结核分枝杆菌Rv2673基因过表达对分枝杆菌细胞壁组分的影响
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摘要
目的分析结核分枝杆菌(Mycobacterium tuberculosis,Mtb)Rv2673基因过表达对分枝杆菌细胞壁组分的影响。方法构建Rv2673在耻垢分枝杆菌(Mycobacterium smegmatis,Msm)中的表达载体,导入到Msm后提取膜蛋白,以抗组胺酸标签的单克隆抗体鉴定表达的Rv2673蛋白。细菌培养时加入~(14)C-葡萄糖,收获Msm后分别以氯仿∶甲醇(2∶1)、氯仿∶甲醇∶水(10∶10∶3)、ε-soak-lovolunnee试剂分步提取全细胞的细胞壁组分,其余的沉淀则用以提取LAM。以HP-TLC及SDS-PAGE方法分离提取的组分,放射自显影。结果生物信息学分析表明Rv2673蛋白为在羧基末端具有8次跨膜结构的膜蛋白;Western bloting方法检测到过表达Rv2673的菌株膜蛋白中具有表达的Rv2673蛋白。TLC方法对细胞壁组分的分步分析表明过表达Rv2673的菌株及其对照菌株间DPA、DPM、Ac2PIM2、AcPIM2和Ac2PIM4/AcPIM4等小分子脂溶性分子没有显著性差异,但Western blot分析的细胞壁中大分子脂类组分显示,Rv2673具有显著性促进Ac2PIM6和AcPIM6生成的作用,以及促进LM转变为LAM。推测Rv2673蛋白能促进大分子脂类分子的生物合成,但对细胞壁中小分子的脂类分子无显著影响。结论 Rv2673参与LAM中阿拉伯聚糖分枝结构的形成,推测Rv2673蛋白对细菌感染宿主细胞及/或宿主细胞杀死Mtb有一定影响,是有发展潜力的药物靶向分子。
Objective To determine the effect of over-expressing the Rv2673 gene of Mycobacterium tuberculosis on mycobacterial cell wall components. Methods The expression vector pVV16-Rv2673/pVV2-Rv2673 was constructed and introduced in M. smegmatis to express Rv2673 protein. The membrane protein Rv2673 was identified with monoclonal antibodies against the polyhistidine tag. Components of the cell wall of whole M. smegmatis cells labeled with ~(14)C-glucose were analyzed via extraction with chloroform:methanol(2:1), chloroform:methanol:water(10:10:3), and E-soak, followed by LAM extraction. The fractions were separated using HP-TLC and SDS-PAGE, and the bands were visualized using autoradiography. Results Rv2673 protein was predicted to be a membrane protein with eight transmembrane structures at the carboxyl end according to bioinformatic analysis. Soluble Rv2673 protein was obtained in the membrane of M. smegmatis strains harboring pVV16/pVV2-Rv2673. Sequential extraction of cell wall components indicated that there were no significant differences in DPA, DPM, Ac2 PIM2, AcPIM2, or Ac2 PIM4/AcPIM4 between strains over-expressing Rv2673 and control strains, but Rv2673 promoted the biosynthesis of Ac2 PIM6 and AcPIM6 and the transformation of LM into LAM. Analysis of cell wall components from whole M. smegmatis cells indicated that Rv2673 protein significantly promoted the biosynthesis of LAM, but it had no marked effect on lipid molecules with a low molecular weight in the cell wall. Conclusion Rv2673 was involved in the formation of arabinan branching of LAM. Therefore, Rv2673 is hypothesized to potentially affect M. tuberculosis infection of host cells and/or killing M. tuberculosis by host cells. These findings provide evidence that Rv2673 has promise as a novel anti-tuberculosis drug target and an essential gene product.
Objective To determine the effect of over-expressing the Rv2673 gene of Mycobacterium tuberculosis on mycobacterial cell wall components. Methods The expression vector pVV16-Rv2673/pVV2-Rv2673 was constructed and introduced in M. smegmatis to express Rv2673 protein. The membrane protein Rv2673 was identified with monoclonal antibodies against the polyhistidine tag. Components of the cell wall of whole M. smegmatis cells labeled with ~(14)C-glucose were analyzed via extraction with chloroform:methanol(2:1), chloroform:methanol:water(10:10:3), and E-soak, followed by LAM extraction. The fractions were separated using HP-TLC and SDS-PAGE, and the bands were visualized using autoradiography. Results Rv2673 protein was predicted to be a membrane protein with eight transmembrane structures at the carboxyl end according to bioinformatic analysis. Soluble Rv2673 protein was obtained in the membrane of M. smegmatis strains harboring pVV16/pVV2-Rv2673. Sequential extraction of cell wall components indicated that there were no significant differences in DPA, DPM, Ac2 PIM2, AcPIM2, or Ac2 PIM4/AcPIM4 between strains over-expressing Rv2673 and control strains, but Rv2673 promoted the biosynthesis of Ac2 PIM6 and AcPIM6 and the transformation of LM into LAM. Analysis of cell wall components from whole M. smegmatis cells indicated that Rv2673 protein significantly promoted the biosynthesis of LAM, but it had no marked effect on lipid molecules with a low molecular weight in the cell wall. Conclusion Rv2673 was involved in the formation of arabinan branching of LAM. Therefore, Rv2673 is hypothesized to potentially affect M. tuberculosis infection of host cells and/or killing M. tuberculosis by host cells. These findings provide evidence that Rv2673 has promise as a novel anti-tuberculosis drug target and an essential gene product.
引文
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[9] 何黎, 齐文静, 郑雪婷, 等. 细粒棘球绦虫EgM123重组耻垢分枝杆菌疫苗在小鼠体内的表达及其免疫应答[J]. 中国病原生物学杂志, 2018, 13(12): 1314-8, 1323.
[10] 李雅琼, 张文利, 董旭. 分枝杆菌细胞壁聚阿拉伯甘露糖脂的结构、合成过程及免疫调节[J]. 中华微生物学和免疫学杂志, 2010, 30(9): 866-70.
[11] Abrahams KA, Besra GS. Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target[J]. Parasitology, 2016, 145(2): 1-18.
[12] Birch HL, Grover S, Jankute M, et al. Genetics of mycobacterial arabinogalactan and lipoarabinomannan assembly[J]. Microbiol Spectr, 2014, 2(4): 10.
[13] Jankute M, Cox JAG, Harrison J, et al. Assembly of the mycobacterial cell wall[J]. Ann Rev Microbiol, 2015, 69(1): 405-23.
[14] Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis[J]. Mol Microbiol, 2010, 48(1): 77-84.
[2] Gandhi NR, Moll A, Sturm AW, et al. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa[J]. Lancet, 2008, 368(9547): 1575-80.
[3] Rombouts Y, Brust B, Ojha AK, et al. exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism[J]. J Bio Chem, 2012, 287(14): 11060-9.
[4] Kaur D, Guerin ME, Henrieta Skovierova, et al. Chapter 2: Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis[J]. Ad App Microb, 2009, 69(69): 23-78.
[5] Grzegorzewicz AE, de Sousad'Auria C, Mcneil MR, et al. Assembling of the Mycobacterium tuberculosis Cell Wall Core[J]. J Biol Chem, 2016, 291(36): 18867-79.
[6] Birch HL, Alderwick LJ, Bhatt A, et al. Biosynthesis of mycobacterial arabinogalactan: identification of a novel alpha(1-->3) arabinofuranosyltransferase[J]. Mol Microbiol, 2010, 69(5): 1191-206.
[7] Liu J, Mushegian A. Three monophyletic superfamilies account for the majority of the known glycosyltransferases[J]. Protein Sci, 2010, 12(7): 1418-31.
[8] Crellin PK, Kovacevic S, Martin KL, et al. Mutations in pimE restore lipoarabinomannan synthesis and growth in a Mycobacterium smegmatis lpqW mutant[J]. J Bacteriology, 2008, 190(10): 3690-9.
[9] 何黎, 齐文静, 郑雪婷, 等. 细粒棘球绦虫EgM123重组耻垢分枝杆菌疫苗在小鼠体内的表达及其免疫应答[J]. 中国病原生物学杂志, 2018, 13(12): 1314-8, 1323.
[10] 李雅琼, 张文利, 董旭. 分枝杆菌细胞壁聚阿拉伯甘露糖脂的结构、合成过程及免疫调节[J]. 中华微生物学和免疫学杂志, 2010, 30(9): 866-70.
[11] Abrahams KA, Besra GS. Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target[J]. Parasitology, 2016, 145(2): 1-18.
[12] Birch HL, Grover S, Jankute M, et al. Genetics of mycobacterial arabinogalactan and lipoarabinomannan assembly[J]. Microbiol Spectr, 2014, 2(4): 10.
[13] Jankute M, Cox JAG, Harrison J, et al. Assembly of the mycobacterial cell wall[J]. Ann Rev Microbiol, 2015, 69(1): 405-23.
[14] Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis[J]. Mol Microbiol, 2010, 48(1): 77-84.