结核分枝杆菌多聚磷酸盐外切酶Rv1026对耻垢分枝杆菌的菌落形态和生物膜的影响

发布时间：2018-05-07 12:30

本文选题：外切多聚磷酸盐水解酶 + 分枝杆菌　；参考：《西南大学》2011年硕士论文

【摘要】：结核病是一种严重危害人类健康的传染病。全球近1/3的人口感染结核菌。随着人类社会和药物治疗的发展,人们对结核病的致病菌——结核分枝杆菌(Mycobacterium tuberculosis)的生物学特性有了进一步的研究,继而研发了链霉素、异烟肼、利福平等治疗性药物,使得人类对结核病的控制取得了可喜的成绩,遏制了结核病的蔓延和传播。然而由于化学药物的不合理应用、艾滋病与结核病联合感染、人口流动的增加以及耐药结核病菌的流行,使得近年来结核病疫情下降缓慢,在部分地区甚至有所回升。当前,在结核病控制工作中急需有效的预防性、治疗性疫苗和新型药物。寻找新的药物作用靶点和开发新型抗结核药物已成为当前最为关注的焦点。多聚磷酸盐(polyP)是由几个到几百个无机磷酸盐单体通过高能磷酸键聚合而成的线性多聚体,广泛分布于自然界和生物体。polyP在生物体中起到重要功能,包括基因表达和调控、DNA的摄取、微生物的运动性、对胁迫和饥饿的应答、病原菌的毒性、以及对乳腺癌细胞的增殖、血液凝固、细胞钙化、线粒体功能的调节等。细胞内参与polyP代谢的酶包括内切酶、外切酶、葡萄糖激酶、NAD激酶和AMP磷酸转移酶等。细胞内合成和分解polyP的两个主要酶分别是多聚磷酸盐激酶(polyphosphate kinase, PPK)和外切聚磷酸酶(exopolyphosphatase, PPX)。结核分枝杆菌中的PPK已经得到一定程度的研究。结核分枝杆菌的ppkl基因的表达下调与药物诱导的转录抑制相关,表明多聚磷酸盐代谢与抑制转录过程的联系。Kamakshi Sureka等报道ppkl涉及结核分枝杆菌在低氧,变性剂压力和氧化压力条件下的存活。其作用机制为：而MprB优先利用多聚磷酸盐将MprA磷酸化,从而促进sigE的转录,产生的SigE调节严紧应答调节子rel的转录。Ppkl的表达下降导致结核菌在巨噬细胞中存活能力的下降,表明ppkl在细菌的细胞内生活起到重要的功能。此外,另一个多聚磷酸盐激酶PPK2能利用多聚磷酸盐将GDP转化为GTP,维持GTP的平衡。核苷酸库的平衡对许多生理过程非常重要,例如,结核分枝杆菌一个重要的细胞壁组份——脂阿拉伯甘露聚糖就是从1-磷酸甘露糖和GTP合成。实验证实ppk2的表达下调损伤其在巨噬细胞中的生存能力,意味着ppk2在分枝杆菌定居于巨噬细胞起到重要的生理功能。然而,作为参与polyP代谢的另一个酶PPX目前的研究还很少,其在分枝杆菌中的生理功能还有待进一步揭示。因此,开展对该蛋白的功能和性质的研究,有助于进一步对结核分枝杆菌胞内的存活和免疫逃逸机制的研究。系统进化分析表明结核分枝杆菌中的Rv0496和Rv1026可能具有外切多聚磷酸酶活性。利用PSIPRED服务器的GenTHREADER程序搜寻,发现风产叶菌的PPX/GPPA蛋白与结核分枝杆菌的Rv1026和Rv0496具有最高的结构相似性。基于风产叶菌的PPX/GPPA的二级结构,我们用已研究的PPX序列与结核分枝杆菌中的同源蛋白进行序列对比。发现结核分枝杆菌外切多聚磷酸酶具有与风产叶菌PPX的活性位点Glu121禾(?)Arg93相对应的残基Glu和Arg,结核分枝杆菌PPX存在焦磷酸结合位点Asp22, Arg267, Glu144和Glu211对应的残基,还具有富含甘氨酸的磷酸盐结合环(P-loop Gly143-Ser146)。目前关于Rv0496和Rv1026基因的研究主要来自于转座子突变筛选研究。两个基因都与结核分枝杆菌的致病性相关,其中Rv0496是T细胞抗原,Rv1026在感染巨噬细胞时被诱导表达。然而,Rv0496和Rv1026是否真正具有外切磷酸酶活性还没有得到实验证实。本研究中,从Genbank数据库中获取H37Rv的Rv1026基因序列,并以此设计一对引物。然后以H37Rv的基因组为模板,PCR扩增出Rv1026基因。PCR产物连接到pMD19-T载体上,并亚克隆到表达载体pMV261上。重组质粒通过PCR反应,限制性酶切反应和测序证实,质粒构建成功。然后将构建成功的重组质粒点转入耻垢分枝杆菌。37℃培养重组耻垢分枝杆菌,诱导表达目的基因,我们发现改变的菌落形态,生物膜形成能力的降低,而且这种改变对非离子变性剂Tween80敏感。由于耻垢分枝杆菌菌落形态和生物膜形成能力与细胞壁脂质成分相关,并提取了重组耻垢分枝杆菌和对照菌的细胞壁脂肪酸,进行气相色谱分析。发现重组和对照菌之间存在四个峰的差异。总之,我们实验证实了Rv1026具有多聚磷酸盐外切酶活性,并发现耻垢分枝杆菌细胞内多聚磷酸盐的减少,导致耻垢分枝杆菌菌落形态的改变和生物膜形成能力的减少,这种改变对Tween80敏感。
[Abstract]:Tuberculosis is an infectious disease which is seriously harmful to human health . With the development of human society and drug therapy , the biological characteristics of Mycobacterium tuberculosis have been further studied .

PolyP ( polyP ) is a linear polymer formed by polymerization of several hundreds of inorganic phosphate monomers through high - energy phosphate bond . It is widely distributed in nature and organism . PolyP plays an important role in organism , including gene expression and regulation , DNA uptake , microorganism ' s motility , response to stress and hunger , toxicity of pathogenic bacteria , and regulation of breast cancer cell proliferation , blood coagulation , cell calcification , NAD kinase and AMP phosphotransferase . PPK in M.tuberculosis has been studied to some extent . The downregulation of ppkl gene of M.tuberculosis is associated with drug - induced transcription inhibition , suggesting that polyphosphate metabolism plays an important role in the inhibition of transcription . The mechanism of action is : while MprB preferentially utilizes polyphosphate to phosphorylate MprA to maintain the balance of GTP . The expression of ppkl is important for many physiological processes . For example , the expression of ppk2 reduces its viability in macrophages , meaning that ppk2 regulates the viability of macrophages in macrophages , meaning that ppk2 plays an important physiological function in the colonization of macrophages .

However , the present study of PPX , another enzyme involved in polyP metabolism , is still rare , and its physiological function in M.tuberculosis remains to be further revealed . Therefore , studies on the function and properties of the protein contribute to the further study of the survival and immune escape mechanisms in M.tuberculosis cells .

The results of phylogenetic analysis showed that Rv0496 and Rv1026 in Mycobacterium tuberculosis could have the activity of exo - polyphosphatase . The PPX / GPPA protein was found to have the highest structural similarity with Rv1026 and Rv0496 of Mycobacterium tuberculosis . Currently , studies on Rv0496 and Rv1026 genes are mainly derived from transposon mutation screening studies . Both genes are associated with the pathogenicity of M . tuberculosis , where Rv0496 is a T cell antigen and Rv1026 is induced to express when macrophages are infected . However , whether Rv0496 and Rv1026 truly have exo - phosphatase activity has not been experimentally confirmed .

In this study , the Rv1026 gene sequence of H37Rv was obtained from the GeneBank database , and a pair of primers were designed . The PCR products were ligated to pMD19 - T vector and subcloned into the expression vector pMV261 .

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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