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。
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 target
Among 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(IC_(50) 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 target
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.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.
引文
1.Rupp ME,Archer GL.Coagulase-negative staphylococci:pathogens associated with medical progress.Clin Infect Dis.1994;19:231-243;quiz 244-235.
2.Thylefors JD,Harbarth S,Pittet D.Increasing bacteremia due to coagulase negative staphylococci:fiction or reality? Infect Control Hosp Epidemiol.1998;19:581-589.
3.Jarlov JO.Phenotypic characteristics of coagulase-negative staphylococci:typing and antibiotic susceptibility.APMIS.1999;Supple 91:1-42.
4.Sanyal D,Johnson AP,George RC et al.Peritonitis due to vancomycin-resistant Staphylococcus epidermidis.Lancet.1991;337:54.
5.Zhang YQ,Ren SX,Li HL et al.Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain(ATCC 12228).Mol Microbiol.2003;49:1577-1593.
6.Gill SR,Fouts DE,Archer GL et al.Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain.J Bacteriol.2005;187:2426-2438.
7.Kim S,Lee SW,Choi EC et al.Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics.Applied microbiology and biotechnology 2003;61:278-88.
8.Casewell MW,Hill RL.In-vitro activity of mupirocin('pseudomonic acid') against clinical isolates of Staphylococcus aureus.The Journal of antimicrobial chemotherapy 1985;15:523-31.
9.Ochsner UA,Young CL,Stone KC et al.Mode of action and biochemical characterization of REP8839,a novel inhibitor of methionyl-tRNA synthetase.Antimicrobial agents and chemotherapy 2005;49:4253-62.
10.Cochereau C,Sanchez D,Bourhaoui A et al.Capsaicin,a structural analog of tyrosine,inhibits the aminoacylation of tRNA(Tyr).Toxicology and applied pharmacology 1996;141:133-7.
11.Anderson JW,Fewden L.1-amino-2-phenylethane 1-phosphonic acid:a specific competitive inhibitor of phenylalanyl-tRNA synthetase.Chemico-biological interactions 1970;2:53-5.
12.Balg C,Blais SP,Bernier S et al.Synthesis of beta-ketophosphonate analogs of glutamyl and glutaminyl adenylate,and selective inhibition of the corresponding bacterial aminoacyl-tRNA synthetases.Bioorganic & medicinal chemistry 2007;15:295-304.
13.Metlitskaya A,Kazakov T,Komrner A et al.Aspartyl-tRNA synthetase is the target of peptide nucleotide antibiotic Microcin C.J Biol Chem 2006;281:18033-42.
14.Hurdle JG,O'Neill AJ,Chopra I.Anti-staphylococcal activity of indolmycin,a potential topical agent for control of staphylococcal infections.The Journal of antimicrobial chemotherapy 2004;54:549-52.
15.Brown MJ,Carter PS,Fenwick AS et al.The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase.Bioorganic & medicinal chemistry letters 2002;12:3171-4.
16.Stephenson K,Hoch J A.Two-component and phosphorelay signal-transduction systems as therapeutic targets.Curr Opin Pharmacol.2002;2:507-512.
17.Zhiqiang Qin,Yang Zhong et al.Bioinformatics analysis of two-component regulatory systems in Staphylococcus epidermidis.Chinese Science Bulletin,2004,49(12):1267-1271.
18.Qin Z,Zhang J,Xu B et al.Structure-based discovery of inhibitors of the YycG histidine kinase:new chemical leads to combat Staphylococcus epidermidis infections.BMC microbiology 2006;6:96
19.陈明亮,学位论文《志贺菌双组分信号转导系统PhoQ/PhoP和EnvZ/OmpR作为药靶的研究》
20.Raad,Ⅱ,Bodey GP.Infectious complications of indwelling vascular catheters.Clin Infect Dis 1992;15:197-208.
21.Hiramatsu K.Vancomycin-resistant Staphylococcus aureus:a new model of antibiotic resistance. Lancet Infect Dis. 2001 ;1:147-155.
22. Yu Y, Liu Y, Shen N et al. Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment: insights into substrate recognition, tRNA binding, and angiogenesis activity. The Journal of biological chemistry 2004; 279: 8378-88.
23.Paley EL, Smelyanski L, Malinovskii V et al. Mapping and molecular characterization of novel monoclonal antibodies to conformational epitopes on NH_2 and COOH termini of mammalian tryptophanyl-tRNA synthetase reveal link of the epitopes to aggregation and Alzheimer's disease. Molecular immunology 2007; 44: 541-57.
24. Otani A, Slike BM, Dorrell MI et al. A fragment of human TrpRS as a potent antagonist of ocular angiogenesis. Proceedings of the National Academy of Sciences of the United States of America 2002; 99: 178-83.
25. Ibba M. Eyeing up tryptophanyl-tRNA synthetase. Trends in biochemical sciences 2002; 27: 227.
26. Werner RG, Thorpe LF, Reuter W et al. Indolmycin inhibits prokaryotic tryptophanyl-tRNA ligase. European journal of biochemistry / FEBS 1976; 68: 1-3.
27. Kuntz ID. Structure-based strategies for drug design and discovery. Science 1992; 257: 1078-82.
28. Kuroda M, Ohta T, Uchiyama I et al. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet. 2001 ;357:1225-1240.
29. Holden MT, Feil EJ, Lindsay JA et al. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad Sci U S A. 2004; 101:9786-9791.
30. Baba T, Takeuchi F, Kuroda M et al. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet. 2002;359:1819-1827.
31. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673-4680.
32. Kumar S, Tamura K, Jakobsen IB et al. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics. 2001; 17:1244-1245.
33. Shen J, Xu X, Cheng F et al. Virtual screening on natural products for discovering active compounds and target information. Curr Med Chem. 2003; 10:2327-2342.
34. Abagyan R, Totrov M. High-throughput docking for lead generation. Curr Opin Chem Biol.2001,5:375-382.
35. Carter CW, Jr., Crumley KV, Coleman DE et al. Direct phase determination for the molecular envelope of tryptophanyl-tRNA synthetase from Bacillus stearothermophilus by X-ray contrast variation. Acta crystallographica 1990; 46: 57-68.
36. InsightII [molecular modeling package] Version 2000 Calif (USA): Molecular Simulations Inc.
37. Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234:779-815.
38. Laskowski RA, MacArthur MW, Moss DS et al. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst. 1993;26:283- 291.
39. Luthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992;356:83-85.
40. Zheng S, Luo X, Chen G et al. A new rapid and effective chemistry space filter in recognizing a druglike database. J Chem Inf Model. 2005;45:856-862.
41. Ewing TJ, Makino S, Skillman AG et al. DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. J Comput Aided Mol Des. 2001 ;15:411-428.
42. Kuntz ID. Structure-based strategies for drug design and discovery. Science. 1992; 257:1078-1082.
43. Sybyl [molecular modeling package] 2000 Version 6.8. St Louis (MO): Tripos Associates.
44. Rarey M, Kramer B, Lengauer T et al. A fast flexible docking method using an incremental construction algorithm. J Mol Biol. 1996;261:470-489.
45. Xu F, Chen X, Xin L et al. Species-specific differences in the operational RNA code for aminoacylation of tRNA(Trp). Nucleic acids research 2001; 29: 4125-33.
46. Clinical and Laboratory Standards Institute: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. In Approved standard M7-A7 Clinical and Laboratory Standards Institute, Villanova, Pa; 2006.
47. Gyo¨ngyi Gunics et al. Interaction between antibiotics and non-conventional antibiotics on bacteria. International Journal of Antimicrobial Agents 2000; 14: 239-242
48. Berna Karakoc, A. Alev Gerceker. In-vitro activities of various antibiotics, alone and in combination with amikacin against Pseudomonas aeruginosa. International Journal of Antimicrobial Agents 2001; 18: 567-570
49. Li M, Guan M, Jiang XF et al. Genetic polymorphism of the accessory gene regulator (agr) locus in Staphylococcus epidermidis and its association with pathogenicity. J Med Microbiol 2004; 53: 545-549.
50. Vuong C, Saenz HL, Gotz F, Otto M. Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis 2000; 182:1688-1693.
51. Fournier B, Klier A, Rapoport G. The two-component system ArlS-ArlR is a regulator of virulence gene expression in Staphylococcus aureus. Mol Microbiol 2001;41:247-261.
52. Reinhold B. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiology Letter 1997; 151:1 -8
53. Maryvonne A, Arnaud C, Michel D. New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria. Applied and Environmental Microbiology 2004;70:6887-6891
54. Staib P, Kretschmar M, Nichterlein T et al. Host versus in vitro signals and intrastrain allelic diferences in the expression of a Candida albicans virulence gene. Mol Microbiol 2002, 44: 1351 -66.
55. Brunskill EW, Bayles KW. Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus. J Bacteriol 1996; 178:611-618.
56. Alejandro TA et al. Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system. Journal of Bacteriology 2005; 187:5318-5329.
57. Vandecasteele SJ, Peetermans WE, Merckx R et al. Expression of biofilm-associated genes in Staphylococcus epidermidis during in vitro and in vivo foreign body infections. J Infect Dis. 2003, 188(5):730-737.
58. Klug D, Wallet F, Kacet S et al. Involvement of adherence and adhesion Staphylococcus epidermidis genes in pacemaker lead-associated infections. J Clin Microbiol. 2003, 41(7):3348-3350.
59. Vandecasteele SJ, Peetermans WE, R Merckx R et al. Reliability of the ica, aap and atlE genes in the discrimination between invasive, colonizing and contaminant Staphylococcus epidermidis isolates in the diagnosis of catheter-related infections. Clin Microbiol Infect. 2003, 9(2): 114-119.
60. Rupp ME, Fey PD, Heilmann C et al. Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model. J Infect Dis. 2001,183(7): 1038-1042.
61. Heilmann C, Hussain M, Peters G et al. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol. 1997,24(5):1013-1024.
62. Qin Z, Ou Y, Yang L et al. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology 2007; 153:2083-2092.
63. Vuong C, Gerke C, Somerville GA et al. Quorum-sensing control of biofilm factors in Staphylococcus epidermidis. J Infect Dis. 2003 ,188(5):706-718.
64. Arciola CR, Baldassarri L, Montanaro L. Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections. J Clin Microbiol. 2001, 39(6): 2151-2156.
65. Arciola CR, Baldassarri L, Montanaro L. In catheter infections by Staphylococcus epidermidis the intercellular adhesion (ica) locus is a molecular marker of the virulent slime-producing strains J Biomed Mater Res. 2002, 59(3):557-562.
66. Frebourg NB, Lefebvre S, Baert S, et al. PCR-Based assay for discrimination between invasive and contaminating Staphylococcus epidermidis strains. J Clin Microbiol. 2000, 38(2):877-880.
67. Arciola CR, Collamati S, Donati E, Montanaro L. A rapid PCR method for the detection of slime-producing strains of Staphylococcus epidermidis and S. aureus in periprosthesis infections. Diagn Mol Pathol. 2001,10(2):130-137.
68. Arciola CR, Campoccia D, Gamberini S et al. Occurrence of ica genes for slime synthesis in a collection of Staphylococcus epidermidis strains from orthopedic prosthesis infections. Acta Orthop Scand. 2003, 74(5):617-621.
69. Heilmann C, Schweitzer O, Gerke C et al. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol Microbiol. 1996, 20(5):1083-1091.
70. McKenney D, Hubner J, Muller E et al. The ica locus of Staphylococcus epidermidis encodes production of the capsular polysaccharide/adhesin. Infect Immun. 1998, 66(10):4711-4720.
71. Gotz F. Staphylococcus and biofilms. Mol Microbiol. 2002, 43(6): 1367-1378. Review.
72. Kies S, Otto M, Vuong C et al. Identification of the sigB operon in Staphylococcus epidermidis: construction and characterization of a sigB deletion mutant. Infect Immun. 2001, 69(12):7933-7936.
73. Rachid S, Cho S, Ohlsen K et al. Induction of Staphylococcus epidermidis biofilm formation by environmental factors: the possible involvement of the alternative transcription factor sigB. Adv Exp Med Biol. 2000,485:159-166.
74. Knobloch JK, Jager S, Horstkotte MA et al. RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR. Infect Immun. 2004, 72(7):3838-3848.
75.Tormo MA,Marti M,Valle J et al.SarA is an essential positive regulator of Staphylococcus epidermidis biofilm development.J Bacteriol.2005,187(7):2348-2356.
76.Conlon KM,Humphreys H,O'Gara JP.Inactivations of rsbU and sarA by IS256represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis.J Bacteriol.2004,186(18):6208-6219.
77.Knobloch JK,Jager S,Horstkotte MA et al.RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR.Infect Immun.2004 Jul,72(7):3838-3848.
78.Fluckiger U,Wolz C,Cheung AL.Characterization of a sar homolog of Staphylococcus epiderrnidis.Infect Immun.1998,66(6):2871-2878.
79.江娟,孙景勇,欧元祝等。医源性表皮葡萄球菌ica操纵子转录水平对生物膜表型的影响。上海医学。2006;29:40-44。 non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Mol Microbiol, 49(6), 1577-1593 (2003).
9. Gill SR, Fouts DE, Archer GL et al. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol, 187(7), 2426-2438 (2005).
10. Mack D, Haeder M, Siemssen N, Laufs R. Association of biofilm production of coagulase-negative staphylococci with expression of a specific polysaccharide intercellular adhesin. J Infect Dis, 174(4), 881-884 (1996).
11. Gerke C, Kraft A, Sussmuth R, Schweitzer O, Gotz F. Characterization of the N-acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesin. J Biol Chem, 273(29), 18586-18593 (1998).
12. Vuong C, Kocianova S, Voyich JM et al. A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J Biol Chem, 279(52), 54881-54886(2004).
13. Cordon KM, Humphreys H, O'Gara JP. icaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis. J Bacteriol, 184(16), 4400-4408 (2002).
14. Chokr A, Watier D, Eleaume H et al. Correlation between biofilm formation and production of polysaccharide intercellular adhesin in clinical isolates of coagulase-negative staphylococci. Int J Med Microbiol, 296(6), 381-388 (2006).
15. Qin Z, Yang X, Yang L et al. Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates. J Med Microbiol, 56(Pt 1), 83-93 (2007).
16. Sadovskaya I, Vinogradov E, Flahaut S, Kogan G, Jabbouri S. Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A. Infect Immun, 73(5), 3007-3017 (2005).
17. Ziebuhr W, Krimmer V, Rachid S, Lossner I, Gotz F, Hacker J. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol, 32(2), 345-356 (1999).
18. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science, 295(5559), 1487 (2002).
19. Moscoso M, Garcia E, Lopez R. Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. J Bacteriol, 188(22), 7785-7795 (2006).
20. Yang L, Barken KB, Skindersoe ME, Christensen AB, Givskov M, Tolker-Nielsen T. Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa. Microbiology, 153(Pt 5), 1318-1328 (2007).
21. Petersen FC, Tao L, Scheie AA. DNA binding-uptake system: a link between cell-to-cell communication and biofilm formation. JBacteriol, 187(13), 4392-4400 (2005).
22. Rice KC, Mann EE, Endres JL et al. The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc Natl Acad Sci U S A, 104(19), 8113-8118(2007).
23. Qin Z, Ou Y, Yang L et al. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis.Microbiology,153(Pt 7),2083-2092(2007).
24.Allesen-Holm M,Barken KB,Yang L et al.A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms.Mol Microbiol,59(4),1114-1128(2006).
25.Vuong C,Gerke C,Somerville GA,Fischer ER,Otto M.Quorum-sensing control of biofilm factors in Staphylococcus epidermidis.J Infect Dis,188(5),706-718(2003).
26.Schneewind O,Fowler A,Faull KF.Structure of the cell wall anchor of surface proteins in Staphylococcus aureus.Science,268(5207),103-106(1995).
27.Bowden MG,Chen W,Singvall J et al.Identification and preliminary characterization of cell-wall-anchored proteins of Staphylococcus epidermidis.Microbiology,151(Pt 5),1453-1464(2005).
28.Hussain M,Herrmann M,von Eiff C,Perdreau-Remington F,Peters G.A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces.Infect Immun,65(2),519-524(1997).
29.Rohde H,Burdelski C,Bartscht K et al.Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases.Mol Microbiol,55(6),1883-1895(2005).
30.Yang XM,Li N,Chen JM et al.Comparative proteomic analysis between the invasive and commensal strains of Staphylococcus epidermidis.FEMS Microbiol Lett,261(1),32-40(2006).
31.Banner MA,Cunniffe JG,Macintosh RL et al.Localized tufts of fibrils on Staphylococcus epidermidis NCTC 11047 are comprised of the accumulation-associated protein.J Bacteriol,189(7),2793-2804(2007).
32.Tormo MA,Knecht E,Gotz F,Lasa I,Penades JR.Bap-dependent biofilm formation by pathogenic species of Staphylococcus:evidence of horizontal gene transfer? Microbiology,151(Pt 7),2465-2475(2005).
33.Hussain M,Heilmann C,Peters G,Herrmann M.Teichoic acid enhances adhesion of Staphylococcus epidermidis to immobilized fibronectin.Microb Pathog,31(6),261-270(2001).
34.Biswas R,Voggu L,Simon UK,Hentschel P,Thumm G,Gotz F.Activity of the major staphylococcal autolysin Atl.FEMS Microbiol Lett,259(2),260-268(2006).
35.Heilmann C,Hussain M,Peters G,Gotz F.Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface.Mol Microbiol,24(5),1013-1024(1997).
36.Rupp ME,Fey PD,Heilmann C,Gotz F.Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model.J Infect Dis,183(7),1038-1042(2001).
37.Lemos JA,Burne RA.Regulation and Physiological Significance of ClpC and ClpP in Streptococcus mutans.J Bacteriol,184(22),6357-6366(2002).
38.O'Toole GA,Kolter R.Initiation of biofilm formation in Pseudomonas fluorescens WCS365proceeds via multiple,convergent signalling pathways:a genetic analysis.Mol Microbiol,28(3),449-461(1998).
39.Frees D,Chastanet A,Qazi Set al.Clp ATPases are required for stress tolerance,intracellular replication and biofilm formation in Staphylococcus aureus.Mol Microbiol,54(5),1445-1462 (2004).
40. Shanks RM, Caiazza NC, Hinsa SM, Toutain CM, O'Toole GA. Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria. Appl Environ Microbiol, 72(7), 5027-5036 (2006).
41. Wang C, Li M, Dong D et al. Role of ClpP in biofilm formation and virulence of Staphylococcus epidermidis. Microbes Infect, 9(11), 1376-1383 (2007).
42. Fuqua WC, Winans SC, Greenberg EP. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol, 176(2), 269-275 (1994).
43. Novick RP, Projan SJ, Kornblum J et al. The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. Mol Gen Genet, 248(4), 446-458 (1995).
44. Ji G, Beavis R, Novick RP. Bacterial interference caused by autoinducing peptide variants. Science, 276(5321), 2027-2030 (1997).
45. Lina G, Jarraud S, Ji G et al. Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus. Mol Microbiol, 28(3), 655-662 (1998).
46. Koenig RL, Ray JL, Maleki SJ, Smeltzer MS, Hurlburt BK. Staphylococcus aureus AgrA binding to the RNAIII-agr regulatory region. J Bacteriol, 186(22), 7549-7555 (2004).
47. Li M, Guan M, Jiang XF et al. Genetic polymorphism of the accessory gene regulator (agr) locus in Staphylococcus epidermidis and its association with pathogenicity. J Med Microbiol, 53(Pt 6), 545-549 (2004).
48. Vuong C, Saenz HL, Gotz F, Otto M. Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis, 182(6), 1688-1693 (2000).
49. Vuong C, Durr M, Carmody AB, Peschel A, Klebanoff SJ, Otto M. Regulated expression of pathogen-associated molecular pattern molecules in Staphylococcus epidermidis: quorum-sensing determines pro-inflammatory capacity and production of phenol-soluble modulins. Cell Microbiol, 6(8), 753-759 (2004).
50. Yao Y, Sturdevant DE, Otto M. Genomewide analysis of gene expression in Staphylococcus epidermidis biofilms: insights into the pathophysiology of S. epidermidis biofilms and the role of phenol-soluble modulins in formation of biofilms. J Infect Dis, 191(2), 289-298 (2005).
51. Xu L, Li H, Vuong C et al. Role of the luxS quorum-sensing system in biofilm formation and virulence of Staphylococcus epidermidis. Infect Immun, 74(1), 488-496 (2006).
52. Rachid S, Cho S, Ohlsen K, Hacker J, Ziebuhr W. Induction of Staphylococcus epidermidis biofilm formation by environmental factors: the possible involvement of the alternative transcription factor sigB. Adv Exp Med Biol, 485, 159-166 (2000).
53. Knobloch JK, Bartscht K, Sabottke A, Rohde H, Feucht HH, Mack D. Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an activator of the sigB operon: differential activation mechanisms due to ethanol and salt stress. J Bacteriol, 183(8), 2624-2633 (2001).
54. Knobloch JK, Jager S, Horstkotte MA, Rohde H, Mack D. RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR. Infect Immun, 72(7), 3838-3848 (2004).
55. Tormo MA, Marti M, Valle J et al. SarA is an essential positive regulator of Staphylococcus epidermidis biofilm development. J Bacteriol, 187(7), 2348-2356 (2005).
56. Trotonda MP, Manna AC, Cheung AL, Lasa I, Penades JR. SarA positively controls bap-dependent biofilm formation in Staphylococcus aureus. J Bacteriol, 187(16), 5790-5798 (2005).
57. Hoch JA. Two-component and phosphorelay signal transduction. Curr Opin Microbiol, 3(2), 165-170(2000).
58. Darouiche RO. Treatment of infections associated with surgical implants. N Engl J Med, 350(14), 1422-1429 (2004).
59. Patel JD, Ebert M, Ward R, Anderson JM. S. epidermidis biofilm formation: effects of biomaterial surface chemistry and serum proteins. J Biomed Mater Res A, 80(3), 742-751 (2007).
60. Shah A, Mond J, Walsh S. Lysostaphin-coated catheters eradicate Staphylococccus aureus challenge and block surface colonization. Antimicrob Agents Chemother, 48(7), 2704-2707 (2004).
61. Kaplan JB, Ragunath C, Velliyagounder K, Fine DH, Ramasubbu N. Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother, 48(7), 2633-2636 (2004).
62. Niemela SM, Lansman S, Ikaheimo I et al. Self-reinforced ciprofloxacin-releasing polylactide-co-glycolide 80/20 inhibits attachment and biofilm formation by Staphylococcus epidermidis: an in vitro study. J Craniofac Surg, 17(5), 950-956 (2006).
63. Luo J, Chen Z, Sun Y. Controlling biofilm formation with an N-halamine-based polymeric additive. J Biomed Mater Res A, 77(4), 823-831 (2006).
64. Cheng G, Zhang Z, Chen S, Bryers JD, Jiang S. Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces. Biomaterials, 28(29), 4192-4199 (2007).
65. Huang XD, Yao K, Zhang H, Huang XJ, Xu ZK. Surface modification of silicone intraocular lens by 2-methacryloyloxyethyl phosphoryl-choline binding to reduce Staphylococcus epidermidis adherence. Clin Experiment Ophthalmol, 35(5), 462-467 (2007).
66. Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc NatlAcadSci USA, 87(23), 9188-9192 (1990).
67. Cerca N, Jefferson KK, Oliveira R, Pier GB, Azeredo J. Comparative antibody-mediated phagocytosis of Staphylococcus epidermidis cells grown in a biofilm or in the planktonic state. Infect Immun, 74(8), 4849-4855 (2006).
68. Schlag S, Nerz C, Birkenstock TA, Altenberend F, Gotz F. Inhibition of staphylococcal biofilm formation by nitrite. J Bacteriol, (2007).
69. Sun D, Accavitti MA, Bryers JD. Inhibition of biofilm formation by monoclonal antibodies against Staphylococcus epidermidis RP62A accumulation-associated protein. Clin Diagn Lab Immunol, 12(1), 93-100 (2005).
70. Gov Y, Bitler A, Dell'Acqua G, Torres JV, Balaban N. RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and function analysis. Peptides, 22(10), 1609-1620 (2001).
71. Balaban N, Stoodley P, Fux CA, Wilson S, Costerton JW, Dell'Acqua G. Prevention of staphylococcal biofilm-associated infections by the quorum sensing inhibitor RIP. Clin Orthop Relat Res, (437), 48-54 (2005).
72. Balaban N, Giacometti A, Cirioni O et al. Use of the quorum-sensing inhibitor RNAIII-inhibiting peptide to prevent biofilm formation in vivo by drug-resistant Staphylococcus epidermidis.J Infect Dis,187(4),625-630(2003).
73.Xavier JB,Picioreanu C,Rani SA,van Loosdrecht MC,Stewart PS.Biofilm-control strategies based on enzymic disruption of the extracellular polymeric substance matrix—a modelling study.Microbiology,151(Pt 12),3817-3832(2005).
74.Boyd A,Chakrabarty AM.Pseudomonas aeruginosa biofilms:role of the alginate exopolysaccharide.J Ind Microbiol,15(3),162-168(1995).
75.Hughes KA,Sutherland IW,Clark J,Jones MV.Bacteriophage and associated polysaccharide depolymerases—novel tools for study of bacterial biofilms.J Appl Microbiol,85(3),583-590(1998).
76.Kaplan JB,Velliyagounder K,Ragunath C et al.Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms.J Bacteriol,186(24),8213-8220(2004).
77.Chaignon P,Sadovskaya I,Ragunah C,Ramasubbu N,Kaplan JB,Jabbouri S.Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition.Appl Microbiol Biotechnol,75(1),125-132(2007).
78.Yakandawala N,Gawande PV,Lovetri K,Madhyastha S.Effect of ovotransferrin,protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria.J Appl Microbiol,102(3),722-727(2007).
79.Thormann KM,Saville RM,Shukla S,Spormann AM.Induction of rapid detachment in Shewanella oneidensis MR-1 biofilms.J Bacteriol,187(3),1014-1021(2005).
80.Martineau L,Dosch HM.Biofilm reduction by a new burn gel that targets nociception.J Appl Microbiol,103(2),297-304(2007).
81.LaPlante KL,Mermel LA.In vitro activity of daptomycin and vancomycin lock solutions on staphylococcal biofilms in a central venous catheter model.Nephrol Dial Transplant,22(8),2239-2246(2007).
82.Rodriguez-Martinez JM,Ballesta S,Garcia I,Conejo MC,Pascual A.[Activity and penetration of linezolid and vancomycin against Staphylococcus epidermidis biofilms].Enferm Infecc Microbiol Clin,25(7),425-428(2007).
83.Gualtieri M,Bastide L,Villain-Guillot P,Michaux-Charachon S,Latouche J,Leonetti JP.In vitro activity of a new antibacterial rhodanine derivative against Staphylococcus epidermidis biofilms.J Antimicrob Chemother,58(4),778-783(2006).
84.Villain-Guillot P,Gualtieri M,Bastide L et al.Structure-activity relationships of phenyl-furanyl-rhodanines as inhibitors of RNA polymerase with antibacterial activity on biofilms.J Med Chem,50(17),4195-4204(2007).
85.Qin Z,Lee B,Yang L et al.Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms.FEMS Microbiol Lett,273(2),149-156(2007).
86.Frank KL,Patel R.Activity of sodium metabisulfite against planktonic and biofilm Staphylococcus species.Diagn Microbiol Infect Dis,57(4),355-359(2007).
87.Nostro A,Roccaro AS,Bisignano G et al.Effects of oregano,carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms.J Med Microbiol,56(Pt 4),519-523(2007).
88.Presterl E,Suchomel M,Eder M et aL Effects of alcohols,povidone-iodine and hydrogen peroxide on biofilms of Staphylococcus epidermidis.J Antimicrob Chemother,60(2),417-420(2007).
89. Wood HL, Holden SR, Bayston R. Susceptibility of Staphylococcus epidermidis biofilm in CSF shunts to bacteriophage attack. Eur J Pediatr Surg, 11 Suppl 1, S56-57 (2001).
90. Cerca N, Oliveira R, Azeredo J. Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K. Lett Appl Microbiol, 45(3), 313-317(2007).
91. Curtin JJ, Donlan RM. Using bacteriophages to reduce formation of catheter-associated biofilms by Staphylococcus epidermidis. Antimicrob Agents Chemother, 50(4), 1268-1275 (2006).
92. Rabinovitch C, Stewart PS. Removal and inactivation of Staphylococcus epidermidis biofilms by electrolysis. Appl Environ Microbiol, 72(9), 6364-6366 (2006).
93. Kamgang JO, Briandet R, Herry JM, Brisset JL, Naitali M. Destruction of planktonic, adherent and biofilm cells of Staphylococcus epidermidis using a gliding discharge in humid air. J Appl Microbiol, 103(3), 621-628 (2007).
94. Donelli G, Francolini I, Romoli D et al. Synergistic activity of dispersin B and cefamandole nafate in inhibition of staphylococcal biofilm growth on polyurethanes. Antimicrob Agents Chemother, 51(8), 2733-2740 (2007).
95. Aslam S, Trautner BW, Ramanathan V, Darouiche RO. Combination of tigecycline and N-acetylcysteine reduces biofilm-embedded bacteria on vascular catheters. Antimicrob Agents Chemother, 51(4), 1556-1558 (2007).
96. Lu TK, Collins JJ. Dispersing biofilms with engineered enzymatic bacteriophage. Proc Natl Acad Sci U S A, 104(27), 11197-11202 (2007).