Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by <i>Toxoplasma gondii</i>

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.刚地弓形虫（Toxoplasma gondii）拥有数量扩增的钙调蛋白（calmodulin, CaM）样蛋白家族，但其成员的具体功能尚不明确。本研究结合CRISPR/Cas9介导的基因编辑技术与植物源生长素诱导降解域（auxin-induced degron, AID）系统，对3种顶端定位的CaM开展功能验证实验。实验结果显示：CaM1与CaM2单基因敲除后寄生虫仍可正常存活，但同时敲除二者会产生合成致死表型；CaM3基因敲除难度极高，提示其为寄生虫生存必需的基因。与此预测一致，通过生长素诱导降解CaM3可显著阻断寄生虫的增殖生长。表型分析进一步表明，这3种CaM均参与调控寄生虫的运动能力、宿主细胞入侵与逸出过程，且其作用环节位于微线体与棒状体分泌下游。超分辨率显微镜成像显示，3种CaM均定位于顶锥（conoid）结构，并与入侵过程必需的动力蛋白肌球蛋白H（myosin H, MyoH）存在共定位区域。通过与CaM融合的BirA生物素连接酶进行生物素标记，我们鉴定出包括MyoH及其轻链MLC7在内的多种顶端蛋白，提示这些CaM与MyoH可能存在相互作用。与此假说相符，敲除MyoH会导致CaM3发生降解，或是引起CaM1与CaM2的重新分布。综上，本研究结果表明，上述CaM可能通过与MyoH相互作用，共同调控寄生虫的运动与宿主细胞入侵过程。