Functional Characterization of 17 Protein Serine/Threonine Phosphatases in Toxoplasma gondii Using CRISPR-Cas9 System

Protein serine/threonine phosphatases (PSPs), found in various plants and protozoa, are involved in the regulation of various biological processes. However, very little is known about the role of PSPs in the pathogenicity of the apicomplexan protozoan Toxoplasma gondii. Herein, the subcellular localization of 17 PSPs (PP5, PP7, EFPP, SLP, PPM3F, PPM4, PPM5A, PPM5B, PPM6, PPM8, PPM9, PPM12, PPM14, PPM18, CTD1, CTD2, and CTD3) was examined by 6x HA tagging of endogenous genes in C-terminal. The PSPs were detected in the cytoplasm (PP5, EFPP, PPM8, and CTD2), dense granules (SLP), nucleus (PPM4 and PPM9), inner membrane complex (PPM12), basal complex (CTD3), and apical pole (PP7). The remaining PSPs exhibited low or undetectable level of expression. To characterize the contribution of these genes to the infectivity of T. gondii, knock-out (KO) strains of type I RH strain deficient in the 17 psp genes and KO type II Pru strain deficient in pp7 and slp genes were constructed. The pathogenicity of individual RH Delta psp mutants was characterized in vitro using plaque, egress, and intracellular replication assays, and mouse infection, while pathogenicity of Pru Delta pp7 and Pru Delta slp mutant strains was evaluated by examining the parasite lytic cycle in vitro and assessment of brain cyst burden in mice. No significant differences were observed between 16 RH Delta psp strains and wild-type (WT) RH strain. However, RH Delta pp7 exhibited significantly lower invasion efficiency and parasitophorous vacuole formation in vitro, and less virulence in mice compared with other RH Delta psp and WT strains. In addition, Pru Delta pp7 exhibited marked attenuation of virulence and significant reduction in the brain cyst burden in mice compared with Pru Delta slp and WT strains, suggesting the key role of PP7 in the virulence of T. gondii. Comparative transcriptomic profiling of the 17 psp genes showed that they may play different roles in the pathogenesis of different genotypes or life cycle stages of T. gondii. These findings provide new insight into the role of PSPs in the pathogenesis of T. gondii.

Automated summary

This study investigated the subcellular localization of 17 PSPs in the apicomplexan protozoan Toxoplasma gondii and examined their role in the pathogenicity of the parasite. The results showed that some PSPs play a crucial role in the invasion and infection process of T. gondii.