【作者】 杨淑凤；

【导师】 马郁芳；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 结核分枝杆菌引起的结核病是全球危害严重的慢性传染病,大多数结核病例呈现慢性感染状态。结核病的慢性和持续性感染状态揭示结核分枝杆菌巨大的适应宿主免疫环境的能力。细胞壁作为细菌与宿主免疫系统相互作用的第一道屏障,细胞壁上的许多生物大分子最先暴露于宿主免疫系统。因此细菌细胞壁成分在细菌毒力和免疫逃逸机制中发挥了重要作用。许多病原菌通过对细胞壁合成代谢过程的肽聚糖或其衍生物胞壁肽的修饰,例如细胞壁肽聚糖的脱乙酰化作用,来逃避机体免疫系统对细胞壁成分的监测,从而使细菌获得在机体免疫细胞中的存活能力。目前在肺炎链球菌、产单核李斯特菌和乳酸乳球菌等均存在细胞壁的脱乙酰化修饰机制,并且在细菌的毒力和免疫逃逸中发挥了重要的作用。慢性或休眠性状态下的结核分枝杆菌主要是在人体肺泡巨噬细胞内存活和增殖,提示这种细菌可能运用多种机制避免肺泡巨噬细胞吞噬体的成熟、阻断溶酶体的降解等。对结核杆菌细胞壁蛋白Rv1096的生物信息学分析显示,Rv1096蛋白与肺炎链球菌的肽聚糖N-乙酰葡糖胺脱乙酰基酶PgdA、产单核细胞李斯特菌中肽聚糖N-乙酰葡糖胺乙酰基酶Lmo0415和乳酸链球菌肽聚糖脱乙酰酶XyD等在C-末端尤其是酶活性区域具有高度相似性。因此,其可能具有脱乙酰基酶功能。同时,Rv1096蛋白是结核分枝杆菌细胞壁上的一种O-甘露糖基化蛋白,具有三个可能的甘露糖基化位点。提示Rv1096蛋白极有可能参与了结核分枝杆菌与肺泡巨噬细胞表面特定受体的相互识别过程,从而在细菌的免疫识别过程和免疫逃逸过程发挥作用。因此,本课题将从以下三方面探讨Rv1096蛋白的功能,研究结果有助于我们深入理解结核分枝杆菌细胞壁大分子的合成与降解规律、细菌与宿主免疫识别过程以及免疫逃逸机制等。一、Rv 1096蛋白的酶学功能研究在大肠杆菌克隆表达结核分枝杆菌Rv1096基因,获得纯化的Rv1 096蛋白。建立脱乙酰基酶活性测定方法,将Rv1 096蛋白与耻垢分枝杆菌细胞壁肽聚糖孵育,检测产物中是否存在乙酰基以验证Rv1096蛋白是否具有脱乙酰酶功能,并且对其酶学特性参数进行测定。二、高表达Rv1096蛋白耻垢分枝杆菌的构建及其抵抗溶菌酶作用和巨噬细胞内杀菌作用的研究构建高表达Rv1096蛋白的耻垢分枝杆菌(M smegma,tis/Rv1096)纯化Rv1096蛋白再次验证其脱乙酰基酶功能,并对M.Smegmatis/Rv1096进行溶菌酶处理和巨噬细胞杀菌实验,来观察细菌形态和存活能力的变化。三、Rv1096蛋白的O-甘露糖基化修饰分析利用甘露糖与ConA-凝集素特异结合的特性来证实Rv1096蛋白是O-甘露糖基化蛋白。对可能的O-甘露糖基化位点进行多点和单点的定向突变,来测定其O-甘露糖基化位点,用质谱方法初步解析其糖链结构。本文获得以下结果:一、Rv1096蛋白具有肽聚糖脱乙酰基酶功能。1.Rv1096蛋白是一种新的肽聚糖脱乙酰基酶,能使肽聚糖脱乙酰基,但对几丁寡糖无作用。2.Rv1096蛋白是一种金属依赖性的脱乙酰基酶,Co++存在下其酶活性最大。3.该酶特征参数Km为0.910±0.007 mM,Vmax为 0.514±0.038 μMmin-1。二、M.smegmatis/Rv1096具有溶菌酶抵抗性和抗巨噬细胞杀菌作用。1.M smegmatis/Rv1096中表达的Rv1096蛋白同样具有脱乙酰基酶功能。2.M smegmatis/Rv1096经溶菌酶处理后细菌抗酸染色阳性、细菌形态完整,具有溶菌酶抵抗性.3.M smegmatis/Rv1096在巨噬细胞内多位于胞质中,存活能力增强。三、Rv1096蛋白是0-甘露糖基化蛋白,其O-甘露糖基化位点是265Thr。1.M 表达的Rv 1096蛋白能被ConA凝集素识别,说明Rv1096蛋白是甘露糖基化蛋白。2.Rv1096蛋白具有三个可能的O-甘露糖基化位点,借助多点和单点定向突变确定了其O-甘露糖基化位点是265Thr。3.用质谱技术对Rv1096蛋白的糖链结构进行了初步分析。结论:1.结核分枝杆菌Rv1096蛋白是一种新的金属依赖性肽聚糖脱乙酰基酶。2.高表达结核分枝杆菌Rv1096的M.smegmatis/Rv1096菌株具有溶菌酶抵抗性和抗巨噬细胞杀菌作用。3.Rv1096蛋白是O-甘露糖基化蛋白,糖基化位点位于265Thr上。未来研究方向:1.深入探讨Rv1096蛋白的脱乙酰基酶催化机制。确定脱乙酰基酶的酶活性位点;确定脱乙酰基酶是催化肽聚糖中N-乙酰葡糖胺的乙酰基还是N-乙酰胞壁酸的乙酰基。2.确定Rv1096蛋白是否具有毒力作用以及识别巨噬细胞上哪种受体。3.进一步解析Rv1096蛋白的糖链结构及确定糖链的连接方式。更多还原

【Abstract】 Tuberculosis caused by Mcobacterium tuberculosis still is a worldwide infectious disease.Most of cases were in the chronic and latent state,which imply that M.tuberculosis has developed the sophisticated strategies to adapt and evade the host immune surveillances.As well as known,bacterial cell wall which composed of many biological macromolecules is the first barrier exposed to the host immune system.There is increasing evidence that bacterial cell wall components are associated with bacterial virulence even immune evasion.Many bacteria modulate and evade the immune defenses of their hosts through cell wall modification such as peptidoglycan deacetylation to gain the ability to survive in host immune cells.The peptidoglycan deacetylases from Streptococcus pneumonia,Listeria monocytogenes and Lactococcus lactis have been characterized.Additionally the peptidoglycan deacetylation plays an important role in the virulence.As we known,Mycobacterium tuberculosis in the chronic and dormant state is mainly survive and replicate in human alveolar macrophage.All these suggest that the bacteria can use various mechanisms to avoid alveolar macrophage phagosome maturation,blocking lysosomal degradation.However,the PG deacetylase of M.tuberculosis has not been identified yet.Bioinformatic analysis of M.tuberculosis Rv1096,a cell wall protein,revealed that it is homologous to the other known deactylases such as the N-acetyl glucosamine from S.pneumioae named PgdA,the N-acetyl glucosamine from L.monocytogenes named Lmo0415and the peptidoglycan deacetylase from L.lactis named XyD.Rv1096 protein is also a member of O-mannosylated proteins of M.tuberculosis and has three possible mannosylation sites.It is suggested that Rv1096 protein maybe involved in the recognition process with the specific surface receptors of the pulmonary alveolar macrophages.Three parts following were performed in our study.1.To determine the deacetylase activity of Rv 1096 protein.The Rv1096 gene from M.tuberculosis H37Rv genome was cloned and Rv1096 protein was expressed in E.coli.The enzyme assay of deacetylase was established.Rv1096 protein was incubated with M.smegmatis PG as substrate to determine its deacetylase activity.The kinetic parameters were also determined.2.To check the bacterial survival ability owing to Rv1096 protein.M.smegmatis with expression of Rv1096 protein(M.smegmatis/Rv1096)as a model bacterium was constructed.M.smegmatis/Rv1096 was treated with lysozyme and cultured with macrophage.3.To determine the mannosylation of Rv 1096 protein by using ConA-lectin and its mannosylation sites by making Rv1096 mutants using site-directed mutagenesis method.Followings results we got in this study:1.Rv1096 protein possessed metallo-dependent PG deacetylase activity.(1)Rv1096 protein was a novel PG deacetylase,which was able to deactylate PG but had no effect on chitin.(2)Rv1096 protein was a metallo-dependent PG deacetylase and its activity was the highest in the presence of Co2+.(3)The kinetic parameters of Rv1096 protein were determined.Km,0.910±0.007 mM;Vmax,0.514±0.038 μMmin-1;and Kcat=0.099±0.007(S-1).2.M.smegmatis/Rv1096 showed resistant to lysozyme and increased survival capacity in microphages.(1)Rv1096 protein expressed in M.had the same deacetylae activity as Rv1096 protein expressed in E.coli.(2)M.smegmatis/Rv1096 was resistant to lysozyme and still had the character of acid-fast.(3)The survival ability of M.smegmatis/Rv1096 was higher than wild yipe M.smegmatis in microphages.3.Rv1096 protein was an O-mannsylated protein which mannosylation site was at 265Thr residue.(1)M.smegmatis was able to carry out the mannosylation of Rv 1096 protein.(2)The O-mannosylated site of Rv1096 protein was at 265Thr residue.(3)The glycan structure of Rv1096 protein was analyzed by mass spectrometry.Conclusions:1.M.tuberculosis Rv1096 protein is a metallo-dependent PG deacetylase.2.M.smegmatis/Rv1096 had the lysozyme resistance and increased survival capacity in microphages owing to the deacetylation of cell wall PG caused by Rv1096 protein.3.Rv1096 protein was an O-mannosylated protein and its mannosylation site was at 265 Thr residue.Further studies:1.To investigate the catalytic mechanism on deacetylase of Rv 1096 protein.To determine the active sites of deacetylase and the specificity of its substrate.2.To deterimine whether Rv1096 protein is a virulent factor or not and which kind of receptor on macrophage interacted with Rv1096 protein.3.To determine the structure and the linkage of the glycan in Rv1096 protein.更多还原