中国兽医杂志2025，Vol.61Issue(10)：1-8,8.DOI:10.20157/j.cnki.zgsyzz.2025.10.001
弓形虫TgMIT蛋白的鉴定和功能分析
Identification and Function analysis of TgMIT protein in Toxoplasma gondii
摘要
Abstract
Mitochondrial iron transporter(MIT)is a class of critical iron transport proteins in eukaryotes that play essential roles in maintaining intracellular iron homeostasis and redox balance.In this study,the amino acid sequence of yeast MIT was aligned against the Toxoplasma gondii(T.gondii)database,and a homologous protein,TgMIT,was identified.To investigate the localization and function of TgMIT in T.gondii,a TgMIT endogenously tagged parasite strain(TgMIT-3HA)was constructed using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)technology.The strain was validated by polymerase chain reaction(PCR),Western blotting(WB),and immunofluorescence assay(IFA).IFA was employed to determine TgMIT localization,while WB was used to analyze TgMIT expression under different iron conditions.Furthermore,a TgMIT knockout strain(Δmit)was generated and validated by PCR.Plaque assays,invasion assays,and proliferation assays were conducted to assess the role of TgMIT in parasite biological activities.The oxidative status of Δmit parasites was examined by measuring reactive oxygen species(ROS)levels and the ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG).The results showed that TgMIT-3HA parasites were successfully constructed,and TgMIT localized to the parasite plasma membrane.Compared with untreated controls,TgMIT expression was significantly downregulated under low-iron conditions(P<0.01)and markedly upregulated under high-iron conditions(P<0.001 or P<0.000 1).The Δmit strain was successfully established,and deletion of the mit gene profoundly inhibited parasite plaque formation(P<0.000 1),invasion(P<0.001),and proliferation(P<0.000 1).Moreover,Δmit parasites exhibited oxidative stress,as indicated by significantly elevated ROS levels(P<0.01)and a decreased GSH/GSSG ratio(P<0.05).In summary,TgMIT is localized to the plasma membrane of T.gondii and functions as an iron-regulatory protein critical for maintaining redox balance and supporting normal parasite viability.关键词
刚地弓形虫/TgMIT/CRISPR/Cas9/铁离子/氧化还原Key words
Toxoplasma gondii/TgMIT/CRISPR/Cas9/iron ion/oxidation reduction分类
农业科技引用本文复制引用
邓舒予,尹萌,应铸,刘晶,刘群..弓形虫TgMIT蛋白的鉴定和功能分析[J].中国兽医杂志,2025,61(10):1-8,8.基金项目
国家重点研发计划(2022YFD1800205) （2022YFD1800205）
