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Background <p>Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of <i>Toxoplasma gondii</i> by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in <i>T. gondii</i> biology, with a focus on their contributions to virulence.</p> Methods <p>Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)–mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the<i> T. gondii</i> RHΔ<i>ku80</i> strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔ<i>rop64</i> and RHΔ<i>rop65</i> knockout strains.</p> Results <p>All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including <i>ROP5</i>, <i>ROP39</i>, <i>TgIST</i>, and <i>PLP1</i>. In addition, RHΔ<i>rop64</i> exhibited broader suppression of ROPs than RHΔ<i>rop65</i>, suggesting it has a more pronounced role in immune modulation.</p> Conclusions <p>ROP64 and ROP65 are critical to <i>T. gondii</i> virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔ<i>rop64</i> mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis.</p> Graphical Abstract <p></p>

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Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月13日

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  • Hong-Yu Song,
  • Hui Cao,
  • Shi-Bo Huang,
  • Hany M. Elsheikha,
  • Zhi Zheng,
  • Xin-Sheng Lu,
  • Xing Tian,
  • Xiao-Nan Zheng,
  • Xing-Quan Zhu

摘要

Background

Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence.

Methods

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)–mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RHΔku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔrop64 and RHΔrop65 knockout strains.

Results

All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5, ROP39, TgIST, and PLP1. In addition, RHΔrop64 exhibited broader suppression of ROPs than RHΔrop65, suggesting it has a more pronounced role in immune modulation.

Conclusions

ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔrop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis.

Graphical Abstract