ236640) and the wild-type (WT) strain in the T. gondii lytic cycle, including plaque formation, invasion, intracellular replication, and egress, as well as in vitro virulence (p > 0.05). However, the RHΔ273150 strain exhibited significantly lower replication efficiency compared to the other seven RHΔzfp strains and the WT strain, while in vivo virulence in mice was not significantly affected. Comparative expression analysis of the eight zfp genes indicates that certain genes may have essential functions in the sexual reproductive stage of T. gondii. Taken together, these findings expand our current understanding of the roles of ZFPs in T. gondii.

Funder

The National Natural Science Foundation of China

Shanxi Provincial Key Research and Development Program

Shanxi Provincial Agricultural and Rural Research Program

the Research Fund of Shanxi Province for Introduced High-level Leading Talents

the Special Research Fund of Shanxi Agricultural University for High-level Talents

Publisher

MDPI AG

Subject

Infectious Diseases,Microbiology (medical),General Immunology and Microbiology,Molecular Biology,Immunology and Allergy

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