【作者】 董燕；

【导师】 王仙园；

【作者基本信息】 第三军医大学 ， 护理学， 2008， 博士

【摘要】 耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)是医院感染的重要病原菌,也是引起烧伤、严重创伤后感染的最常见致病菌之一。在金黄色葡萄球菌感染中,MRSA所致的医院感染越来越流行,尤其在ICU、烧伤病房等,MRSA的检出率都有明显增高趋势,最高可达90%以上,给临床治疗护理工作带来了极大的困难。其中,老年患者、危重病患者、免疫功能低下者及新生儿等是MRSA感染的高危人群;长期使用抗生素,侵入性操作以及消毒隔离措施等医源性因素均可导致MRSA感染率增加。MRSA的耐药机制主要与其mecA基因大量表达产生的一种特殊青霉素结合蛋白PBP2a有关,该蛋白由668个氨基酸组成,分子量为76.1 kDa,对所有β-内酰胺类抗生素亲和力低。水溶性的PBP2a为去除N-末端23个氨基酸后的蛋白质(不影响PBP2a与β-内酰胺类抗生素的结合),含有两个功能域:N-末端非青霉素结合域(24~326 aa),负责蛋白的延伸和锚定;C-末端功能域(327~668 aa),具有转肽酶活性和β-内酰胺酶的作用。当β-内酰胺类抗生素抑制了由敏感金葡菌产生的PBPs时,PBP2a可以替代其转肽酶功能,继续维持细菌细胞壁的合成,表现出耐药性。MRSA临床分离株大多是多重耐药,仅对糖肽类抗生素如万古霉素敏感。然而,随着其广泛应用,已出现一些耐万古霉素金黄色葡萄球菌的报道。1996年在日本分离到万古霉素中度敏感的MRSA,随后2002年在美国发现了万古霉素耐药的MRSA,至今尚无其它治疗MRSA感染的有效药物。所以,更新研究策略、寻找新的药物来抑制PBP2a的活性,恢复MRSA对抗生素的敏感性或降低MRSA耐药性具有十分重要的临床意义。目的本研究基于PBP2a在MRSA耐药机制中的重要性,拟从MRSA临床分离株中获得多重耐药蛋白PBP2a,以此为靶点应用生物传感器技术筛选具有与PBP2a高结合力的中药,从中药中寻找抗菌增敏剂,恢复抗生素的敏感性,为MRSA感染的防治研究提供新的策略。方法1.可溶性重组蛋白PBP2a的表达、纯化与鉴定:采用PCR方法从MRSA临床分离株中扩增编码PBP2a可溶性全片段的mecA基因(76~2007 bp),构建原核表达载体pQE30-mecA,经酶切鉴定后进行DNA测序;将pQE30-mecA转化入大肠杆菌M15并诱导表达PBP2a可溶性全片段,对重组蛋白PBP2a进行Ni2+亲和层析纯化后,测定其纯度;并进行氨基酸测序与分子量鉴定。然后,对PBP2a的生物学活性与结合活性进行评价。采用分光光度法对PBP2a分别进行转肽酶及β-内酰胺酶活性测定,并通过二倍微孔稀释法与激光共聚焦技术观察PBP2a与抗生素的体外结合作用。2.以PBP2a为靶点筛选拮抗MRSA的中药:将PBP2a包被于生物传感器的样品池作为固相配基,通过对26种中药水煎液与PBP2a结合活性的测定,选择结合力较高的10种中药,再结合直接抗菌实验,以及10种中药与苯唑西林的联合抗菌实验,筛选出对MRSA具有抗菌增敏作用的中药。3.夏枯草中对MRSA抗菌增敏组分的分离提取及活性评价:首先通过生物传感器测定夏枯草不同部位水煎液与PBP2a的结合活性,再经联合抗菌实验对它们的抗菌效果进行初步测定;利用AB-8大孔吸附树脂分离富集夏枯草全草水煎液中的有效组分,追踪检测其与PBP2a的结合力,通过抗菌实验与联合抗菌实验确定抗菌增敏组分,并对其物质属性进行鉴定;观察其与苯唑西林联用的抑菌曲线、与β-内酰胺类抗生素的联合抗菌作用,然后通过透射电镜观察其对MRSA细菌形态的影响,评价可能的抗菌作用机制。结果1.成功扩增出mecA基因片段(76~2007bp),构建了重组质粒pQE30-mecA,基因序列分析表明,mecA基因片段的大小与预期一致,但序列中出现了9个碱基突变,所编码蛋白质与GenBank收录的蛋白质同源性为96%;在大肠杆菌M15中实现了可溶性PBP2a的高效表达,经分离纯化后得到的重组蛋白PBP2a纯度达到90.6%;蛋白质N端15个氨基酸测序结果为:MRGSH HHHHH GSKDK,与预期一致;质谱分析其相对分子质量约为74 kDa,与预期相符;所获得的重组蛋白PBP2a具有转肽酶活性与β-内酰胺酶活性,并且在体外与β-内酰胺类抗生素具有一定的结合活性。2.成功地将PBP2a包被于生物传感器的羧甲基葡聚糖样品池上,从26种中药中筛选了10种与PBP2a有较高结合力的中药;体外抗菌实验显示,黄芩、夏枯草、黄连对MRSA的单独抗菌效果较好;10种中药与苯唑西林的联合抗菌实验显示,夏枯草与苯唑西林的联合抗菌效果最好。3.夏枯草的全草与果穗中均含有与PBP2a具较高结合力的活性成分,其抗菌效果差别不明显;夏枯草全草水煎液经过AB-8大孔吸附树脂分离富集,得到SP2组分;SP2组分不仅与PBP2a具有较高的结合力,而且与苯唑西林联用能够明显抑制MRSA的生长速度;可以使苯唑西林、青霉素、舒氨西林、氨苄西林对MRSA的MIC分别降低256、8、16及4倍,SP2与苯唑西林、青霉素、舒氨西林、氨苄西林的FIC分别为0.007、0.151、0.064、0.276;SP2能够通过破坏MRSA的细胞壁结构发挥抗菌增敏作用;SP2为水溶性组分,含有大量的酚羟基类物质,其有效成分的分子量范围大约为3~5kDa。结论采用基因重组技术,成功实现了MRSA临床分离株中可溶性PBP2a的高效表达;以多重耐药蛋白PBP2a为靶点,应用生物传感器建立了筛选拮抗MRSA中药的技术平台。利用该平台,我们从26种中药中筛选出与PBP2a有较高结合力、与苯唑西林联合抗MRSA效果最好的夏枯草;进一步从夏枯草全草水煎液中分离得到水溶性有效组分SP2,SP2具有恢复β-内酰胺类抗生素对MRSA敏感性的作用;SP2组分中有效成分的分子量范围大约为3~5kDa,有望通过进一步的分离纯化得到有活性的小分子化合物作为抗菌增敏剂,为MRSA感染防治提供新的方法。更多还原

【Abstract】 Methicillin-resistant Staphylococcus aureus (MRSA) is a very important pathogen in nosocomial infection, and one common cause of deep burns infections and severe trauma infections. Nosocomial infection caused by MRSA, among Staphylococcus aureus, become more and more popular, especially in intensive care units and burn wards. The detective rate of MRSA is increasing gradually, even up to over 90%. This issue brings great challenges to the clinical treatmant and nursing. Actually, the senile, the severe, the immunological- deficient and the neonatal, are high-risk groups with MRSA infection. The iatrogenic factors such as longtime administration of antibiotics, invading manipulation and sterilisation and isolation, can result in the growth of MRSA infection.The mechanism of MRSA resistance is mainly associated with the abundant PBP2a, a special penicillin-binding protein that encoded by mecA gene. PBP2a is composed of 668 amino acids, and its molecular weight is 76.1 kDa. PBP2a has low affinity for all beta-lactam antibiotics. The soluble PBP2a (644 amino acids, 74 kDa) has two domains. One is an N-terminal lobe, a centralized non-penicillin binding domain of unknown function, the other is C-terminal transpeptidase domain. Of particular interest is the C-terminal transpeptidase domain, which has a folding pattern that is typical of the PBP transpeptidases and the serine beta-lactamases. PBP2a provides complementary transpeptidase activity for the cells so that cell walls can be synthesized although other PBPs are inhibited by beta-lactams, thus result in drug resistance. MRSA clinical isolates are typically multidrug resistant and could only be treated with glycopeptides such as vancomycin. However, vancomycin-resistant MRSA strain had emerged in Japan in 1996, followed by similar strains isolated in the United States in 2002, and no effective drug can cure this kind of infection caused by MRSA. Therefore, it is very important and significant to search for new agents that inhibit PBP2a in order to restore the susceptibility of antibiotics in MRSA or decrease MRSA resistance.ObjectiveConsidering that PBP2a plays an important role in MRSA resistance, we undertake the current study to express the soluble PBP2a from clinical MRSA isolate. Using the biosensor technology to screen traditional Chinese herbs with high affinity for PBP2a, and then search for anti-MRSA potentiator to restore antibiotics susceptibility in order to provide a new treatment for MRSA.Methods1. The expression, purification and identification of the soluble PBP2a.The mecA gene fragments including bases 76 to 2007, encoding soluble PBP2a, were amplified by PCR to construct prokaryotic expression vector pQE30-mecA. After the positive recombinant identified by enzyme digestion was sequenced correctly, the recombinant was transformed into E.coli M15. The bacteria were induced with 1 mmol/L IPTG, and the soluble PBP2a was expressed. After the protein was purified with Ni-NTA agarose, its purity, amino acid sequences and molecular wieght were identified. Subsequently, biological activities of the PBP2a such as the transpeptidase and beta-lactamase activities were respectively detected by spectrophotometric method, and binding activities to antibiotics in vitro were observed through double micropore dilution method and confocal technology.2. Screening of anti-MRSA traditional Chinese herbs targeting PBP2a.The soluble PBP2a was immobilized onto the surface of carboxymethyl dextran cuvette as a target. According to the affinity capacities of 26 traditional Chinese herbs for PBP2a, 10 herbs were selected to test their antibacterial efficiency, then the specific anti-MRSA herb was chosen out.3. Isolation of the anti-MRSA components from Spica Prunellae and evaluation of their activities.The affinities of aqueous extractions for PBP2a from different parts of Spica Prunellae were firstly measured by biosensor. Their antibacterial effects were evaluated through synergetic antibacterial experiment in order to confirm the active components for the next study. The anti-MRSA components from entire plants of Spica Prunellae were isolated through macroporous adsorptive resins AB-8, followed by affinity tests, antibacterial and synergetic antibacterial experiments. At the same time, the material attribute was identified. Then the growth curves of MRSA and synergetic antibacterial effects of the active component combined with beta-lactams were observed respectively. Finally, the possible mechanism of the component, as anti-MRSA potentiator, was approached by observing the morphology of MRSA through transmission electron microscope.Results1. After the mecA gene fragment including bases 76 to 2007 had been amplified by PCR, the recombinant pQE30-mecA was successfully constructed. By gene sequencing it showed that the mecA fragments consisted of 1932 bp as expected, with 9 site-mutations. The recombinant protein was 96% homology with the one in GenBank. Then, soluble PBP2a was efficiently expressed in E.coli M15. The purity of recombinant PBP2a was up to 90.6%. The N-terminal amino acid sequences were MRGSH HHHHH GSKDK as expectated. Its molecular wieght was identified as 74 kDa by mass spectrography analysis. The recombinant PBP2a had transpeptidase and beta-lactamase activities, and possessed some binding effects to beta-lactam antibiotics in vitro.2. The soluble PBP2a was successfully immobilized onto the surface of carboxymethyl dextran cuvette. 10 traditional Chinese herbs with high affinity for PBP2a were selected out from 26 herbs. Scutellaria, Spica Prunellae and Coptis exerted better anti-MRSA effects, while Spica Prunellae showed the best synergetic antibacterial effect with oxacillin among 10 herbs.3. The active components of Spica Prunellae were mainly from the ear of plants, but there was no difference from the entire plants in anti-MRSA. Therefore, SP2 fraction was isolated from entire plants of Spica Prunellae through macroporous adsorptive resins AB-8. This fraction not only had high affinity for PBP2a, but also could decrease the MICs of oxacillin, penicilin, ampicillin and sulbactam sodium-ampicillin sodium on MRSA by 256, 8, 16 and 4 times respectively. Their FIC were 0.007, 0.151, 0.064 and 0.276. SP2 fraction was water-soluble and consisted of some phenolic hydroxyl groups, with the molecular wieght range of 3~5 kDa. SP2 could intensify antibacterial effects of antibiotics on MRSA by destroying their cell walls.ConclusionsThe soluble PBP2a from clinical MRSA isolates has been successfully expressed by gene recombination technique. It is feasible to sreen anti-MRSA traditional Chinese herbs targeting the multidrug-resistant protein PBP2a through biosensor technology. Using this screening technique, Spica Prunellae, with high affinity for PBP2a and the best synergetic anti-MRSA effect with oxacillin, is selected out of 26 traditional Chinese herbs. Furthermore, water-soluble active component SP2 has been derived from entire plants of Spica Prunellae, which could enhance the antibacterial effects of beta-lactams on MRSA. Molecular weight of the active component in SP2 ranges from 3 to 5 kDa. The compound as an anti-MRSA potentiator may be discovered by further isolation and purification in the future.更多还原