微生物对抗菌肽耐受的诱导及耐受机理初步研究
文献类型：学位论文
| 作者 | 宋玉竹 |
| 学位类别 | 博士 |
| 答辩日期 | 2009-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赖仞 |
| 关键词 | 抗菌肽 耐受 蛋白酶 蛋白质组学 系统命名法 |
| 其他题名 | Experimental induction of microorganism to antimicrobial peptides and research on the resistant mechanisms |
| 中文摘要 | 抗菌肽是一类具有强大杀菌能力的肽类分子，同时还具有离子调节、免疫调节、蛋白酶抑制剂和自由基清除等其他生物活性。现已鉴定的抗菌肽超过1,200种，几乎存在于所有生物种类中。在抗生素耐受严重的今天，抗菌肽极有潜力成为新型的有效抗菌药物，许多抗菌肽已进入临床前研究或临床研究。 在本论文中，我们选择了无指盘臭蛙（Odorrana grahami）来源的三种抗菌肽（Brevinin 2E-OG1、Nigrocin-OG4和Palustrin-OG1），单独或组合使用，以藤黄微球菌（Micrococcus luteus）、枯草芽孢杆菌（Bacillus subtilis）和白假丝酵母菌（Candida albicans）为研究对象，进行微生物对抗菌肽耐受性的实验诱导；并通过检测胞外蛋白酶活性、蛋白质组学等方法对微生物耐受抗菌肽机制进行初步的研究。 将微生物培养于含有低浓度抗菌肽（单独使用或组合使用）培养基中，每日转接一次，每十次酌情提高抗菌肽浓度。80次转接后，除藤黄微球菌未对Palustrin-OG1产生耐受外，其余所有的实验菌株均表现出对所用三种抗菌肽的耐受。但是Palustrin-OG1与Brevinin 2E-OG1或Nigrocin-OG4联合使用能在一定程度上降低耐受性。将诱导后细菌于不含抗菌肽条件下培养，转接5次后，对耐受现象无影响，说明这种耐受是可以稳定遗传的。 抗菌肽耐受机制之一是分泌蛋白酶水解胞外抗菌肽，我们通过两种方式检测胞外蛋白酶活性，一种是检测发酵液的酪蛋白水解活性，另一种是检测发酵液处理抗菌肽后对抗菌活性的影响。结果发现枯草芽孢杆菌和藤黄微球菌发酵液存在着蛋白酶活性，推测胞外蛋白酶可能与二者对抗菌肽的耐受有关；而白假丝酵母菌发酵液中未检测到蛋白酶活性。 另外，我们还通过蛋白质组学的手段对枯草芽孢杆菌耐受机制进行了初步的研究，鉴定了5个差异表达的蛋白，表达上调的蛋白有yraA（功能未知）、Tpx（巯基过氧化物酶，Thiol peroxidase）、pdhD（二氢硫辛酰胺脱氢酶，dihydrolipoa- mide dehydrogenase），表达下调的有cotN/TasA（芽孢膜相关蛋白，spore coat-associated protein）和gapA（三磷酸甘油醛脱氢酶，Glyceraldehyde 3-phosphate dehydrogenase 1 ，GAPDH）。yraA和Tpx都由Spx调控，yraA可以水解小肽增加自由氨基酸，而自由氨基酸增多时gapA、tasA表达水平会下降，Spx是由sigma-M因子调控的，所以我们推测sigma-M因子在B. subtiis对抗菌肽耐受中起到了重要的作用。 总之，本研究发现抗菌肽的联合作用会减缓微生物对其耐受的程度，为抗菌肽类药物研发提供了一种新思路；同时对抗菌肽耐受机制的初步研究也为今后的深入研究打下了基础。 另外，我们还设计了一种新型的抗菌肽系统命名方法，并构建了昆明动物研究所抗菌肽数据库。 |
| 英文摘要 | Antimicrobial peptides (AMPs) are a group of molecules with potent antimicrobial activity and other activities such as iron regulation, immunity regulation, protease inhibition, free radicals clearance and so on. More than 1,200 AMPs have been identified from nearly all groups of organisms, including bacteria, fungi, plants and animals. AMPs are considered as the promising potent antimicrobial agents in the era of antibiotic resistance and many of these are conducting the preclinical or clinical trials. However, some studies suggested that the therapeutic use of AMPs might result in the resistance of microorganisms. In this study, we designed a selection protocol, where Micrococcus luteus, Bacillus subtilis and Candida albicans were grown for several hundred generations in media containing sole AMPs or combined AMPs identified from Odorrana grahami. The used AMPs are Brevinin 2E-OG1、Nigrocin-OG4 and Palustrin-OG1. And then, we studied the resistance mechanisms using protease assay and proteomics. We inoculated the selected strains in the culture media with low concentrations of AMPs, sole or combined, and transferred them daily. We increased the concentrations of AMPs every ten transfers cautiously. After 80 transfers, all strains of M. luteus remained sensitive to Palustrin OG-1. All the other tested strains, including those grown in combined AMPs, were resisted to the AMPs used in the selection protocol. However, the combinations of Palustrin-OG1 with Brevinin 2E-OG1 or Nigrocin-OG4 could decrease the resistance to some extent. After five transfers in the medium without AMPs, the tested strains could maintain the resistance, which means the induced resistance by AMPs can transmit to further generations stably. The secretion of extracellular proteases is one of the mechanisms that microorganism resist to AMPs. So we studied the extracellular proteases activity through casein degradation assay and MIC assay of AMPs pretreated by culture supernatant. The results indicated the extracellular proteases might play important roles in the resistance of B. subtilis and M. luteus, but not in C. albicans. Furthermore, we studied the resistance mechanism of B.subtilis to AMPs using proteomic technology and identified five proteins with differential expression. The up-regulated proteins are yraA with unknown function, Tpx belonging to Thiol peroxidase family and pdhD which is an indentified dihydrolipoamide dehydrogenase. The down-regulated proteins are spore coat-associated protein-cotN/tasA and Glyceraldehyde-3-phosphate dehydrogenase 1-gapA. Tpx and yraA are regulated by the Spx which is regulated by sigma-M factor. GapA and cotN/tasA can be down-regulated by increasing of free amino acids that may be produced from the degradation of peptides by yraA. We suggested that the sigma-M factor play an important role in the mechanisms of B. subtilis resist to AMPs. In conclusion, the combined application of AMPs can postpone the resistance of microorganism. Our studies provided a new thread to the therapeutic use of AMPs. At the same time, the research on resistance mechanisms can be a basis of the coming study. In addition, we designed a systematic nomenclature of antimicrobial peptides and established a database to exhibit it. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6301]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 宋玉竹. 微生物对抗菌肽耐受的诱导及耐受机理初步研究[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
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