Table2_Functional Characterization of 17 Protein Serine/Threonine Phosphatases in Toxoplasma gondii Using CRISPR-Cas9 System.DOCX

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 6× 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 RHΔpsp mutants was characterized in vitro using plaque, egress, and intracellular replication assays, and mouse infection, while pathogenicity of PruΔpp7 and PruΔslp 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 RHΔpsp strains and wild-type (WT) RH strain. However, RHΔpp7 exhibited significantly lower invasion efficiency and parasitophorous vacuole formation in vitro, and less virulence in mice compared with other RHΔpsp and WT strains. In addition, PruΔpp7 exhibited marked attenuation of virulence and significant reduction in the brain cyst burden in mice compared with PruΔslp 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.

丝氨酸/苏氨酸蛋白磷酸酶（Protein serine/threonine phosphatases, PSPs）广泛分布于多种植物与原生生物中，参与调控诸多生物学过程。然而，目前学界对顶复门原生动物刚地弓形虫（Toxoplasma gondii）中PSPs的致病作用仍知之甚少。本研究通过对17种PSPs（PP5、PP7、EFPP、SLP、PPM3F、PPM4、PPM5A、PPM5B、PPM6、PPM8、PPM9、PPM12、PPM14、PPM18、CTD1、CTD2及CTD3）的内源基因进行C端6×血凝素（6× HA）标签标记，对其亚细胞定位展开检测。结果显示，这些PSPs分别定位于细胞质（PP5、EFPP、PPM8及CTD2）、致密颗粒（SLP）、细胞核（PPM4与PPM9）、内膜复合物（PPM12）、基底复合体（CTD3）以及顶极（PP7）；其余PSPs的表达水平较低或未被检测到。为明确上述基因对刚地弓形虫感染性的贡献，本研究构建了缺失17个psp基因的I型RH株基因敲除（KO）菌株，以及缺失pp7与slp基因的II型Pru株KO菌株。针对单个RHΔpsp突变株的致病力，本研究通过体外噬斑实验、逸出实验、细胞内增殖实验以及小鼠感染模型进行了表征；而PruΔpp7与PruΔslp突变株的致病力，则通过体外检测寄生虫裂解周期以及评估小鼠脑囊荷量完成评估。16株RHΔpsp菌株与野生型（WT）RH株之间未观察到显著差异。但与其余RHΔpsp菌株及WT RH株相比，RHΔpp7的体外侵袭效率与纳虫空泡形成能力显著降低，小鼠体内毒力也更弱。此外，与PruΔslp及WT菌株相比，PruΔpp7的毒力显著减弱，小鼠脑囊荷量也显著降低，这表明PP7在刚地弓形虫的致病过程中发挥关键作用。对17个psp基因的比较转录组分析显示，它们可能在不同基因型或生活周期阶段的刚地弓形虫致病过程中发挥不同功能。本研究结果为理解PSPs在刚地弓形虫致病机制中的作用提供了新的视角。