本文選題：表皮葡萄球菌 + 生物膜��； 參考：《復(fù)旦大學(xué)》2008年博士論文

【摘要】： 凝固酶陰性的表皮葡萄球菌(Staphylococcus epidermidis)為人體皮膚表面常見的共生菌,通常不致病。但近年來隨著各種植入性醫(yī)療材料的廣泛使用,表皮葡萄球菌已成為院內(nèi)感染的主要條件致病菌,主要原因是其能在這些醫(yī)療材料表面形成生物膜(biofilm,BF)樣結(jié)構(gòu),該結(jié)構(gòu)能夠更好地保護細(xì)菌抵抗抗生素的治療和人體免疫系統(tǒng)的攻擊,并從中不斷釋放細(xì)菌,從而造成機體的反復(fù)感染,最終不得不將被污染的植入性醫(yī)療材料通過外科手術(shù)摘除,對患者造成極大的痛苦和對社會造成巨大的經(jīng)濟損失。此外,由于抗生素的大量使用導(dǎo)致表皮葡萄球菌多重耐藥株(multi-drug resistant strains)的發(fā)生率日趨增高,急需開發(fā)新型的抗葡萄球菌感染的藥物,尤其是能有效殺傷生物膜包被細(xì)菌的抗生素。 本課題的目的是發(fā)現(xiàn)抗表皮葡萄球菌的藥靶,為開發(fā)新型抗葡萄球菌感染的藥物奠定理論和實踐基礎(chǔ),主要采用兩種策略:一種策略是通過生物信息學(xué)的方法尋找細(xì)菌必須蛋白質(zhì),通過同源模建和虛擬篩選技術(shù)獲得針對該蛋白的潛在的小分子抑制劑,結(jié)合分子生物學(xué)和微生物學(xué)的方法對小分子化合物的抑酶和抗菌活性進行驗證;另一種策略是通過傳統(tǒng)的微生物遺傳學(xué)和分子生物學(xué)方法深入研究表皮葡萄球菌臨床菌株生物膜形成的相關(guān)分子機制,發(fā)現(xiàn)潛在藥物靶標(biāo),以期設(shè)計出針對生物膜的新型藥物。 本論文分兩部分:第一部分通過生物信息學(xué)方法分析了表皮葡萄球菌色氨酰-tRNA合成酶作為抗菌靶標(biāo)的可行性,通過蛋白結(jié)構(gòu)同源模建和高通量虛擬篩選技術(shù)獲得潛在的小分子抑制劑并對其生物學(xué)活性進行研究,在體外驗證了其抗菌活性;第二部分探討了表皮葡萄球菌雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)ArlRS對生物膜形成的調(diào)控作用,對其分子機制進行了較深入的研究,認(rèn)為ArlRS可以作為抗生物膜的藥靶而發(fā)展出抗生物膜藥物。 第一部分表皮葡萄球菌色氨酰tRNA合成酶作為抗菌靶標(biāo)的研究 氨酰tRNA合成酶(ARS)是一類古老而保守、廣泛存在于動物、植物、細(xì)菌、病毒等各種生物體中的酶。其功能是催化特定氨基酸與相應(yīng)tRNA之間的連接反應(yīng),以及水解錯誤的連接并加以校正,從而保證核酸、蛋白質(zhì)之間信息傳遞的準(zhǔn)確性。由于氨酰tRNA合成酶是生物體中不能或缺的組成成分,而在真核生物和原核生物中又存在很大差異,使得在細(xì)菌多重抗藥性普遍越來越強、開發(fā)新藥的需求越來越迫切的今天,人們把目光投向了這類極具潛力的藥靶上來。色氨酰-tRNA合成酶(WRS)屬于氨酰tRNA合成酶Ⅰ類家族。目前針對WRS的有效抑制劑僅有吲哚霉素及其衍生物,但都未能進入臨床實驗。我們運用生物信息學(xué)方法確認(rèn)在表皮葡萄球菌全基因組中僅存在一個WRS的編碼基因trpS。通過對表皮葡萄球菌色氨酰-tRNA合成酶(SeWRS)的空間結(jié)構(gòu)進行同源模建,并在此基礎(chǔ)上運用高通量虛擬篩選技術(shù)共發(fā)現(xiàn)111個潛在的小分子抑制劑(先導(dǎo)化合物),其中有三個化合物(Compound 1-3)能明顯抑制靶蛋白的酶活性(半數(shù)抑制率濃度IC_(50)范圍在15.1～42.2μM),證實這3個化合物為SeWRS的抑制劑;而對人色氨酰-tRNA合成酶(HWRS)活性的影響很弱(IC_(50)范圍在89.3～＞100μM)。進一步的研究發(fā)現(xiàn)這3個化合物能在體外與靶蛋白SeWRS相結(jié)合(結(jié)合平衡常數(shù)K_d范圍在3.76-13.9μM)。體外抑菌實驗表明其中3個SeWRS抑制劑能明顯抑制表皮葡萄球菌的生長(MIC范圍在6.25～100 gM),其中Compound 1和Compound 2能明顯抑制金黃色葡萄球菌的生長;而上述3個SeWRS抑制劑在實驗中的最高濃度(200μM)下對大腸桿菌的生長均無抑制作用。此外,這3個SeWRS抑制劑對哺乳動物細(xì)胞(Vero細(xì)胞)無明顯細(xì)胞毒性,提示了這些化合物作為新型抗葡萄球菌感染藥物的開發(fā)前景。 第二部分表皮葡萄球菌雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)ArlRS在生物膜形成中的作用:作為抗生物膜藥靶的可行性研究 表皮葡萄球菌能夠形成生物膜(Biofilm),該結(jié)構(gòu)是其抵抗抗生素治療和宿主免疫系統(tǒng)的攻擊、以及導(dǎo)致感染遷延不愈的主要原因,因此研發(fā)抗生物膜的藥物也是對抗表皮葡萄球菌感染的策略之一。 表皮葡萄球菌生物膜的形成及其調(diào)控是非常復(fù)雜的過程。表皮葡萄球菌生物膜的形成主要分為兩個階段:單個細(xì)菌初始黏附到材料表面;細(xì)菌之間互相黏附,形成多細(xì)胞層的結(jié)構(gòu)。目前己發(fā)現(xiàn)了參與這兩個階段的一些功能基因(如atlE、icaADBC、aap、bap等),其中icaADBC編碼細(xì)胞間多糖黏附素(Polysaccharide Intercellular Adhesion,PIA),而PIA是表皮葡萄球菌生物膜的主要成分之一,但在表皮葡萄球菌臨床分離株中也發(fā)現(xiàn)部分菌株的基因組中雖然存在icaADBC基因,但不形成生物膜(即ica~+/BF~-菌株)。很多調(diào)控因子(如sarA、sigB、agr等)在生物膜形成的兩個階段對上述功能性基因的表達起調(diào)控作用。目前,國外文獻和本實驗室的相關(guān)研究均提示雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)(Two-component Signal Transduction Systems,TCSs)參與了生物膜形成的調(diào)控。本實驗室前期研究在表皮葡萄球菌基因組中發(fā)現(xiàn)了16對TCSs,本論文主要探討了其中的ArlRS對生物膜形成的調(diào)控機制,以期從中發(fā)現(xiàn)新的抗生物膜的靶點。 首先使用溫度敏感性穿梭質(zhì)粒pBT2,采用同源重組法在表皮葡萄球菌1457株基因組中刪除了雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)ArlRS的組氨酸激酶編碼基因arlS,獲得了arlS基因刪除株(WW06株);通過PCR、RT-PCR確認(rèn)arlS基因刪除株構(gòu)建成功,并采用梅里埃公司API-Staph細(xì)菌鑒定系統(tǒng)確認(rèn)WW06為表皮葡萄球菌。Western Blot結(jié)果表明WW06株不表達ArlR蛋白,因此WW06株是ArlRS系統(tǒng)缺失株。WW06株的生長曲線與野生株相似,但不形成生物膜。對WW06株喪失生物膜形成能力的機制進行了深入研究,發(fā)現(xiàn)其初始黏附能力并未發(fā)生變化,而通過基因芯片以及Real-Time RT-PCR等方法,發(fā)現(xiàn)參與生物膜形成第二階段的一些基因在突變株中的轉(zhuǎn)錄水平明顯降低,如icaADBC轉(zhuǎn)錄水平降低5-10倍、sigB轉(zhuǎn)錄水平降低約10倍、sarA轉(zhuǎn)錄水平降低約3-5倍。凝膠遷移阻滯實驗(EMSA)證明ArlR可以與icaADBC的啟動子區(qū)特異性結(jié)合,提示ArlRS對icaADBC的轉(zhuǎn)錄起到直接調(diào)控作用。在WW06株中過表達icaADBC可以恢復(fù)形成生物膜的表型,而過表達sigB則不能恢復(fù),上述結(jié)果證明icaADBC是ArlRS調(diào)節(jié)的下游功能基因,ArlRS通過直接調(diào)節(jié)icaADBC的轉(zhuǎn)錄對生物膜的形成起正調(diào)控作用。進一步檢測9株ica~+/BF~-的表皮葡萄球菌在對數(shù)生長中期(4hrs)的icaADBC轉(zhuǎn)錄水平和arlRS的轉(zhuǎn)錄水平,結(jié)果顯示與SE1457相比,icaADBC和arlRS的轉(zhuǎn)錄水平均顯著降低,提示兩者之間存在相關(guān)性。實驗結(jié)果表明ArlRS可作為潛在的抗生物膜靶標(biāo)。 本論文采用兩種策略,一方面通過生物信息學(xué)、結(jié)構(gòu)生物學(xué)和細(xì)菌分子生物學(xué)等方法獲得了具有抑菌活性的表皮葡萄球菌色氨酰tRNA合成酶抑制劑;另一方面通過細(xì)菌遺傳學(xué)和分子生物學(xué)等方法深入研究了表皮葡萄球菌雙組分信號轉(zhuǎn)導(dǎo)系統(tǒng)ArlRS對生物膜形成的調(diào)控作用,認(rèn)為ArlRS可以作為抗生物膜的藥靶而發(fā)展出抗生物膜藥物。本研究為抗表皮葡萄球菌藥物的研發(fā)奠定了一定的基礎(chǔ)。
[Abstract]:The coagulase negative Staphylococcus epidermidis (Staphylococcus epidermidis) is a common symbiotic bacteria on the skin surface of the human body, which is usually not pathogenic. But in recent years, with the extensive use of various implant medical materials, Staphylococcus epidermidis has become the main pathogenic bacteria in hospital infection, mainly because it can be formed on the surface of these medical materials. The structure of the biofilm (biofilm, BF), which can better protect the bacteria from the treatment of antibiotics and the attack of the human immune system, and release bacteria from them, resulting in repeated infection of the body, and eventually have to be removed by surgical removal of contaminated implant materials, causing great pain to the patients and to the society. In addition, the incidence of Staphylococcus epidermidis multiple resistant strains (multi-drug resistant strains) is increasing because of the extensive use of antibiotics, and it is urgent to develop a new drug for anti staphylococcal infection, especially the antibiotics that can effectively kill the biofilm coated bacteria.
The purpose of this project is to discover the drug target of anti Staphylococcus epidermidis, and to lay a theoretical and practical basis for the development of a new type of anti staphylococcal drug. Two strategies are adopted mainly: a strategy is to find the necessary protein by bioinformatics, and obtain the potential of the protein by homologous modeling and virtual screening. Small molecular inhibitors, combined with molecular and microbiological methods, verify the inhibition and antibacterial activity of small molecular compounds. Another strategy is to study the molecular mechanism of the biofilm formation of Staphylococcus epidermidis by traditional Microbiological Genetics and molecular biology methods, and find potential drugs. The target is to design new drugs for biofilm.
This thesis is divided into two parts: the first part analyses the feasibility of the Staphylococcus epidermidis tryptophan -tRNA synthetase as an antiseptic target by bioinformatics method. The potential small molecular inhibitors are obtained by the homologous protein structure construction and high throughput virtual screening technology, and the bioactivity of the Staphylococcus epidermidis is studied. The antibacterial activity is verified in vitro. In the second part, the second part explored the regulatory role of the bicomponent signal transduction system of Staphylococcus epidermidis to the formation of biofilm, and studied its molecular mechanism in depth. It was considered that ArlRS could be used as a drug target for anti biological membrane to develop anti biofilm drugs.
The first part is the study of Staphylococcus epidermidis tryptophan tRNA synthetase as an antibacterial target.
Aminoacyl tRNA synthetase (ARS) is an old and conservative enzyme that widely exists in various organisms such as animals, plants, bacteria, viruses and other organisms. Its function is to catalyze the connection between specific amino acids and corresponding tRNA, and to correct the error of hydrolysis, so as to ensure the accuracy of the transmission of information between nucleic acids and proteins. Acyl tRNA synthetase (acyl) synthase is an indispensable component in organism, and there is a great difference in eukaryotes and prokaryotes, which makes the multidrug resistance of bacteria more and more strong. Today, the demand for developing new drugs is becoming more and more urgent. People focus on this kind of potential drug target. Tryptophan -tRNA synthetase (WRS). It belongs to the family of the amyl tRNA synthetase type I. The current effective inhibitor for WRS is only indometamycin and its derivatives, but they have failed to enter clinical trials. We used bioinformatics to confirm that there is only one WRS encoding gene trpS. in the whole genome of Staphylococcus epidermidis, which has passed to the tryptophan -tRNA synthetase of Staphylococcus epidermidis. The spatial structure of (SeWRS) is built by homologous model, and on this basis, 111 potential small molecular inhibitors (lead compounds) are found by high throughput virtual screening technology, of which three compounds (Compound 1-3) can significantly inhibit the enzyme activity of the target protein (half of the inhibitory rate of IC_ (50) range from 15.1 to 42.2 M), and the 3 compounds are confirmed. The substance was an inhibitor of SeWRS, and the effect on the activity of tryptophan -tRNA synthetase (HWRS) was very weak (IC_ (50) range from 89.3 to 100 mu M). Further studies found that these 3 compounds could be combined with target protein SeWRS in vitro (the K_d range of equilibrium constant K_d in 3.76-13.9 Mu M). In vitro bacteriostasis experiments showed that 3 SeWRS inhibitors could be obvious Inhibition of the growth of Staphylococcus epidermidis (MIC range from 6.25 to 100 gM), of which Compound 1 and Compound 2 significantly inhibit the growth of Staphylococcus aureus, and the above 3 SeWRS inhibitors have no inhibitory effect on the growth of Escherichia coli at the highest concentration (200 u M) in the experiment. In addition, the 3 SeWRS inhibitors are on mammalian cells (Vero). The cells showed no obvious cytotoxicity, suggesting the development prospect of these compounds as new anti staphylococcal drugs.
The second part: the role of ArlRS, a two component signal transduction system of Staphylococcus epidermidis, in biofilm formation: a feasibility study as an anti biofilm target.
Staphylococcus epidermidis can form a biofilm (Biofilm), which is the main cause of resistance to antibiotic therapy and host immune system and the main cause of infection. Therefore, the research and development of anti biofilm drugs is also a strategy against Staphylococcus epidermidis infection.
The formation and regulation of Staphylococcus epidermidis biofilm is a very complex process. The formation of Staphylococcus epidermidis biofilm is divided into two stages: single bacteria adhered to the surface of the material; bacteria adhered to each other to form a multi cell layer structure. At present, some functional genes (such as atlE, ICA) have been found. ADBC, AAP, BAP, etc., in which icaADBC encodes the intercellular polysaccharide adhesion (Polysaccharide Intercellular Adhesion, PIA), and PIA is one of the main components of the biofilm of Staphylococcus epidermidis, but the icaADBC gene exists in the genome of some strains of Staphylococcus epidermidis, but it does not form a biofilm (i.e. ica~+/BF). A number of regulatory factors (such as sarA, sigB, agr, etc.) play a regulatory role in the expression of the above functional genes in the two stages of biofilm formation. At present, both foreign literature and related studies in our laboratory suggest that the dual component signal transduction system (Two-component Signal Transduction Systems, TCSs) is involved in the regulation of biofilm formation. In our previous study, 16 pairs of TCSs were found in the genome of Staphylococcus epidermidis. This paper mainly discussed the regulation mechanism of ArlRS on the formation of biofilm, in order to find new targets for anti biofilm.
First, using the temperature sensitive shuttle plasmid pBT2, the homologous recombination method was used to delete the histidine kinase encoding gene arlS of the two component signal transduction system ArlRS in the 1457 strains of Staphylococcus epidermidis, and the arlS gene deletion strain (WW06 strain) was obtained. The construction of the deletion strain of the arlS gene was confirmed by PCR and RT-PCR, and the MRI company AP was adopted. The I-Staph bacterial identification system confirmed that WW06 was.Western Blot of Staphylococcus epidermidis indicating that the WW06 strain did not express ArlR protein, so the WW06 strain was similar to the wild strain of the.WW06 strain of the ArlRS system, but did not form the biofilm. The mechanism of the loss of the biofilm formation ability of the WW06 strain was deeply studied and the initial adhesion was found. The ability did not change, and by gene chip and Real-Time RT-PCR, the transcriptional level of some genes involved in the second stage of biofilm formation was obviously reduced, such as the decrease of icaADBC transcriptional level by 5-10 times, the decrease of sigB transcriptional level about 10 times, and the decrease of sarA transcriptional level by 3-5 times. EMSA) demonstrated that ArlR can specifically bind to the promoter region of icaADBC, suggesting that ArlRS plays a direct role in the transcription of icaADBC. The overexpression of icaADBC in the WW06 strain can restore the phenotype of the biofilm, and the over expression sigB can not be restored. The above results prove that icaADBC is a downstream functional gene of ArlRS regulation, ArlRS through direct regulation. The transcription of icaADBC plays a positive role in the formation of biofilm. Further detection of icaADBC transcriptional level and arlRS transcriptional level of 9 strains of Staphylococcus epidermidis at the middle of logarithmic growth (4hrs) of ica~+/BF~- showed that the transcriptional level of icaADBC and arlRS decreased significantly compared with SE1457, suggesting a correlation between the two. It is indicated that ArlRS can be used as a potential anti biological membrane target.
In this paper, two strategies were adopted. On the one hand, the inhibitory activity of Staphylococcus epidermidis tryptophan tRNA synthetase inhibitor was obtained by bioinformatics, structural biology and bacterial molecular biology. On the other hand, the two component signal conversion of Staphylococcus epidermidis was studied through bacterial genetics and molecular biology. The regulatory effect of ArlRS on the formation of biofilm has been considered as a drug target for anti biological membrane. This study has laid a foundation for the research and development of anti Staphylococcus epidermidis.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R378

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本文編號：2018619