Functional Characterization of 11 Tentative Microneme Proteins in Type I RH Strain of Toxoplasma gondii Using the CRISPR-Cas9 System

Author:

Ma Zhi-Ya12ORCID,Wu Xiao-Jing12,Li Chuan12,Gao Jin12,Kou Yong-Jie12ORCID,Wang Meng2,Zhu Xing-Quan1ORCID,Zheng Xiao-Nan1

Affiliation:

1. Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, 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

Abstract

Toxoplasma gondii, a pathogenic apicomplexan parasite, infects approximately one third of the world’s population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii.

Funder

National Natural Science Foundation of China

Shanxi Provincial Key Research and Development Program

National Key Research and Development Program of China

Shanxi Provincial Agricultural and Rural Research Program

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

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

Publisher

MDPI AG

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