No abstract

Translationally controlled tumor protein (TCTP) is a highly conserved, multifunctional protein that has been implicated in a range of cell physiologic processes, especially cell growth and development. A TCTP-like gene has been identified in the Toxoplasma genome [ Toxoplasma gondii TCTP ( TgTCTP)], although its function remains unknown. The sequence analysis of TgTCTP indicated that it is a highly conserved protein in eukaryotes. We found that the expression level of TgTCTP in the virulent RH strain was significantly higher than that in the avirulent PLK strain. Indirect immunofluorescence showed that TgTCTP was expressed in the parasite cytoplasm. The localization of TgTCTP was unchanged during the replication of the parasite. We expressed a functional recombinant TgTCTP (r TgTCTP) protein in Escherichia coli and found that the recombinant protein could form a multimer. We then evaluated the function of TgTCTP using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout (KO) system. Phenotypic analysis of the KO strain (Δ TgTCTP) revealed that TgTCTP is required for the robust growth of the parasites. TgTCTP deficiency also led to early egress of the parasites and subsequent impairment in their invasion and attachment abilities. We subsequently found that the multimer form of TgTCTP might not be necessary for the growth and replication of the parasite. Then the expression profiling of genes in the Δ TgTCTP and complement strains were analyzed. The results revealed that 988 genes were regulated in Δ TgTCTP compared with the complement strain. Overall, although not essential, TgTCTP is required for the fast growth of Tg and maintenance of its intracellular development.-Zheng, J., Chen, Y., Li, Z., Cao, S., Zhang, Z., Jia, H. Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Toxoplasma gondii is an obligate intracellular parasite of phylum Apicomplexa. To facilitate high-efficiency invasion of host cells, T. gondii secretes various proteins related to the moving junction (MJ) complex from rhoptries and micronemes into the interface between the parasite and host. AMA1/RON2/4/5/8 is an important MJ complex, but its mechanism of assembly remains unclear. In this study, we used the CRISPR-Cas9 system to generate a derivative of T. gondii strain RH with a null mutation in TgRON4, thought to be an essential MJ component. Deficiency of TgRON4 moderately decreased invasion ability relative to that of the wild-type parasite. In addition, expression of the endogenous N-terminal fragment of RON5 decreased in the mutant. Together, the results improve our understanding of the assembly mechanism of the MJ complex of T. gondii and raise the possibility of developing new therapeutic drugs that target this complex.