AUTHOR=Liang Qin-Li , Nie Lan-Bi , Li Ting-Ting , Elsheikha Hany M. , Sun Li-Xiu , Zhang Zhi-Wei , Zhao Dan-Yu , Zhu Xing-Quan , Wang Jin-Lei 

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

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2021.738794

DOI=10.3389/fcell.2021.738794

ISSN=2296-634X

ABSTRACT=<p>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 <italic>Toxoplasma gondii</italic>. 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 <italic>T. gondii</italic>, knock-out (KO) strains of type I RH strain deficient in the 17 <italic>psp</italic> genes and KO type II Pru strain deficient in <italic>pp7</italic> and <italic>slp</italic> genes were constructed. The pathogenicity of individual RHΔ<italic>psp</italic> mutants was characterized <italic>in vitro</italic> using plaque, egress, and intracellular replication assays, and mouse infection, while pathogenicity of PruΔ<italic>pp7</italic> and PruΔ<italic>slp</italic> mutant strains was evaluated by examining the parasite lytic cycle <italic>in vitro</italic> and assessment of brain cyst burden in mice. No significant differences were observed between 16 RHΔ<italic>psp</italic> strains and wild-type (WT) RH strain. However, RHΔ<italic>pp7</italic> exhibited significantly lower invasion efficiency and parasitophorous vacuole formation <italic>in vitro</italic>, and less virulence in mice compared with other RHΔ<italic>psp</italic> and WT strains. In addition, PruΔ<italic>pp7</italic> exhibited marked attenuation of virulence and significant reduction in the brain cyst burden in mice compared with PruΔ<italic>slp</italic> and WT strains, suggesting the key role of PP7 in the virulence of <italic>T. gondii</italic>. Comparative transcriptomic profiling of the 17 <italic>psp</italic> gene<italic>s</italic> showed that they may play different roles in the pathogenesis of different genotypes or life cycle stages of <italic>T. gondii</italic>. These findings provide new insight into the role of PSPs in the pathogenesis of <italic>T. gondii</italic>.</p>