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Effect of deleting four Toxoplasma gondii calcium-binding EGF domain-containing proteins on parasite replication and virulence

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Abstract 

Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress from the host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated.

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Acknowledgements

We thank Professor Bang Shen, Huazhong Agricultural University for providing the pSAG1-Cas9-SgUPRT and pUPRT-DHFR-D vectors.

Funding

Project support was provided by the National Natural Science Foundation of China (Grant No. 32002306), Fundamental Research Funds of the Chinese Academy of Agricultural Sciences (Grant No. 1610032021017), the Fund for Shanxi “1331 Project” (Grant No. 20211331–13), the Agricultural Science and Technology Innovation Program (ASTIP) of China (Grant No. CAAS-ASTIP-2016-LVRI-03), the Yunnan Expert Workstation (Grant No. 202005AF150041), and the Veterinary Public Health Innovation Team of Yunnan Province (Grant No. 202105AE160014).

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Authors and Affiliations

  1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, 730046, People’s Republic of China

    Xin-Cheng Wang, Ting-Ting Li, Dan-Yu Zhao, Jin-Lei Wang, Meng Wang & Xing-Quan Zhu

  2. Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu, Sichuan Province, 610213, People’s Republic of China

    Ting-Ting Li, Jin-Lei Wang & Meng Wang

  3. Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, Loughborough, LE12 5RD, UK

    Hany M. Elsheikha

  4. Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi Province, 030801, People’s Republic of China

    Xiao-Nan Zheng & Xing-Quan Zhu

  5. Key Laboratory of Veterinary Public Health of Higher Education of Yunnan Province, College of Veterinary Medicine, Yunnan Agricultural University, Kunming, People’s Republic of China

    Xing-Quan Zhu

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  1. Xin-Cheng Wang
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  2. Ting-Ting Li
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  3. Hany M. Elsheikha
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  4. Xiao-Nan Zheng
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Contributions

Xing-Quan Zhu, Meng Wang, Jin-Lei Wang, and Hany M. Elsheikha conceived and designed the study. Xin-Cheng Wang performed the experiments, analysed the data, and wrote the manuscript. Ting-Ting Li, Xiao-Nan Zheng, and Dan-Yu Zhao participated in the implementation of the study. Meng Wang and Jin-Lei Wang contributed reagents/materials/analysis tools. Hany M. Elsheikha, Xing-Quan Zhu, and Meng Wang critically revised the manuscript. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Xing-Quan Zhu.

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All animal studies were carried out in accordance with protocols reviewed and approved by the Research Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All animals were handled strictly according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China. All efforts were made to minimize the number of mice used in the study.

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Supplemental Fig. 5

The schematic diagram of the predicted functional domains in each CBDP protein. EGF, epidermal growth factor-like domain; EGF-CA, calcium- binding EGF-like domain; EGF-like, EGF domain, unclassified subfamily. The putative functional domains of the CBDP proteins were predicated by the SMART algorithm (http://smart.embl-heidelberg.de). The blue rectangles represent the transmembrane helix region, as detected by the TMHMM v2.0 program. (PNG 496 kb)

Supplementary file1 (TIF 828 KB)

Supplemental Fig. 6

The expression profiles of Toxoplasma gondii CBDPs. A The expression profile of 3 CBDP genes of T. gondii RH strain presented by the cell cycle phases. B The expression profiles of 3 CBDP genes in Type I (RH and GT1), Type II (Pru and ME49), and Type III (CTG and VEG) strains. C The expression profiles of 3 CBDP genes related to the parasite life cycle stages (oocyst, tachyzoite and bradyzoite). Expression profile of 3 CBDP genes of the oocysts recovered from cat feces at 0 day (unsporulated), 4 days (4 days sporulated), and 10 days (10 days sporulated), tachyzoites grown for 2 days in HFF cells (2 days in vitro), bradyzoites grown in HFF cells for 4 days and 8 days (4 day in vitro and 8 days in vitro), and 21 days tissue cyst-containing bradyzoites harvested from infected mouse brains (21 days in vivo). Each line represents the expression value of the corresponding gene. (PNG 528 kb)

Supplementary file2 (TIF 1465 KB)

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Wang, XC., Li, TT., Elsheikha, H.M. et al. Effect of deleting four Toxoplasma gondii calcium-binding EGF domain-containing proteins on parasite replication and virulence. Parasitol Res 122, 441–450 (2023). https://doi.org/10.1007/s00436-022-07739-6

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