摘要
目的通过实验和生物信息学分析,初步探究Rv0305c(PPE6)蛋白在结核分枝杆菌(Mycobacterium tuberculosis,Mtb)致病过程中的作用,为深入阐明其作用机制及发现抗结核药物新靶点奠定基础。方法基于CRISPR-Cas辅助的非同源末端连接基因编辑技术在Mtb H37Rv野生株(wild type,WT)中构建Rv0305c基因敲除株(ΔRv0305c),进而电转入Rv0305c表达载体构建基因敲除回补株(ΔRv0305c::Rv0305c);采用扫描电镜分析Mtb菌株形态;通过紫外可见分光光度计测定培养菌液OD600值并绘制生长曲线;采用逆转录定量PCR动态分析3种菌株感染THP-1巨噬细胞后炎症相关细胞因子的表达差异;分别采用AlphaFold进行蛋白质三维结构建模、STRING进行互作网络分析、SignalP预测信号肽、DeepTMHMM预测跨膜结构、InterPro分析家族和结构域、DeepLocPro分析亚细胞定位以及ELM分析真核样短线性模体。结果成功构建了ΔRv0305c和ΔRv0305c::Rv0305c菌株。Rv0305c基因敲除后不影响Mtb的形态和生长速度,但ΔRv0305c相比WT和ΔRv0305c::Rv0305c能诱导巨噬细胞在感染后2、24、48 h产生更多IL-6和TNF-α,与此同时诱导产生更少的IL-10(P<0.05)。生物信息学分析显示:Rv0305c与PPE5在基因组中紧密相邻且转录方向一致,Rv0305c蛋白属于富含谷氨酰胺蛋白2家族,具有特定结构域,三维结构模型置信度平均分值为78.19,互作网络蛋白包括多个PE/PPE家族成员以及CpnT、Rv2082和Rv1004c,Rv0305c蛋白可能含有真核样短线性模体,其本身不含信号肽、为非跨膜胞外球蛋白,定位于胞外的概率最大(0.7218)。结论Rv0305c蛋白能够协助Mtb抑制感染巨噬细胞的炎症反应,该蛋白被预测为真核样非典型分泌蛋白。
Objective To preliminarily investigate the role of the Rv0305c(PPE6)protein in Mycobacterium tuberculosis(Mtb)pathogenesis through experimental and bioinformatics analyses,thereby laying the groundwork for elucidating its molecular mechanism and identifying novel targets for anti-tuberculosis drug development.Methods The Mtb Rv0305c gene knockout strain(ΔRv0305c)was constructed from the Mtb H37Rv wild-type(WT)strain using CRISPR-Cas-assisted non-homologous end joining gene editing technology.Subsequently,the complemented strain(ΔRv0305c::Rv0305c)was generated by electroporation with an Rv0305c expression vector.Bacterial morphology was examined by scanning electron microscopy.Growth curves were plotted by measuring the optical density at 600 nm(OD600)of the bacterial cultures using an ultraviolet-visible spectrophotometer.Dynamic expression differences of inflammation-related cytokines in THP-1 macrophages infected with the three strains were analyzed by reverse transcription quantitative PCR(RT-qPCR).For bioinformatic characterization of Rv0305c,the following tools were employed:AlphaFold for protein three-dimensional(3D)structure modeling,STRING for protein-protein interaction network analysis,SignalP for signal peptide prediction,DeepTMHMM for transmembrane structure prediction,InterPro for family and domain analysis,DeepLocPro for subcellular localization prediction,and ELM for eukaryotic-like short linear motif analysis.Results TheΔRv0305c andΔRv0305c::Rv0305c strains were successfully constructed.Deletion of the Rv0305c gene did not affect the morphology or growth rate of Mtb.However,ΔRv0305c induced significantly upregulated expression of IL-6 and TNF-αand significantly downregulated expression of IL-10 in macrophages at 2 h,24 h,and 48 h post-infection,compared to the WT andΔRv0305c::Rv0305c(P<0.05).Bioinformatic analyses revealed the following:Rv0305c is genomically adjacent to PPE5 and is transcribed in the same direction;the Rv0305c protein belongs to the glutamine-rich protein 2 family and contains specific domains;its predicted 3D structure model has an average confidence score of 78.19;its interaction network includes several PE/PPE family members,as well as CpnT,Rv2082,and Rv1004c;Rv0305c may contain eukaryotic-like short linear motifs;it lacks a signal peptide,is predicted to be an non-transmembrane extracellular globulin,and shows the highest probability of extracellular localization(0.7218).Conclusion Rv0305c contributes to the suppression of the inflammatory response in Mtb-infected macrophages and is predicted to be a eukaryotic-like non-classically secreted protein.
作者
吴亚东
刘唯夷
朱传智
张蓝月
杜博平
胡一凡
贾红彦
陈琳
张宗德
潘丽萍
李自慧
WU Yadong;LIU Weiyi;ZHU Chuanzhi;ZHANG Lanyue;DU Boping;HU Yifan;JIA Hongyan;CHEN Lin;ZHANG Zongde;PAN Liping;LI Zihui(Beijing Chest Hospital,Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)
出处
《中国热带医学》
北大核心
2026年第3期323-330,共8页
China Tropical Medicine
基金
国家自然科学基金项目(82070012,32394013)。
