【作者】 李学军；

【导师】 杨和平；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 金黄色葡萄球菌(Staphylococcus aureus)是目前下呼吸道感染常见的革兰阳性球菌,其致病机制与其能分泌多种胞外酶和外毒素有关,它可以引起多个器官组织感染,并有较高的发病率和死亡率[1,2]。近年来,由于其感染率的上升、产生耐药速度的加快以及其易于从急性感染向慢性、持久性、复发性感染转变等特点,金黄色葡萄球菌导致的感染逐渐成为人们关注的焦点。其附属基因调节子(accessory gene regulator, agr)是一种重要的密度感应(quorum sensing,QS)信号系统和毒力因子调节系统,参与金黄色葡萄球菌生物膜(biofilm,BF)的形成与分散(dispersion)以及毒力因子(virulence factor)的表达调控[3-5]。许多研究表明金葡菌agr系统是在毒力因子调节过程中起中心作用的密度感应系统。AgrC是金葡菌二元信号系统中的信号转导因子,它是一个受体组氨酸蛋白激酶,包含一个氨基酸的跨膜区和一个细胞内的组氨酸蛋白激酶域,能够被同源或非同源自诱导肽(autoinducing peptides,AIPs)激活或抑制[6,7]。AgrC属于比较罕见的组氨酸蛋白激酶亚科,包括5-6个跨膜片段。AgrC通过对AIP的特异识别,激活蛋白激酶胞内域部分活性,诱导转录操纵子RNAII和RNAIII的转录,从而调控agr系统,在agr调节系统中对毒力调节起到关键作用[8]。由于抗生素的广泛使用,使多数细菌产生了抗药性,因此,应用传统的筛选方法获得具有新型抗菌机制的抗生素越来越困难。研究发现一些群体感应信号分子类似物能够抑制致病因子的生成或葡萄球菌的生长[9,10],因此通过干扰QS系统而减少金黄色葡萄球菌毒素、致病因子表达及生物膜形成,已成为预防和治疗金葡菌感染的新的突破口,为解决传统抗生素耐药问题提供了新的靶点[11,12]。因此,发现可以与agrC特异性结合的短肽,阻断agr系统活化,以agrC作为治疗金葡菌感染的靶分子,从而减轻或抑制金葡菌毒力因子产生,达到控制金葡菌感染的目的具有重要意义。近年来,以噬菌体展示(phage display)技术筛选肽类药物的蓬勃发展为金葡菌感染的防治提供了新的思路。噬菌体展示技术的原理是将外源多肽或蛋白与噬菌体的衣壳蛋白融合表达,融合蛋白展示在噬菌体表面,而编码这个融合子的DNA则位于该噬菌体内。噬菌体展示技术使大量随机多肽与其DNA编码序列之间建立了直接联系,使各种靶分子的多肽配体通过淘洗(panning)得以快速鉴定。本实验拟利用噬菌体展示肽库技术,筛选可与agrC特异结合的小分子多肽,并研究它们对金葡菌毒力和生物膜形成能力的影响。研究方法:1、构建金黄色葡萄球菌N315标准株agrC的原核表达质粒pET28α(+)-agrC,经测序鉴定后转化大肠杆菌,IPTG诱导后,SDS-PAGE鉴定agrC融合蛋白的表达,磁珠分选纯化法获得纯化的agrC蛋白。2、采用经典的亲和富集法对噬菌体12肽库进行4轮亲和筛选,以agrC为靶分子,经过4轮筛选,获得可与agrC特异性结合的阳性噬菌体克隆。ELISA实验进一步确证。3、通过ELISA法对筛选出来的克隆进行鉴定,获得能与agrC特异性结合的阳性噬菌体克隆,将获得的阳性噬菌体克隆进行DNA测序,根据噬菌体克隆基因所插入的外源DNA序列推导出呈现的外源多肽氨基酸序列。应用Blast软件将多肽序列与agrC的一级结构序列进行比较分析。4、检测阳性克隆噬菌体与agrC的亲和力及其剂量依赖性,筛选出亲和力最高的阳性克隆噬菌体。5、人工合成筛选得到agrC结合多肽,观察其对金葡菌生物膜形成能力的影响及其对生物膜形成相关基因和毒素分泌的影响。研究结果:1、重组克隆PAT2-agrC经测序鉴定与GenBank上金葡菌N315株的agrC核苷酸序列一致性为98.0%,氨基酸序列一致性为98.5%。SDS-PAGE结果表明,重组质粒pET28α(+)-agrC在大肠杆菌中成功表达,磁珠分选系统有效纯化了目的蛋白。2、采用亲和筛选法,以agrC为靶分子,经过4轮筛选,从噬菌体12肽库中获得了可与agrC特异结合的噬菌体克隆。3、从上述获得的噬菌体克隆中随机挑取60个噬菌体克隆进行扩增和纯化,发现22个克隆与agrC结合力显著强于对照组。提示它们与agrC结合可能是特异的。4、将这些阳性克隆进行DNA序列分析及氨基酸序列推导,发现5个不同的DNA序列,其中3个有共同的核心序列,这些克隆的核心序列为(XX)L(X)Q(XX)L(XX)L(X)。5、人工合成agrC结合多肽N1、N2、N4及无关多肽N0,观察到N1在细菌生物膜的粘附、聚集期对金葡菌N315及ATCC25923生物膜形成能力有明显抑制效应;N1多肽还能影响生物膜相关粘附聚集因子atlE和ica转录水平及其蛋白表达,同时N1可以减少N315菌株TSST-1毒素的分泌。而N0、N2、N4对生物膜形成能力及其相关因子没有明显影响。经ELISA检测证实N1是与agrC结合力最高的特异性多肽,其氨基酸序列为CRLEQERLSPLI。结论:1、本实验获得了agrC蛋白的表达与纯化。2、从噬菌体随机12肽库中筛选获得了22个可与agrC特异结合的噬菌体克隆,经DNA序列分析及氨基酸序列推导,发现5个不同的氨基酸序列,其中3个有共同的核心序列,这些克隆的核心序列为(XX)L(X)Q(XX)L(XX)L(X)。ELISA检测发现N1是与agrC结合力最高的多肽,其氨基酸序列为CRLEQERLSPLI。3、人工合成N1多肽能显著抑制金葡菌生物膜形成能力、降低生物膜相关结构基因的活性、减少金葡菌毒素分泌作用。4、N1多肽用于预防和治疗早期金葡菌生物膜相关感染可能更为有效,对分化成熟期生物膜的抑制效果不明显。上述研究结果对于阐明agrC对金葡菌毒力和生物膜形成的调节作用,设计以agrC为靶分子的抗金葡菌多肽提供了有力的实验依据,为金葡菌生物膜感染的治疗提供了新型候选制剂。更多还原

【Abstract】 Staphylococcus aureus is the most frequent positive coccobacteria in lower respiratory tract infection, which pathogenic mechanism is correlated with its secretion of extracellular enzymes and exotoxins, which can causes multi-organs infection and therefore result in high morbidity and mortality. It becomes focal point of infection with Staphylococcus aureus because of the characteristics of high infection rate, drug fast and easy to shift from acute infection to chronicity, persistency and recurrent infection. Accessory gene regulator( agr) is an important quorum sensing system and regulatory system of virulence factors, which participate the biofilm formation and dispersion, as well as control the expression of virulence factors. Many researches indicate that agr system of Staphylococcus aureus is a quorum sensing system which plays a great part in regulating to the virulence factors.AgrC is a signal transduction factor in the tow-component systems of Staphylococcus aureus, which is a histidine protein kinase, including a transmembrane domain and a histidine protein kinase region intracellular, which can be activated or inhibited by homologization or heterogenesis AIPs. AgrC can regulate the transcription of operons RNAII and RNAIII because of its specificity recognization of AIP, thus to regulating the toxicity in the agr system.Because of widespread using and abusing the antibiotics, many bacteria emerge drug resistance, which results in difficulties to exploitation of neotype antibiotics with new antibiosis mechanisms. Studies revealed that many analogues of quorum sensing signals could inhibit Staphylococcus aureus to produce causative agent or to growth. For this reason, to interfere with the quorum sensing system of Staphylococcus aureus to degrade its toxicity, prevent causative agent producing and biofilms formation becomes a new breakthrough to prevent and treat the Staphylococcus aureus infection, which provides a target to solve the drug resistance to traditional antibiotics. So, it is important to taking agrC as a target to finding short peptides specific binding to agrC to blocking agr system activation, so as to suppress the Staphylococcus aureus toxicity, and to treat Staphylococcus aureus infection ultimately.It provides an original route to screening polypeptide drugs to prevention and cure Staphylococcus aureus infection with the technology of phage display. The objective of the present study was to screening peptides conjugated with agrC from phage random 12-mer peptide library and determine the function of agrC.Methods:1、The DNA of Staphylococcus aureus was amplified by PCR and cloned into expression vector pET28α(+). The verified recombinant was transformed into E.coli DH5α. After inducing with isopropyl-β-D-thio- galaetopyranoside(IPTG), the recombinant protein was purified via His Tag magnetic stock.. The recombinant proteins were analyzed with SDS-PAGE and Western blot.2、Phage display technique was employed in our study. After 4 sequential rounds of biopanning to phage random 12-mer peptide library, some phage clones which specific conjugated with agrC were obtained.3、They were selected randomly and amplified. The binding activity of these phage clones were identified by ELISA. Single stranded genomic DNA from the positive clones were extracted for sequencing as described in manual, and then were sequenced. The peptide sequences were deduced from DNA sequences of these phage clones. Trough Blast, the peptide sequences were compared for homology analysis.4、The screening bioactive peptide was synthesized by the solid-phage method, and its further explored functional properties were identified.Results:1、The agrC was successfully constructed and the recombinant agrC protein was expressed in E.coli at a high level . And the purified protein had reached 90%.2、Using agrC as target, 4 rounds of biopanning were performed as described in methods. The bound phages were eluted by the glycine solution at the first tow rounds, and were concentrated at the third and fourth round.3、60 phage clones were selected randomly and amplified. 22 phage clones had high affinity to agrC. Single stranded genomic DNA from the positive clones were extracted and then were sequenced. The peptides sequences of 22 clones were deduced for 5 different sequences. The core peptide sequence of the 5 was (XX)L(X)Q(XX)L(XX)L(X). By homology analysis, one high homologous sequence was found as CRLEQERLSPLI.4、The synthetic peptide with the sequence of CRLEQERLSPLI could inhibit the ability of biofilm formation and the toxcity of Staphylococcus aureus.Conclusions:1、agrC protein was expressed in E.coli at a high level and purified.2、The core peptide sequence of the positive phage clones was (XX)L(X)Q(XX)L(XX)L(X).and the peptide sequence with high ability to bind to agrC was CRLEQERLSPLI.3、The synthetic peptide with the sequence of CRLEQERLSPLI could inhibit the ability of biofilm formation and the toxcity of Staphylococcus aureus. These results may lead to a better understanding of how agrC regulate the toxicity of Staphylococcus aureus and provide a new pathway to explore peptide antibiotics to solve drug resistance in Staphylococcus aureus infection.更多还原