金黄色葡萄球菌糖肽水解酶LytM表达调控及其生物学功能的初步研究

发布时间：2018-04-08 22:18

本文选题：金黄色葡萄球菌　切入点：LytM　出处：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：金黄色葡萄球菌(Staphylococcus aureus)是一种广泛存在的革兰氏阳性致病菌,是导致医院内感染和后天免疫性群体感染的最主要的原因之一。它可以引发多种疾病,如皮肤、黏膜的化脓性炎症,甚至危及生命的败血症、心内膜炎、肺炎和脑膜炎等;此外,金黄色葡萄球菌还可以引起异物相关感染、尿路感染、骨髓炎、关节炎和肠炎等感染性疾病。目前临床上抗金黄色葡萄球菌感染主要通过联合应用抗生素的方法,但耐药性金黄色葡萄球菌菌株MRSA(methicillin resistant Staphylococcus aureus)和VRSA(vancomycin resistant Staphylococcus aureus)的出现加重了金黄色葡萄球菌对人类健康的威胁。金黄色葡萄球菌致病过程中有多种分子的参与,深入研究这些分子的功能,将为金黄色葡萄球菌的防治提供理论基础。细菌能够产生多种自溶素,目前普遍认为自溶素参与细菌的繁殖、分离、隔膜的生成、鞭毛的生成等生理活动,但是其生理学和生态学的具体功能并不十分明确。根据作用于细胞壁肽聚糖化学键的位点差异,细菌自溶素主要分为以下几类:①糖苷酶和糖基转移酶,水解N-乙酰葡萄糖胺和N-乙酰胞壁酸之间的糖苷键;②酰胺酶,水解N-乙酰胞壁酸和肽桥之间的酰胺键;③肽链内切酶,水解氨基酸侧链的肽键或肽桥上氨基酸之间的肽键。LytM蛋白是金黄色葡萄球菌产生的一种自溶素,属于肽链内切酶,在细菌生长过程中被分泌到细胞外,专一性水解葡萄球菌细胞壁肽聚糖甘氨酸与甘氨酸之间的肽键,导致细菌溶解,因此具有潜在的抗葡萄球菌感染的药用价值。LytM蛋白与模仿葡萄球菌(S. simulan)来源的溶葡萄球菌素(Lysostaphin)具有较高的同源性,是目前唯一一个解析晶体结构的Lysostaphin型Zn~(2+)金属蛋白酶。体外研究结果表明,全长形式的LytM蛋白没有水解活性,只有其截短型C末端具有水解活性,但天然状态下,是否存在具有水解活性的LytM并不清楚;同时,LytM自身表达调控的相关机制及其相互作用蛋白也不明确。本研究以LytM蛋白为研究对象,旨在寻找金黄色葡萄球菌中LytM蛋白直接发挥水解作用的天然活性体形式,并进一步探讨调控LytM表达的分子机制以及寻找和鉴定LytM相互作用蛋白。本论文的主要内容如下: 1. LytM天然形式活性体的寻找。我们利用原核表达系统体外重组表达了LytM及其C端185-316aa,并对其水解活性进行了检测,结果显示:全长形式的LytM蛋白没有水解活性,不能裂解金黄色葡萄球菌的细胞壁,而其C端(LytM_(185-316))有水解活性。分别制备了高效价的LytM特异性鼠源和兔源多克隆抗体,并证实两种多克隆抗体均可以与全长形式的LytM及LytM185-316特异性结合。在此基础上,进一步通过Western Blot及免疫共沉淀的方法寻找和鉴定LytM的截短型天然活性体,尽管进行了多种条件的摸索和尝试,但最终没有成功钓取到金黄色葡萄球菌中LytM的天然活性体。分析原因,一方面可能是需要结合条件的进一步优化,另一方面的原因可能是天然状态下LytM活性体痕量存在,利用现有的方法难以获取达到检测水平的目的蛋白。 2. LytM与TRAP蛋白相互作用的研究。我们已有的研究结果表明金黄色葡萄球菌中的信号分子TRAP蛋白能够特异性结合Lysostaphin。本研究中,我们通过ELISA和免疫共沉淀的方法证实了LytM可以与TRAP蛋白相互作用。同时我们通过基因重组的方法在金黄色葡萄球菌8325-4菌株中构建了lytM缺失突变体,进一步比较了野生型、lytM突变体及traP突变体的表型,探索了LytM与TRAP蛋白相互作用的可能的生物学意义。 3. RNAⅢ调控LytM表达的分子机制研究。RNAⅢ是金黄色葡萄球菌中一种重要的具有调控作用的sRNA,其通过序列互补的方式调控靶基因的表达。在本研究中,RT-PCR及Western Blot的结果表明金黄色葡萄球菌RNAⅢ负调控LytM的表达。我们进一步将lytM的5’UTR与lacZ报告基因融合构建了报告载体,发现RNAⅢ可以通过作用于lytM的5’UTR负调控LytM的表达。同时我们将lytM 5’UTR与RNAⅢ相互作用区段中的特异位点突变,进一步证实了RNAⅢ与lytM的5’UTR的相互作用。综上所述,本研究重组表达了金黄色葡萄球菌LytM蛋白,并通过多种方法尝试寻找其天然形式的截短型活性体;同时我们发现了金黄色葡萄球菌TRAP蛋白是LytM的结合蛋白,两种基因突变菌株具有相似的表型改变;另外,我们的研究结果证实RNAⅢ作用于lytM的5’UTR负调控LytM的表达。以上研究结果将会为LytM功能的深入研究和新的抗金黄色葡萄球菌药物靶标寻找奠定基础。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is a kind of gram positive pathogens, is the leading cause of nosocomial infections and community acquired infection is one of the most important reasons. It can cause a variety of diseases, such as skin, mucous purulent inflammation, even life-threatening septicemia, endocarditis, pneumonia and meningitis; in addition, Staphylococcus aureus can also be caused by foreign body infections, urinary tract infections, arthritis and osteomyelitis, enteritis and other infectious diseases. At present the clinical anti infection of Staphylococcus aureus mainly through the combined application of antibiotics, but the drug resistance of Staphylococcus aureus strain MRSA (methicillin resistant Staphylococcus aureus) and VRSA (vancomycin resistant Staphylococcus aureus) were aggravated with Staphylococcus aureus threat to human health. The pathogenic Staphylococcus aureus. The involvement of a variety of molecules in the process and the in-depth study of the functions of these molecules will provide a theoretical basis for the prevention and control of Staphylococcus aureus.
Bacteria can produce a variety of autolysins, generally in autolysin bacteria, separation, membrane formation, formation of physiological activity of flagella, but the specific function of ecology and physiology is not very clear. According to the difference of site effect on the cell wall peptidoglycan chemical bond, bacterial autolysin mainly divided into the following class: glycosidases and glycosyltransferases, hydrolysis of the glycosidic bond between N- acetyl glucosamine and N- n-acetylmuramicacid; the amidase, hydrolysis of the amide bonds between N- n-acetylmuramicacid and peptide bridge; the endopeptidase, amino acid bond between.LytM protein hydrolysis amino acid side chain peptide or peptide bridge is a kind of autolysis of Staphylococcus aureus producing pigment, belonging to the endopeptidase, to be secreted in the bacterial growth process between cells, catalyze the hydrolysis of staphylococcal peptidoglycan of glycine and glycine. The peptide bond, leading to bacterial dissolution, so it is a potential anti medicinal value of.LytM protein of Staphylococcus and Staphylococcus (S. simulan) source of lysostaphin (Lysostaphin) with high homology, is currently the only one of the crystal structures of Lysostaphin Zn~ (2+). The results show that metal protease in vitro the full-length form of LytM protein, no hydrolytic activity, only the truncated C terminal with hydrolytic activity, but the natural state, whether there is the hydrolysis activity of LytM is not clear; at the same time, the related mechanism of LytM expression regulation and interaction of protein is not clear.
In this study, LytM protein as the research object, in order to find the natural activity of Staphylococcus aureus LytM protein play direct hydrolysis form, and further explore the molecular mechanism of the regulation of LytM expression and the search and identification of proteins interacting with LytM. The main contents of this paper are as follows:
For 1. LytM natural form active body. We use the prokaryotic expression system of recombinant expression of LytM and C terminal 185-316aa, and the hydrolysis activity was detected. The results showed that the full-length form of LytM protein had no hydrolytic activity, the cell wall not lyse Staphylococcus aureus, and C (LytM_ (the end 185-316)) were prepared by hydrolysis activity. High titer of LytM specific murine and rabbit polyclonal antibody, and confirmed that two polyclonal antibodies can be combined with LytM and LytM185-316 specific with the full-length form. On this basis, the truncated natural activity further through the method of Western Blot and immune co precipitation search and identification of LytM, in spite of a variety of conditions of exploring and trying, but ultimately did not succeed to the natural fishing activity of LytM in Staphylococcus aureus. Analysis of the reasons, one may require a combination of conditions On the other hand, the reason for further optimization may be the presence of trace LytM in the natural state. It is difficult to obtain the target protein that reaches the detection level by the existing methods.
Study on 2. LytM interact with TRAP. Our previous study results showed that Staphylococcus aureus in the signaling molecule TRAP protein could specifically bind to Lysostaphin. in this study, we adopted the method of ELISA and co immunoprecipitation confirmed that LytM can interact with TRAP protein. At the same time we through gene recombination method in gold 8325-4 strains of Staphylococcus aureus were constructed lytM deletion mutant, further compared with the wild type, lytM mutant and traP mutant, explored the LytM interaction with TRAP protein may have biological significance.
3. RNA III regulates the expression of LytM and the molecular mechanism of.RNA III is one of the important sRNA play a role in the regulation of Staphylococcus aureus, its expression by the complementary sequence regulation of target genes. In this study, RT-PCR and Western Blot results showed that the expression of Staphylococcus aureus RNA in the negative regulation of LytM. We will further fusion gene lytM 5 'UTR and lacZ report to construct a reporter plasmid RNA, III can effect expressed in lytM 5' UTR negative regulation of LytM. At the same time we will lytM 5 'UTR and RNA III interaction in a segment specific locus mutation, further confirmed the interaction between RNA and III lytM 5 "UTR.
In summary, the study of recombinant expression of LytM protein in Staphylococcus aureus, and through a variety of methods to try to find the natural activity of truncated forms; at the same time, we found that Staphylococcus aureus TRAP protein binding protein LytM, two phenotypic gene mutation strains had similar change; in addition, our results confirm the expression of RNA III the role of lytM in the 5 'UTR negative regulation of LytM. The above results will provide LytM function research and new anti staphylococcal drug targets for the foundation.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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