| 徐书琴,沈莉芳,刘含梅,唐雨婷,张 鹭.耻垢分枝杆菌中LprO蛋白过表达促进巨噬细胞凋亡[J].,2022,(21):4001-4008 |
| 耻垢分枝杆菌中LprO蛋白过表达促进巨噬细胞凋亡 |
| Overexpression of LprO Protein in Mycobacterium smegmatis Promotes Apoptosis of Macrophages |
| 投稿时间：2022-06-06 修订日期：2022-06-30 |
| DOI：10.13241/j.cnki.pmb.2022.21.001 |
| 中文关键词: 耻垢分枝杆菌 脂蛋白 LprO 抗原表位 凋亡 |
| 英文关键词: Mycobacterium smegmatis Lipoprotein LprO T-cell epitope prediction Apoptosis |
| 基金项目:十四五国家重点研发计划项目（2021YFC2301500） |
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| 中文摘要: |
| 摘要 目的：探究分枝杆菌脂蛋白LprO对分枝杆菌-巨噬细胞相互作用的影响。方法：使用在线网站分析耻垢分枝杆菌（Mycobacterium smegmatis, M. smeg）LprO蛋白的CD4+T、CD8+T以及细胞毒性T细胞（Cytotoxic T Lymphocytes, CTL）抗原表位数量，评估LprO蛋白的免疫原性。构建lprO过表达的重组耻垢分枝杆菌M. smeg::pMV261-lprO，以转入空载质粒pMV261的M. smeg::pMV261菌株作为对照，分析过表达lprO对M. smeg菌株以及细菌-巨噬细胞互作的影响。结果：LprO蛋白中预测的CD4+T、CD8+T以及CD8+CTL细胞表位数与Ag85A蛋白相当，具有较好的研究潜力。经实时荧光定量PCR（Quantitative real-time PCR, qRT-PCR）验证，M. smeg::pMV261-lprO菌株中，lprO表达量显著高于对照菌株，过表达菌株构建成功。lprO过表达不改变M. smeg菌落形态、细菌形态、细菌体外生长能力和巨噬细胞内生长能力。细菌侵染巨噬细胞Raw264.7，流式细胞技术检测显示，M. smeg::pMV261-lprO在细胞侵染前期能显著促进巨噬细胞凋亡。结论：分枝杆菌LprO蛋白可能具备与Ag85A蛋白相当的T细胞表位数，能在激起宿主的免疫反应中发挥较为重要的作用。在M. smeg中过表达LprO后能诱导侵染前期的巨噬细胞凋亡，参与细菌-宿主相互作用。综上，LprO蛋白或许有作为新型疫苗成分或药物靶标的潜力。 |
| 英文摘要: |
| ABSTRACT Objective: To explore the effect of mycobacterial lipoprotein LprO on the interaction between mycobacteria and macrophages. Methods: The number of CD4+T, CD8+T and cytotoxic T lymphocytes (CTL) cell antigenic epitopes of LprO protein of Mycobacterium smegmatis (M. smeg) was analyzed to evaluate the immunogenicity of LprO protein using the online websites NetMHC II 2.3 Server, NetMHCcons 1.1 Server and CTLPred, respectively. Recombinant M. smeg with lprO overexpression (M. smeg::pMV261-lprO) was constructed to explore the effects of lprO overexpression on M. smeg strains and the interactions between bacteria and macrophages. During the whole study, M. smeg strain with the empty plasmid pMV261, named M. smeg::pMV261, was used as the control strain. Results: The predicted CD4+T, CD8+T and CD8+CTL cell epitopes of LprO protein were comparable to those of Ag85A protein which is a well-known tuberculosis protein with strong immunogenicity. This suggested the good research potential of LprO protein. Expression level of lprO in M. smeg::pMV261-lprO was significantly higher than that in M. smeg::pMV261 verified by quantitative real- time PCR (qRT-PCR). Our study showed that overexpression of lprO did not alter colony morphology, bacterial morphology, bacterial length, in vitro growth and in vivo growth in macrophage Raw264.7 of M. smeg strain. However, the macrophage uptake rate of recombinant M.smeg stains with lprO overexpression was significantly increased. Macrophages Raw264.7 were infected with recombinant M. smeg strains at MOI=10, apoptosis rate of macrophages infected by M. smeg::pMV261-lprO was significantly higher than that of M. smeg::pMV261 infection group. Conclusion: Mycobacterial LprO protein has equivalent T cell epitopes to Ag85A protein and may play an important role in evoking the host's immune response. Overexpression of lprO in M. smeg also induces apoptosis of macrophages at the early stage of infection through bacteria-host interactions. Therefore, LprO protein may have the potential as a novel vaccine component or drug target. |
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