虎耳草抗金黄色葡萄球菌活性成分分析及其诱导耐药性的基因组与蛋白组学研究

Bacteria continue to cause noteworthy infections world-wide, whose treatment by existing antibiotics is increasingly hampered by the emergence of drug-resistant strains. The development of new antibacterial drugs must therefore be a focus of new drug research and development. The hotspots of antibacterial research are analysis and discovery of new active compounds (preferably with a novel mechanism of antibacterial action) and the molecular mechanism of drug resistance. Our previous research focused on antibacterial activities of traditional Chinese medical plant extracts. The extracts of 60 medicinal plants were tested in a disk diffusion assay on Bacillus cereus, Bacillus subtilis, Lactococcus lactis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella typhimurium, Shigella flexneri, and Yersinia enterocolitica. In addition, we showed that extracts prepared with different solvents of Saxifraga stolonifera have reproducible strong inhibitory activity in liquid culture on Staphylococcus aureus for all wild type and clinical drug- resistant strains tested. It reveals that the active compound(s) may have different target(s) and mechanism(s) compared with currently used antibiotics. This project will focus on compounds from Saxifraga stolonifera showing activity on Staphylococcus aureus, and the mechanism by which resistance to these compounds can be generated. First, the activity-oriented separation, purification and identification will be carried out by using various kinds of modern techniques. Qualitative and quantitative analysis will be carried out in the meantime to establish the fingerprint and a quality control method. Then the mechanism of action of purified active compounds will be studied by selecting resistant bacteria and sequencing their genome, as well as proteomics comparison. Specific drug-resistant mutants will be selected by repeatedly sub-culturing bacteria grown in the presence of increasing concentrations of an active compound. The genomic DNA and distinct proteins of resistant strains and the wild type strain will be sequenced and compared, respectively. Thus we will find the key genes and/or proteins related to drug resistance, and reveal the mechanism of antibacterial action by using genomic method. This research will not only reveal the anti-Staphylococcus aureus compound(s) of Saxifraga stolonifera, but also find new target(s) and mechanism for curing diseases caused by Staphylococcus aureus.

由于细菌耐药的普遍性与严重性，抗菌药历来是新药研发的重点。发现新的抗耐药菌成分，尤其是可能具有全新作用机制天然成分，更是当前研究热点。我们对60种常用中药的不同溶剂提取部位进行了体外抗菌活性初筛，发现虎耳草的乙醇、丙酮和正己烷提取物对金黄色葡萄球菌标准株与临床耐药株均表现出了可重复的强抑制活性，提示其作用靶点与机制可能与当前常用药物不尽一致。故本项目拟以虎耳草为研究对象，利用各种分离分析技术，对其开展以活性为导向的抗菌成分分离、纯化和鉴定，并进行多成分定性定量分析，建立多维指纹谱及质控方法；通过逐渐提高药物浓度、连续传代培养获得虎耳草活性成分耐药诱导株，并对其进行全基因测序比对与差异蛋白分析，找出产生耐药性的关键基因位点或变异蛋白。旨在分析虎耳草抗金黄色葡萄球菌活性物质基础的同时，从基因及蛋白角度阐述其发挥抗菌作用的通路与机制，发现拮抗金黄色葡萄球菌可能的新作用靶点。

由于细菌耐药的普遍性与严重性，抗菌药历来是新药研发的重点。发现新的抗耐药菌成分，尤其是可能具有全新作用机制天然成分，更是当前研究热点。虎耳草的70%乙醇提取物对金黄色葡萄球菌标准株与临床耐药株均表现出了可重复的强抑制活性，提示其作用靶点与机制可能与当前常用药物不尽一致。故本项目以虎耳草为研究对象，利用各种分离分析技术，纯化得到27个化合物，运用LC-MS/MS技术开展化学物质基础及体内代谢产物解析，分别鉴定40个化学成分与47个代谢产物；通过逐渐提高药物浓度、连续传代培养获得虎耳草活性成分耐药诱导株，并对其进行差异蛋白分析及全基因测序比对，找出产生耐药性的8个关键变异蛋白、2条相关通路及6个基因位点。在首次全面分离分析虎耳草化学物质基础及体内代谢产物的同时，发现虎耳草拮抗金黄色葡萄球菌的作用机制与金黄色葡萄球菌感染信号通路及相关蛋白与基因密切关联，而产生耐药性的机制与阳离子抗菌肽耐药信号通路、蛋白、基因相关。