表皮葡萄球菌色氨酰tRNA合成酶和ArlRS双组分信号转导系统作为抗菌靶标的研究

发布时间：2018-06-14 18:57

本文选题：表皮葡萄球菌 + 生物膜　；参考：《复旦大学》2008年博士论文

【摘要】： 凝固酶阴性的表皮葡萄球菌(Staphylococcus epidermidis)为人体皮肤表面常见的共生菌,通常不致病。但近年来随着各种植入性医疗材料的广泛使用,表皮葡萄球菌已成为院内感染的主要条件致病菌,主要原因是其能在这些医疗材料表面形成生物膜(biofilm,BF)样结构,该结构能够更好地保护细菌抵抗抗生素的治疗和人体免疫系统的攻击,并从中不断释放细菌,从而造成机体的反复感染,最终不得不将被污染的植入性医疗材料通过外科手术摘除,对患者造成极大的痛苦和对社会造成巨大的经济损失。此外,由于抗生素的大量使用导致表皮葡萄球菌多重耐药株(multi-drug resistant strains)的发生率日趋增高,急需开发新型的抗葡萄球菌感染的药物,尤其是能有效杀伤生物膜包被细菌的抗生素。本课题的目的是发现抗表皮葡萄球菌的药靶,为开发新型抗葡萄球菌感染的药物奠定理论和实践基础,主要采用两种策略:一种策略是通过生物信息学的方法寻找细菌必须蛋白质,通过同源模建和虚拟筛选技术获得针对该蛋白的潜在的小分子抑制剂,结合分子生物学和微生物学的方法对小分子化合物的抑酶和抗菌活性进行验证;另一种策略是通过传统的微生物遗传学和分子生物学方法深入研究表皮葡萄球菌临床菌株生物膜形成的相关分子机制,发现潜在药物靶标,以期设计出针对生物膜的新型药物。本论文分两部分:第一部分通过生物信息学方法分析了表皮葡萄球菌色氨酰-tRNA合成酶作为抗菌靶标的可行性,通过蛋白结构同源模建和高通量虚拟筛选技术获得潜在的小分子抑制剂并对其生物学活性进行研究,在体外验证了其抗菌活性;第二部分探讨了表皮葡萄球菌双组分信号转导系统ArlRS对生物膜形成的调控作用,对其分子机制进行了较深入的研究,认为ArlRS可以作为抗生物膜的药靶而发展出抗生物膜药物。第一部分表皮葡萄球菌色氨酰tRNA合成酶作为抗菌靶标的研究氨酰tRNA合成酶(ARS)是一类古老而保守、广泛存在于动物、植物、细菌、病毒等各种生物体中的酶。其功能是催化特定氨基酸与相应tRNA之间的连接反应,以及水解错误的连接并加以校正,从而保证核酸、蛋白质之间信息传递的准确性。由于氨酰tRNA合成酶是生物体中不能或缺的组成成分,而在真核生物和原核生物中又存在很大差异,使得在细菌多重抗药性普遍越来越强、开发新药的需求越来越迫切的今天,人们把目光投向了这类极具潜力的药靶上来。色氨酰-tRNA合成酶(WRS)属于氨酰tRNA合成酶Ⅰ类家族。目前针对WRS的有效抑制剂仅有吲哚霉素及其衍生物,但都未能进入临床实验。我们运用生物信息学方法确认在表皮葡萄球菌全基因组中仅存在一个WRS的编码基因trpS。通过对表皮葡萄球菌色氨酰-tRNA合成酶(SeWRS)的空间结构进行同源模建,并在此基础上运用高通量虚拟筛选技术共发现111个潜在的小分子抑制剂(先导化合物),其中有三个化合物(Compound 1-3)能明显抑制靶蛋白的酶活性(半数抑制率浓度IC_(50)范围在15.1～42.2μM),证实这3个化合物为SeWRS的抑制剂;而对人色氨酰-tRNA合成酶(HWRS)活性的影响很弱(IC_(50)范围在89.3～＞100μM)。进一步的研究发现这3个化合物能在体外与靶蛋白SeWRS相结合(结合平衡常数K_d范围在3.76-13.9μM)。体外抑菌实验表明其中3个SeWRS抑制剂能明显抑制表皮葡萄球菌的生长(MIC范围在6.25～100 gM),其中Compound 1和Compound 2能明显抑制金黄色葡萄球菌的生长;而上述3个SeWRS抑制剂在实验中的最高浓度(200μM)下对大肠杆菌的生长均无抑制作用。此外,这3个SeWRS抑制剂对哺乳动物细胞(Vero细胞)无明显细胞毒性,提示了这些化合物作为新型抗葡萄球菌感染药物的开发前景。第二部分表皮葡萄球菌双组分信号转导系统ArlRS在生物膜形成中的作用:作为抗生物膜药靶的可行性研究表皮葡萄球菌能够形成生物膜(Biofilm),该结构是其抵抗抗生素治疗和宿主免疫系统的攻击、以及导致感染迁延不愈的主要原因,因此研发抗生物膜的药物也是对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调控是非常复杂的过程。表皮葡萄球菌生物膜的形成主要分为两个阶段:单个细菌初始黏附到材料表面;细菌之间互相黏附,形成多细胞层的结构。目前己发现了参与这两个阶段的一些功能基因(如atlE、icaADBC、aap、bap等),其中icaADBC编码细胞间多糖黏附素(Polysaccharide Intercellular Adhesion,PIA),而PIA是表皮葡萄球菌生物膜的主要成分之一,但在表皮葡萄球菌临床分离株中也发现部分菌株的基因组中虽然存在icaADBC基因,但不形成生物膜(即ica~+/BF~-菌株)。很多调控因子(如sarA、sigB、agr等)在生物膜形成的两个阶段对上述功能性基因的表达起调控作用。目前,国外文献和本实验室的相关研究均提示双组分信号转导系统(Two-component Signal Transduction Systems,TCSs)参与了生物膜形成的调控。本实验室前期研究在表皮葡萄球菌基因组中发现了16对TCSs,本论文主要探讨了其中的ArlRS对生物膜形成的调控机制,以期从中发现新的抗生物膜的靶点。首先使用温度敏感性穿梭质粒pBT2,采用同源重组法在表皮葡萄球菌1457株基因组中删除了双组分信号转导系统ArlRS的组氨酸激酶编码基因arlS,获得了arlS基因删除株(WW06株);通过PCR、RT-PCR确认arlS基因删除株构建成功,并采用梅里埃公司API-Staph细菌鉴定系统确认WW06为表皮葡萄球菌。Western Blot结果表明WW06株不表达ArlR蛋白,因此WW06株是ArlRS系统缺失株。WW06株的生长曲线与野生株相似,但不形成生物膜。对WW06株丧失生物膜形成能力的机制进行了深入研究,发现其初始黏附能力并未发生变化,而通过基因芯片以及Real-Time RT-PCR等方法,发现参与生物膜形成第二阶段的一些基因在突变株中的转录水平明显降低,如icaADBC转录水平降低5-10倍、sigB转录水平降低约10倍、sarA转录水平降低约3-5倍。凝胶迁移阻滞实验(EMSA)证明ArlR可以与icaADBC的启动子区特异性结合,提示ArlRS对icaADBC的转录起到直接调控作用。在WW06株中过表达icaADBC可以恢复形成生物膜的表型,而过表达sigB则不能恢复,上述结果证明icaADBC是ArlRS调节的下游功能基因,ArlRS通过直接调节icaADBC的转录对生物膜的形成起正调控作用。进一步检测9株ica~+/BF~-的表皮葡萄球菌在对数生长中期(4hrs)的icaADBC转录水平和arlRS的转录水平,结果显示与SE1457相比,icaADBC和arlRS的转录水平均显著降低,提示两者之间存在相关性。实验结果表明ArlRS可作为潜在的抗生物膜靶标。本论文采用两种策略,一方面通过生物信息学、结构生物学和细菌分子生物学等方法获得了具有抑菌活性的表皮葡萄球菌色氨酰tRNA合成酶抑制剂;另一方面通过细菌遗传学和分子生物学等方法深入研究了表皮葡萄球菌双组分信号转导系统ArlRS对生物膜形成的调控作用,认为ArlRS可以作为抗生物膜的药靶而发展出抗生物膜药物。本研究为抗表皮葡萄球菌药物的研发奠定了一定的基础。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R378

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