【作者】 秦智强；

【导师】 瞿涤； Soeren Molin； 周道国；

【作者基本信息】 复旦大学 ， 病原生物学， 2007， 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，原因是其能在这些医疗材料表面形成生物膜（biofilm，BF）样结构，该结构能够更好的保护细菌抵抗抗生素的治疗和人体免疫系统的攻击，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，尤其是那些能有效地杀伤生物膜包被细菌的抗生素。因此，本课题的目的在于较深入的研究表皮葡萄球菌临床株生物膜的动态形成过程，相关的分子机制并从中发现潜在的药物靶标，设计新型抗生物膜药物。目前缺乏观察表皮葡萄球菌生物膜结构的体外模型，96孔板结合结晶紫染色的方法虽然简便，但只能进行生物膜的定量分析，不能对生物膜的内部结构及细菌的生理状态进行详细的分析。Flow-chamber系统结合荧光染料染色和激光共聚焦显微镜技术已被成功运用于很多生物膜相关细菌的研究（如铜绿假单胞菌），其优点是能实时观察生物膜的形成过程、内部结构变化、各种成分分布及细菌的生理状态等。然而，该系统应用于表皮葡萄球菌生物膜的观察尚未见报道，且不同细菌形成生物膜要求的培养条件也不同，因此尚需建立一个合适的培养条件来观察表皮葡萄球菌生物膜的形成，并且这个培养条件应该适用于大部分表皮葡萄球菌株（包括临床分离株）。已有的研究发现表皮葡萄球菌的生物膜形成分为两个主要阶段：单个细菌初始黏附到材料表面和细菌间的互相黏附形成多细胞层的结构，并且已发现了参与这两个阶段的一些生物膜相关基因（如atlE、ica、aap等）。而作为表皮葡萄球菌生物膜主要成分的胞外多聚物质（extracellular polymeric substance，EPS），目前仅局限于细胞间多糖黏附素（Polysaccharide Intercellular Adhesion，PIA）的研究，它由ica操纵子编码的蛋白合成，主要介导了细菌间的互相黏附，但对EPS其它成分如胞外DNA（extracellular DNA）在生物膜形成中的作用尚不得而知。本论文在建立了flow-chamber和static-chamber观察表皮葡萄球菌生物膜的体外培养系统的基础上，对表皮葡萄球菌两类主要的临床分离株（ica^-／BF⁺和ica⁺／BF⁺）的生物膜三维结构的动态变化及对抗生素处理的耐受性进行了较全面的研究。对EPS的重要成分胞外DNA在表皮葡萄球菌生物膜形成过程中的作用及与胞外DNA释放相关的分子机制进行了较深入的研究。借助于蛋白结构模建和高通量虚拟筛选技术，以表皮葡萄球菌组氨酸激酶YycG的保守功能域为靶标，寻找潜在的小分子抑制物并对其生物学活性进行研究，在体外验证了其抗菌和抗生物膜活性，为开发新型的抗葡萄球菌感染的药物奠定理论和实践的基础。第一章表皮葡萄球菌生物膜三维结构体外观察模型的建立及临床株生物膜结构的研究为更好的观察表皮葡萄球菌形成生物膜的内部结构形态、成分分布及细菌状态等，我们建立了flow-chamber和static-chamber观察表皮葡萄球菌生物膜的体外培养系统。利用这些体外培养系统可以实时观察表皮葡萄球菌生物膜的形成过程，经各种不同荧光染料的染色后可以在激光共聚焦显微镜下清晰的观察到生物膜的形态结构及内部细菌的生理状态。通过反复比较确定了适用于各系统的培养条件，为以后的研究制定了统一的实验操作标准。利用上述两种体外培养系统，我们首先观察了两株ica阴性生物膜阳性（ica^-／BF⁺）的表皮葡萄球菌临床株SE1和SE4的生物膜形成情况，并将其与ica阳性生物膜阳性（ica⁺／BF⁺）的实验室标准株RP62A进行了比较。实验结果表明，无论在flow-chamber系统还是在static-chamber系统中SE1和SE4均表现出与RP62A在生物膜形态上的明显差异（甚至SE1和SE4之间也存在一定的差异），且PIA特异性染色再次证明SE1和SE4形成的生物膜中不含有PIA。SE1和SE4形成的生物膜能抵抗NaIO₄处理，但能被Proteinase K处理所破坏；而RP62A形成的生物膜对NaIO₄和Proteinase K的反应恰恰与之相反。虽然SE1和SE4形成的生物膜对万古霉素处理具有一定的抗性，但这种抵抗能力不如RP62A形成的生物膜。此外SE1和SE4浮游状态下生长的细菌与RP62A相比对溶葡萄球菌素和万古霉素的耐受性更强，这与SE1和SE4菌株下降的自溶性有关。这些结果说明ica^-／BF⁺临床株代表了一类在抗生素选择压力下逐渐进化的新亚型，但目前尚处于进化的早期阶段，其生物膜结构及保护作用正在不断完善中。我们同时利用flow-chamber系统对4株ica⁺／BF⁺表皮葡萄球菌临床株的生物膜形成进行了一个长时程的观察，结果发现这些临床株生物膜的一个显著的特征是具有很强的“自我更新”能力，具体表现为培养1天左右均能形成完整的生物膜结构且在微菌落（microcolony）的中心部分出现大量死细菌；培养2天左右这些部位的死细菌（也包括一小部分活细菌）从生物膜结构中脱落，形成空泡状结构；培养3～4天左右在脱落的部位通过残留细菌的分裂增殖又能够形成新的生物膜，且其中基本上不含有死细菌；培养5～6天左右在微菌落的中心部分再度出现大量死细菌，开始新一轮的循环。而这种在生物膜结构中出现的反复细菌死亡／脱落／再增殖现象与病人表现出的反复感染症状存在密切关联。第二章胞外DNA在表皮葡萄球菌生物膜形成中的作用胞外DNA作为生物膜基质的主要成分已被证明在很多细菌的生物膜形成中发挥着重要的作用，但其在表皮葡萄球菌生物膜中的存在及具体作用不得而知。DNA酶（DNaseⅠ）处理发现能抑制表皮葡萄球菌实验室标准株和临床株生物膜的形成，但成熟的生物膜可以抵抗这种作用，进一步的研究发现DNaseⅠ可以明显抑制表皮葡萄球菌的初始黏附（为表皮葡萄球菌形成生物膜的第一步），这些结果均说明胞外DNA参与表皮葡萄球菌生物膜形成且与细菌的初始黏附密切相关。在分子水平实验发现胞外DNA的释放与自溶素蛋白AtlE相关，因为atlE突变株胞外DNA的量、细菌初始黏附和生物膜形成能力与野生株相比均显著下降（分别下降约90％，95％和97％），而atlE突变回复株的这些能力又可以恢复到或接近野生株的水平。在flow-chamber和static-chamber系统中野生株和atlE突变回复株均能形成完整的生物膜结构（很多微菌落microcolony），而atlE突变株仅能形成一些很小的细菌团块其厚度远不如野生株的微菌落；DDAO染色表明在野生株和atlE突变回复株的生物膜结构中存在大量的胞外DNA，而atlE突变株的细菌团块中只存在很少量的胞外DNA，并且这种胞外DNA释放的明显下降与atlE突变株形成细菌团块几乎不含有死细菌密切相关，这是因为atlE突变株细菌的自溶性与野生株相比出现明显降低（诱导4小时后野生株的自溶率达到95％，而atlE突变株的自溶率只有约30％）。目前表皮葡萄球菌生物膜中AtlE介导的胞外DNA释放的调控机制尚不明确，但已有的结果说明胞外DNA同样在以表皮葡萄球菌为代表的革兰阳性细菌生物膜形成中发挥着重要的作用，虽然其释放的分子机制与革兰阴性菌如铜绿假单胞菌存在明显的不同。第三章表皮葡萄球菌组氨酸激酶YycG小分子抑制物的筛选及其活性的研究细菌的双组分信号转导系统广泛存在于革兰阳性和阴性细菌中，参与调控很多重要的生理功能，并且与很多病原菌的毒力和致病性密切相关，因而被认为是潜在的药物靶标。我们运用生物信息学分析在表皮葡萄球菌全基因组中共发现了16对双组分信号转导系统，功能分析预测发现大部分双组分信号转导系统参与调控细菌离子交换、外蛋白分泌、黏附和自溶等重要的生理功能，其中有两对双组分信号转导系统YycG／YycF和YhcS／YhcR调控细菌生长。以组氨酸激酶YycG的保守功能域HATPase_c为靶标，模建该功能域的三维结构并在此基础上运用高通量虚拟筛选技术共发现76个潜在的小分子抑制物（先导化合物），生物学实验表明其中有7个化合物（compound 1—7）能明显抑制表皮葡萄球菌的生长（MIC范围在0.2～100μM），它们中的5个（compound 1—5）还具有杀菌作用（MBC范围在25～200μM）。进一步的研究发现除了compound 6，其余6个化合物能在体外与靶标蛋白YycG的片断相互结合（结合平衡常数K_D范围在2.3～40.4），且能不同程度的抑制靶蛋白的磷酸化活性(半数抑制率浓度IC₅₀范围在6.5～48μM)，证实这6个化合物为组氨酸激酶YycG的小分子抑制物。此外，这些小分子抑制物在工作浓度对哺乳动物细胞（Vero细胞）无明显细胞毒性（MTT法），也不引起健康人红细胞的溶血，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。利用static-chamber系统我们观察了其中两个YycG小分子抑制物（compound2和compound 5）在MBC浓度对表皮葡萄球菌生物膜中细菌的杀伤作用，并以万古霉素作为对照。实验结果表明compound 2和compound 5对生物膜中的表皮葡萄球菌（包括实验室标准株和临床株）具有很好的杀伤效果，但二者的作用有所不同，compound 2对位于生物膜中微菌落底部的细菌具有较好的杀伤效果，而compound 5则对生物膜中的所有细菌均有明显的杀伤作用。与之相比，临床上常用于葡萄球菌耐药株感染的万古霉素不但对生物膜中的细菌无明显杀伤作用且有轻微刺激生物膜形成的作用，这在表皮葡萄球菌临床株上表现得更加明显。对表皮葡萄球菌浮游细菌的time-killing assay也显示compound 2和compound5具有比万古霉素更快更有效的杀伤效率。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 require an urgent need to design novel antibiotics against staphylococcus infections, especially in relation to biofilm development.The crystal violet staining represents a simple and rapid method to measure the biofilm formation ability of S. epidermidis strains in 96-well microtiter plates. However, based on this method it can not tell us the detailed structure of bacterial biofilms, nor the components distribution or bacterial physiological features in biofilms. The flow-chamber cultivation system combined with fluorescent staining and confocal laser scanning microscopy technology have been successfully used to study all kinds of features mentioned above in some bacterial biofilms （such as in Pseudomonas aeruginosa）. However, there is few data describing its application in S. epidermidis biofilm formation, so it needs to be found the appropriate cultivation conditions for S. epidermidis biofilm formation in this system.S. epidermidis biofilm formation has been described as a two-step process. The first stage involves attachment of cells to a surface （initial attachment phase）. The second stage includes cell-cell aggregation and the formation of a multilayered architecture （accumulative phase）. Much attention has been focused on the Polysaccharide Intercellular Adhesion （PIA） component of the extracellular polymeric substance （EPS） matrix of S. epidermidis, which is encoded by ica operon and considered as a major cell-to-cell interconnecting compound during biofilm formation. However, it is possible that other matrix components may be important for biofilm development of S. epidermidis, such as extracellular DNA which was shown to be important for biofilm formation of Pseudomonas aeruginosa and other bacteria （but remains unknown functions in S. epidermidis biofilm formation）.In the present work, two cultivation systems flow-chamber and static-chamber were established to study S. epidermidis biofilm development in vitro. Based on these systems, two types of clinical isolates （ica^-/BF⁺ and ica⁺/BF⁺） were investigated including their biofilm development process, structural components, resistance to antibiotics treatment etc. The role of extracellular DNA and the molecular mechanism of release were also further investigated in S. epidermidis biofilm. At last, a series of novel inhibitors of the histidine kinase YycG protein of S. epidermidis were discovered first using structure-based virtual screening from a small molecular leading-compound library, followed by experimental validation of their antibacterial activities.ChapterⅠEstablishment of S. epidermidis biofilm cultivation systems and investigation of different S. epidermidis clinical isolates biofilm developmentTwo cultivation systems flow-chamber and static-chamber were established to study the features of S. epidermidis biofilms in vitro （such as dynamic structural development, Live/Dead cells distribution）. Based on these systems, several S. epidermidis clinical isolates were chosen to be investigated. First, two ica^-/BF⁺ S. epidermidis clinical strains SE1 and SE4 exhibit their heterogeneity in biofilm architecture under static and flow conditions, compared with the ica⁺/BF⁺ RP62A strain. This kind of biofilms absence of PIA displays resistant to NaIO₄ but not Proteinase K, whereas the biofilm formed by RP62A can be disrupted by NaIO₄ but resistant to Proteinase K. More importantly, the cells of both SE1 and SE4 stains show more tolerant than those of RP62A after exposure to lysostaphin and vancomycin, which is associated with the decreased autolysis ability of SE1 and SE4 cells. Based on these results, it suggests that the ica^-/BF⁺ strains may represent a newly emergent subpopulation of S. epidermidis clinical strains by the selection of antibiotics in the nosocomial milieu, which displays survival advantage in host environment.The long-term biofilm development process of 4 ica⁺/BF⁺ S. epidermidis clinical isolates （SE698, SE847, SE886 and SEG203-2） and the laboratory standard strain RP62A （ica⁺/BF⁺） were recorded in the flow-chamber system. The common feature of biofilms formed by these 5 strains is the rebirth ability shown by the cells embedded in biofilm microcolonies: At the 1st day, there are lots of dead cells in the center of microcolony structure especially in those clinical isolates; at the 2nd day, most of dead cells detach from the biofim structure, leaving the vacuolar architecture in microcolonies; at the 3rd/4th day, the residual cells proliferate and occupy the vacuolar architecture, forming a "renew" biofilm in which few dead cells are present; at the 5th/6th day, reappearance of many dead cells in the center of microcolonies. This kind of "rebirth" phenomenon is associated with the repeated infection occurred in patients, which greatly increases the risk of bacteremia.ChapterⅡThe role of extracellular DNA in S. epidermidis biofilm formation and associated mechanism of extracellular DNA releaseAs a component of extracellular polymeric substance （EPS） matrix, extracellular DNA has been shown to be important for biofilm formation of Pseudomonas aeruginosa and other bacteria, but in S. epidermidis its role remains unknown. In this work, DNaseⅠtreatment is found to inhibit S. epidermidis initial attachment to surface and subsequent biofilm formation, but resisted by the established biofilms. The results indicate that extracellular DNA is a major component required for bacterial initial attachment to surfaces, as well as for the subsequent early phase of biofilm development by S. epidermidis. Moreover, evidence is presented that release of extracellular DNA from S. epidermidis mainly is caused by the activity of the autolysin AtlE. For instance, the atlE mutant strain displays dramatically decreased extracellular DNA release （～90%）, initial attachment （～95%） and biofilm formation （97%）, compared with the wild type （wt） strain, whereas the atlE complementary strain can recover all the features to the level of wt strain. In both flow and static chamber systems, the wt and atlE complementary strains form intact biofilm structure （microcolonies）, in which abundant extracellular DNA （shown by DDAO staining） is present. In contrast, the atlE mutant strain only forms a few small cell-clumpings with much less extracellular DNA than its parental strain. In addition, almost no dead cells （shown by PI staining） are observed in cell-clumpings formed by the atlE mutant strain, which is in accord with the decreased autolysis in the atlE mutant cells.ChapterⅢStructure-based discovery of inhibitors of the YycG histidine kinase: New leading-compounds to combat Staphylococcus epidermidis infectionsMany two-component signal transduction systems （TCSTSs） are ubiquitous in bacteria and are integral components of the adaptive regulatory processes utilized by pathogenic bacteria to sense the environment and coordinate the expression of the genes encoding virulence factors, making them attractive targets for antimicrobial therapy. Based on bioinformatics analysis, 16 pairs of TCSTSs are found in the whole genome of S. epidermidis, most of which regulate multiple functions such as exoprotein production, adhesion and autolysis. Among these TCSTSs, two pairs （YycG/YycF and YhcS/YhcR） are required for bacterial growth. Subsequently, a series of novel inhibitors of the histidine kinase YycG protein of S. epidermidis were discovered first using structure-based virtual screening （SBVS） from a small molecular leading-compound library, followed by experimental validation. Of the 76 candidates derived by SBVS targeting of the homolog model of the YycG HATPase_c domain of S. epidermidis, seven compounds （compound 1-7） displayed significant activity in inhibiting S. epidermidis growth （MIC values are 0.2～100μM）. Furthermore, five of them （compound 1-5） displayed bactericidal effects on S. epidermidis cells （MBC values are 25～200μM）. Except for one （compound 6）, the other six compounds were found to bind to the recombinant YycG protein （K_D values are 2.3～40.4） and to inhibit its auto-phosphorylation (IC₅₀ values are 6.5～48μM) in vitro, indicating that they are potential inhibitors of the YycG/YyeF TCSTS, which is essential in S. epidermidis. Importantly, all these compounds did not affect the stability of mammalian cells nor hemolytic activities at the concentrations used in our study, indicating they are of potential value for developing new antibiotics against infecting staphylococci. Based on a static-chamber system, we have assessed the bactericidal effect of two leading-compounds active as YycG inhibitors （compound 2 and 5） on biofilm cells of S. epidermidis laboratory and clinical strains by confocal laser scanning microscopy （CLSM） combined with viability staining. In young biofilms （6-h-old）, the two compounds killed the majority of the embedded cells at concentrations of 100μM and 25μM, respectively （MBC values）. In mature biofilms （24-h-old）, compound 5 was still effectively killing biofilm cells, whereas compound 2 mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC （8μg/mL）, especially to those clinical isolates. Even at a high concentration （128μg/mL）, vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure （6 hours）. The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.更多还原