Ferroptosis is important for Toxoplasma gondii replication and virulence in vitro and in vivo

The protozoan parasite T. gondii employs intricate mechanisms to exploit host cells while sustaining their viability, yet its interaction with ferroptosis – an iron-dependent cell death driven by lipid peroxidation – remains poorly defined. Here, we show T. gondii infection induces ferroptotic hallmarks in RAW264.7 macrophages, including elevated lactate dehydrogenase release, labile Fe2 + accumulation, reactive oxygen species (ROS) generation, and lipid peroxidation. Molecular analyses revealed infection-induced downregulation of ferroptosis suppressor GPX4 and upregulation of pro-ferroptotic ACSL4 in macrophages and mice. Mechanistically, the SLC7A11/GPX4 axis governed parasite growth: knockdown of these genes promoted T. gondii replication, whereas overexpression restricted proliferation. Pharmacological studies showed ferroptosis inhibitor Fer-1 suppressed intracellular parasite proliferation. Notably, GPX4 inhibitor RSL3 exhibited context-dependent effects: pre-infection treatment enhanced replication, while post-infection administration inhibited growth. Direct RSL3 exposure induced time-dependent growth arrest in extracellular tachyzoites, associated with disrupted transcriptomes, increased lipid ROS, and downregulated parasite antioxidant genes (TgPRX2, TgTPX1/2, TgNXN), indicating redox homoeostasis impairment. In vivo murine studies corroborated this biphasic effect: therapeutic RSL3 administration post-infection significantly reduced parasite burdens across multiple organs (spleen, liver, kidney, brain) and improved survival rates, while prophylactic pretreatment exacerbated disease progression. We propose RSL3 exerts direct parasiticidal effects via oxidative damage but also enables early nutrient acquisition from ferroptosis-compromised host cells. These findings establish ferroptosis as a critical node in T. gondii pathogenesis, highlighting the parasite’s hijacking of host iron-lipid metabolism. The dual role of ferroptosis regulators underscores the host-pathogen metabolic complexity and positions the SLC7A11/GPX4 axis as a promising therapeutic target.原生动物寄生虫刚地弓形虫（Toxoplasma gondii，下文简称T. gondii）具备复杂的宿主细胞劫持机制，可在维持宿主细胞存活的同时完成自身增殖，但其与铁死亡（ferroptosis）——一种由脂质过氧化驱动的铁依赖性细胞死亡方式——之间的相互作用仍有待阐明。本研究证实，T. gondii感染可在RAW264.7巨噬细胞中诱导铁死亡相关特征，包括乳酸脱氢酶释放增加、不稳定Fe²+蓄积、活性氧（ROS）生成以及脂质过氧化。分子水平分析显示，感染会下调巨噬细胞与小鼠体内的铁死亡抑制因子谷胱甘肽过氧化物酶4（GPX4），并上调促铁死亡的酰基辅酶A合成酶长链家族成员4（ACSL4）。机制研究表明，SLC7A11/GPX4信号轴调控寄生虫的增殖：敲低该轴相关基因可促进T. gondii复制，而过表达则会抑制其增殖。药理学实验证实，铁死亡抑制剂Fer-1可抑制细胞内寄生虫的增殖。值得注意的是，GPX4抑制剂RSL3表现出情境依赖性的调控效应：感染前给药可增强寄生虫复制，而感染后给药则会抑制其生长。直接暴露于RSL3可使细胞外速殖子出现时间依赖性的生长停滞，该现象伴随转录组紊乱、脂质ROS水平升高以及寄生虫抗氧化基因（TgPRX2、TgTPX1/2、TgNXN）的下调，提示寄生虫的氧化还原稳态遭到破坏。体内小鼠实验验证了这种双相效应：感染后给予治疗剂量的RSL3可显著降低脾脏、肝脏、肾脏、大脑等多个器官中的寄生虫负荷，并提高小鼠存活率；而预防性预处理RSL3则会加重疾病进展。本研究提出，RSL3一方面可通过氧化损伤发挥直接的杀虫作用，另一方面也可通过从铁死亡受损的宿主细胞中获取早期营养物质。上述研究结果确立了铁死亡在T. gondii致病过程中的关键节点地位，揭示了寄生虫对宿主铁-脂质代谢的劫持行为。铁死亡调控因子的双重作用凸显了宿主-病原体代谢互作的复杂性，并将SLC7A11/GPX4轴定位为极具潜力的治疗靶点。