Geng Xiao-Ling, Li Jing-Yu, Xu Huan-Yu, Wu Jiang-Ping, Tao De-Liang, Chen Jin-Ming, Fan Ying-Ying, Yang Xin, Song Jun-Ke, Zhao Guang-Hui
Department of Parasitology, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, China.
Parasit Vectors. 2025 Jul 29;18(1):309. doi: 10.1186/s13071-025-06966-x.
The pathogenicity of Toxoplasma gondii is closely associated with its intracellular lytic cycle in host cells. Currently, the mechanisms by which T. gondii completes the lytic cycle remain unclear. The proline metabolism has been reported to be crucial for intracellular growth of pathogens by providing energy and nutrients. However, it remains unclear whether the intracellular growth and pathogenicity of T. gondii are related to proline metabolism.
The gene-edited strains of proline dehydrogenase (Tgprodh) were constructed by using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. The effects of the Tgprodh gene on the growth in vitro and pathogenicity in vivo of the tachyzoites for T. gondii were studied through proliferation, plaque, invasion, egress and virulence assays. The effects of the Tgprodh gene on mitochondrial function were studied by using reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψm), adenosine triphosphate (ATP) assay kits, mitochondrial DNA (mtDNA) copy numbers, transmission electron microscopy (TEM) analysis, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The effects of the Tgprodh gene on proline metabolism were studied by using L-proline (L-Pro), L-glutamic acid (L-Glu), L-glutamine (L-Gln) assay kits, and RT-qPCR.
TgPRODH, the first rate-limiting enzyme in proline metabolism, was identified to be encoded by T. gondii and localized in the cytoplasm of T. gondii. Deletion of the Tgprodh gene resulted in significant growth inhibition in vitro and reduced pathogenicity in vivo of T. gondii. Further study found that deletion of the Tgprodh gene caused damage to the mitochondrial morphology, decreased membrane potential, mtDNA copy numbers, and the production of ATP and ROS. The expression of genes for maintaining mitochondrial integrity was downregulated in the Tgprodh-knockout strain of T. gondii, while complementation of the Tgprodh gene restored these defects in this parasite. Meantime, the deletion of the Tgprodh gene resulted in the accumulation of proline, reduced the contents of glutamate and glutamine, and affected the expression of genes related to proline catabolism in T. gondii.
The present study found the significance of the Tgprodh gene for the intracellular growth and pathogenicity of T. gondii through regulating mitochondrial function and the proline metabolism and provided a novel insight to reveal intracellular survival strategies of T. gondii.
刚地弓形虫的致病性与其在宿主细胞内的裂解周期密切相关。目前，刚地弓形虫完成裂解周期的机制尚不清楚。据报道，脯氨酸代谢通过提供能量和营养物质，对病原体的细胞内生长至关重要。然而，刚地弓形虫的细胞内生长和致病性是否与脯氨酸代谢有关仍不清楚。
利用成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR-Cas9）技术构建脯氨酸脱氢酶（Tgprodh）基因编辑菌株。通过增殖、噬斑、侵袭、逸出和毒力试验，研究Tgprodh基因对刚地弓形虫速殖子体外生长和体内致病性的影响。利用活性氧（ROS）、线粒体膜电位（∆Ψm）、三磷酸腺苷（ATP）检测试剂盒、线粒体DNA（mtDNA）拷贝数、透射电子显微镜（TEM）分析和逆转录定量聚合酶链反应（RT-qPCR），研究Tgprodh基因对线粒体功能的影响。利用L-脯氨酸（L-Pro）、L-谷氨酸（L-Glu）、L-谷氨酰胺（L-Gln）检测试剂盒和RT-qPCR，研究Tgprodh基因对脯氨酸代谢的影响。
脯氨酸代谢中的首个限速酶TgPRODH被鉴定为由刚地弓形虫编码，并定位于刚地弓形虫的细胞质中。Tgprodh基因的缺失导致刚地弓形虫体外生长显著抑制，体内致病性降低。进一步研究发现，Tgprodh基因的缺失导致线粒体形态受损、膜电位、mtDNA拷贝数以及ATP和ROS的产生减少。在刚地弓形虫的Tgprodh基因敲除株中，维持线粒体完整性的基因表达下调，而Tgprodh基因的互补恢复了该寄生虫的这些缺陷。同时，Tgprodh基因的缺失导致脯氨酸积累，谷氨酸和谷氨酰胺含量降低，并影响刚地弓形虫中与脯氨酸分解代谢相关基因的表达。
本研究通过调节线粒体功能和脯氨酸代谢，发现了Tgprodh基因对刚地弓形虫细胞内生长和致病性的重要性，并为揭示刚地弓形虫的细胞内存活策略提供了新的见解。
