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

【作者】 吴旸；

【导师】 瞿涤； 蒋华良； 龙健儿； 叶荣；

【作者基本信息】 复旦大学 ， 病原生物学， 2008， 博士

【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IC₅₀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(IC₅₀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。更多还原

【Abstract】 Staphylococcus epidermidis is a normal inhabitant of human skin and mucous membranes that rarely causes pyogenic infections in healthy individuals.However, during the past two decades S.epidermidis has emerged as one of the major pathogens in nosocomial infections.The primary pathogenicity trait of S. epidermidis is associated with its ability to form biofilms on surfaces of medical devices,limiting severely the efficacy of many conventional antibiotics,and biofilms may also protect the bacteria against attacks from the host defence system.In parallel,the appearance of multi-drug resistant S.epidermidis strains has increased quickly due to the increasing use of antibiotics in hospitals,which urgently require design novel antibiotics against staphylococcus infections,especially in relation to biofilm development.The aim of this study is to look for the drug targets against S.epidermidis,which is consisted of two major parts:one is the discovery of lead compounds against S. epidermidis;the other is the discovery of molecular basis of biofilms formation in clinical isolates and the evaluation of feasibility of the potential anti-biofilm drug target.In this study,we have found three inhibitors of the S.epidermidis tryptophanyl-tRNA synthetase that have bacteriostatic activites.Besides,we investigated the role of TCS ArlRS in the regulation of biofilm formation in S. epidermidis,and find ArlRS is an anti-biofilm drug target.Our findings will contribute to the development of anti-staphylococci drugs.Part 1.S.epidermidis tryptophanyl-tRNA synthetase as an antibacterial targetAmong bacterial essential proteins,prokaryotic aminoacyl-tRNA synthetases （ARSs） have attracted attention as potential antibacterial targets.6 As a group,ARSs are universal enzymes that exist in all living organisms because they catalyse the attachment of amino acids to transfer RNAs（tRNAs）,which are the adaptors required for the information flux from the messenger RNA templates to the polypeptide chains.Tryptophanyl-tRNA synthetase（WRS） belongs to ARS class I family.S.epidermidis WRS（SeWRS） and cytoplasmic human WRS（HWRS） share very low sequence similarity,which may be a potential drug target.In this work,we apply an approach combining structure-based discovery in silico with biochemical and biological experiments in vitro to screen S.epidermidis WRS inhibitors.A 3D structural model of SeWRS was constructed by using the homologous modelling approach using the B.stearothermophilus enzyme as the reference.SBVS resulted in the identification of 111 candidates as potential SeWRS inhibitors using SPECS chemical lead-compound database.Among them,three compounds were potent inhibitors in vitro,blocking the activity of SeWRS(IC₅₀ are between 15.1～42.2μM, K_d are between 3.76～13.9μM) as well as the growth of S.epidermidis（MIC values are between 6.25～100μM）,while showing low inhibition of the human tryptophanyl-tRNA synthetase and displaying low cytotoxicity to mammalian cells. These compounds are good leads to develop new antibiotics.Part 2.Study of the two component signal transduction system ArlRS of S.epidermidis,a potential anti-biofilm targetThe primary pathogenicity trait of S.epidermidis is associated with its ability to form biofilms on surfaces of medical devices,limiting severely the efficacy of many conventional antibiotics,and biofilms may also protect the bacteria against attacks from the host defence system.Thus,the discovery of anti-biofilm drugs is one of the strategies to combat with S.epidermidis infections.Biofilm formation is a very complicated process,which is composed of two steps: single bacterial cells attaches to the material surface;intercellular adhesion of the bacterial cells forms multiple layers structures.So far many genes are found to be involved in the biofilm formation,including several functional genes（atlE,ica,aap, bap,etc.） and some regulatory genes（sarA,sigB,agr,etc.）.Previous studies have revealed the potential role of some two-component signal transduction systems（TCSs） in regulating biofilm formation.So far,sixteen pairs of TCSs have been found in S.epidermidis,in which ArlRS attracts our attentions.In this part of the study,we investigate the molecular basis of the regulation of biofilm formation by ArlRS,which may contribute to the discovery of new anti-biofilm targets.Firstly,we constructed an arlS gene deletion mutant of the S.epidermidis strain 1457,which was named with WW06.Several methods,including PCR and RT-PCR, were used to confirm the arlS gene deletion and the successful construction of WW06.By western blot,we found that the translation of ArlR protein was deficient in the strain of WW06.Thus,what we got is an ArlRS deficient strain.The growth curve of WW06 was similar to that of SE1457;however,biofilms were no longer forms in the mutant.We further investigated the molecular mechanism of biofilm-forming ability loss in WW06,and found that the initial attachment ability remained in this mutant.By using the methods of DNA microarray and real-time RT-PCR,we found that the transcription of several genes involved in the second step of biofilm formation,such as icaADBC,sigB,sarA,etc,decreased in WW06.The biofilm forming phenotype can be restored by overexpressing icaADBC in WW06, but not by overexpressing sigB,indicating that ArlRS regulates biofilm formation through the regulation of icaADBC.ArlR can bind to the promoter region of ica operon,which was confirmed by electrophoretic mobility shift assay（EMSA）, indicating ArlRS directly regulates the transcription of icaADBC.We further investigated the relationship between ica transcription and arlRS transcription in the 9 ica⁺/BF^- S.epidermidis strains,and found that the transcription of ica operon in these nine strains was dramatically lower that in SE1457.In these S.epidermidis strains,seven displayed transcriptional loss of arlR,and two of them showed lower arlR transcription than SE1457.Based on these findings,we think two component signal transduction system ArlRS is an anti-biofilm target.Two strategies were used in the present study.Our findings will contribute to the development of anti-staphylococci drugs.更多还原