本文選題：金黃色葡萄球菌　切入點：LytM　出處：《中國人民解放軍軍事醫(yī)學科學院》2011年碩士論文

【摘要】：金黃色葡萄球菌(Staphylococcus aureus)是一種廣泛存在的革蘭氏陽性致病菌,是導致醫(yī)院內(nèi)感染和后天免疫性群體感染的最主要的原因之一。它可以引發(fā)多種疾病,如皮膚、黏膜的化膿性炎癥,甚至危及生命的敗血癥、心內(nèi)膜炎、肺炎和腦膜炎等;此外,金黃色葡萄球菌還可以引起異物相關感染、尿路感染、骨髓炎、關節(jié)炎和腸炎等感染性疾病。目前臨床上抗金黃色葡萄球菌感染主要通過聯(lián)合應用抗生素的方法,但耐藥性金黃色葡萄球菌菌株MRSA(methicillin resistant Staphylococcus aureus)和VRSA(vancomycin resistant Staphylococcus aureus)的出現(xiàn)加重了金黃色葡萄球菌對人類健康的威脅。金黃色葡萄球菌致病過程中有多種分子的參與,深入研究這些分子的功能,將為金黃色葡萄球菌的防治提供理論基礎。 細菌能夠產(chǎn)生多種自溶素,目前普遍認為自溶素參與細菌的繁殖、分離、隔膜的生成、鞭毛的生成等生理活動,但是其生理學和生態(tài)學的具體功能并不十分明確。根據(jù)作用于細胞壁肽聚糖化學鍵的位點差異,細菌自溶素主要分為以下幾類:①糖苷酶和糖基轉(zhuǎn)移酶,水解N-乙酰葡萄糖胺和N-乙酰胞壁酸之間的糖苷鍵;②酰胺酶,水解N-乙酰胞壁酸和肽橋之間的酰胺鍵;③肽鏈內(nèi)切酶,水解氨基酸側(cè)鏈的肽鍵或肽橋上氨基酸之間的肽鍵。LytM蛋白是金黃色葡萄球菌產(chǎn)生的一種自溶素,屬于肽鏈內(nèi)切酶,在細菌生長過程中被分泌到細胞外,專一性水解葡萄球菌細胞壁肽聚糖甘氨酸與甘氨酸之間的肽鍵,導致細菌溶解,因此具有潛在的抗葡萄球菌感染的藥用價值。LytM蛋白與模仿葡萄球菌(S. simulan)來源的溶葡萄球菌素(Lysostaphin)具有較高的同源性,是目前唯一一個解析晶體結(jié)構(gòu)的Lysostaphin型Zn~(2+)金屬蛋白酶。體外研究結(jié)果表明,全長形式的LytM蛋白沒有水解活性,只有其截短型C末端具有水解活性,但天然狀態(tài)下,是否存在具有水解活性的LytM并不清楚;同時,LytM自身表達調(diào)控的相關機制及其相互作用蛋白也不明確。 本研究以LytM蛋白為研究對象,旨在尋找金黃色葡萄球菌中LytM蛋白直接發(fā)揮水解作用的天然活性體形式,并進一步探討調(diào)控LytM表達的分子機制以及尋找和鑒定LytM相互作用蛋白。本論文的主要內(nèi)容如下: 1. LytM天然形式活性體的尋找。我們利用原核表達系統(tǒng)體外重組表達了LytM及其C端185-316aa,并對其水解活性進行了檢測,結(jié)果顯示:全長形式的LytM蛋白沒有水解活性,不能裂解金黃色葡萄球菌的細胞壁,而其C端(LytM_(185-316))有水解活性。分別制備了高效價的LytM特異性鼠源和兔源多克隆抗體,并證實兩種多克隆抗體均可以與全長形式的LytM及LytM185-316特異性結(jié)合。在此基礎上,進一步通過Western Blot及免疫共沉淀的方法尋找和鑒定LytM的截短型天然活性體,盡管進行了多種條件的摸索和嘗試,但最終沒有成功釣取到金黃色葡萄球菌中LytM的天然活性體。分析原因,一方面可能是需要結(jié)合條件的進一步優(yōu)化,另一方面的原因可能是天然狀態(tài)下LytM活性體痕量存在,利用現(xiàn)有的方法難以獲取達到檢測水平的目的蛋白。 2. LytM與TRAP蛋白相互作用的研究。我們已有的研究結(jié)果表明金黃色葡萄球菌中的信號分子TRAP蛋白能夠特異性結(jié)合Lysostaphin。本研究中,我們通過ELISA和免疫共沉淀的方法證實了LytM可以與TRAP蛋白相互作用。同時我們通過基因重組的方法在金黃色葡萄球菌8325-4菌株中構(gòu)建了lytM缺失突變體,進一步比較了野生型、lytM突變體及traP突變體的表型,探索了LytM與TRAP蛋白相互作用的可能的生物學意義。 3. RNAⅢ調(diào)控LytM表達的分子機制研究。RNAⅢ是金黃色葡萄球菌中一種重要的具有調(diào)控作用的sRNA,其通過序列互補的方式調(diào)控靶基因的表達。在本研究中,RT-PCR及Western Blot的結(jié)果表明金黃色葡萄球菌RNAⅢ負調(diào)控LytM的表達。我們進一步將lytM的5’UTR與lacZ報告基因融合構(gòu)建了報告載體,發(fā)現(xiàn)RNAⅢ可以通過作用于lytM的5’UTR負調(diào)控LytM的表達。同時我們將lytM 5’UTR與RNAⅢ相互作用區(qū)段中的特異位點突變,進一步證實了RNAⅢ與lytM的5’UTR的相互作用。 綜上所述,本研究重組表達了金黃色葡萄球菌LytM蛋白,并通過多種方法嘗試尋找其天然形式的截短型活性體;同時我們發(fā)現(xiàn)了金黃色葡萄球菌TRAP蛋白是LytM的結(jié)合蛋白,兩種基因突變菌株具有相似的表型改變;另外,我們的研究結(jié)果證實RNAⅢ作用于lytM的5’UTR負調(diào)控LytM的表達。以上研究結(jié)果將會為LytM功能的深入研究和新的抗金黃色葡萄球菌藥物靶標尋找奠定基礎。
[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.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R378

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相關期刊論文 前1條

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本文編號：1723598