白假丝酵母菌表现型耐药滞留菌相关基因的单细胞表达分析及协助清除滞留菌的小分子佐剂的筛选

Drug resistance is the main obstacle for anti-infection therapy .Drug resistance mutants reslut from gene variation and phenotypic drug tolerance are two equally important aspects for clincial anti-infection therapy. Persisters are the subpopulation which can tolerant fatal concentration of antibiotics,The important characteristic of persisters is that the ability of drug toleracne can not be inherited to their offspring.our research and others scientist have proved that the subpoplulation of these perisiter cells play a major role in the recalcitrance of chronic infections to antibiotics.Saccharomyces albicans(Candida albicans) is one of the most important opportunistic pathogens which can form biofilm and then produce persisters.We have established a dircet link between the ability of S.albicans producing persisters and clincial behaviors.We also preliminarily identified the possible target genes which may directed result in persister cell formation, although this conclusion still need more sudy for identification.They are GIN4 and FLO8.In this research ,by using report gene technology,microfluidic devices and single-cell workstation, we try to observe and analysis dynamicly the related genes expression at the single cell level.At the same time, we alsotry to search the suitable small molecule compounds which can help antifungal drugs (such as AmB) to eliminate persister cells.The aimed compounds can be called antifungal drug adjuvants. Four groups of molecule compounds should be consider firstly, including (i)compounds which can inhibit the drug efflux of S.abicans (ii)compounds which can awake the dormant cells and increase the antifungal drug absorption (iii)compounds which can promote the dissociation of biofilm or interfere the biofilm formation.(iiii) Newly discovered natural compound which have proved antifungal active.Secondly we will also screen the compound bank. Two ways were used to search the suitable antifungal drug adjuvant. (1) high-throughout protocol by using 384-well cell culture plates to form biofilm ,treating by combiantion of antifungal drugs and adjuvant candidates. The collagues of Prof.K.lewis successly identified some suitable compounds which can help to eliminate persister cells . (2) Gene expression screening method for searching the compounds which can interfere the possible key genes(GIN4 and FLO8) for persisters formation. The suitable adjuvant together with antifungal drugs were pharmacodynamic evaluated by C.elegens research model which were established by our group.At last the suibale combination of adjuvant and antifungal drugs for get rid of biofilm including persisters will be defined. Our resaerch group try to uncover the mechanism of phenotypic tolerance by single cell technology, and also identify the suitable compound which can help to eliminate S.albicans persiters ,at last help to control chronic infectious diseases.

微生物耐药性是威胁人类健康的严重问题，从机制上分为两大类：一是基因变异产生耐药菌株（resistance），二是微生物种群自发和由环境因素诱导"分化"出来的少数难以被常规药物浓度杀灭的、耐药性没有遗传性的表现型耐药滞留菌（persisters）造成的药物耐受（tolerance）。相关研究提示滞留菌的产生与慢性感染性疾病迁延不愈直接相关。在上一个基金资助下，我们初步确定了白假丝酵母菌滞留菌产生的相关激酶（GIN4）和转录因子(FLO8)，本研究拟采用微流池，在单细胞水平上动态观察相关基因的表达波动，来验证这两个基因在滞留菌产生中作用；依托目前成熟的滞留菌清除方法，利用384孔体外生物膜模型和报告基因技术，大通量筛选联合抗真菌药物清除滞留菌的小分子物质为抗真菌药物佐剂；利用前期建立起来的酵母菌感染秀丽隐杆线虫作为药效学评价模型进一步筛选，达到研发直接清除滞留菌的药物组合的目的。

本项目主要的目的是研究白假丝酵母菌滞留菌的清除策略，利用单细胞技术研究其相关基因的表达与滞留菌产生的相关性。. 主要研究成果包括：第一，根据改良过的高通量384孔加生物染料的筛选手段，对国家化合物库的51520个小分子化合物进行高通量筛选，初步筛选出10个小分子化合物对药效提高率>30% ，之后通过菌落计数法(CFU)复筛，最终确定小分子化合物F0371-0041 、F0760-2126可以协同两性霉素B清除滞留菌，验证小分子化合物的协同作用。两种小分子化合物本身对白假丝酵母菌没有抑菌作用，但当与抗真菌药物两性霉素B联用时，却能够提高两性霉素B对滞留菌的清除作用，符合小分子佐剂的特点。. 第二，对两种小分子化合物进行了细胞毒性实验和动物实验。细胞毒性实验中，3号小分子化合物在很大范围内都没有显示细胞毒性，10号小分子化合物则出现浓度依赖的细胞毒性，但是其IC50为4.130ug/ml,这个浓度超过了实验中实际使用的小分子化合物浓度。动物实验的结果表明，两性霉素和小分子化合物的治疗，可以明显减少动物肝脏肾脏等主要脏器的孢子及菌丝数量，可以明显提高治疗效果。. 第三，Illumina–Solexa 转录组测序技术对小分子化合物F0371-0041（3号）及F0760-2126（10号） 的作用进行了转录组测序，利用荧光定量PCR进行验证，发现两组共9个基因与小分子化合物协同两性霉素B的作用相关。两组中表达差异显著的基因并没有重复，证明两种小分子化合物作用于白假丝酵母菌不同的位点 。. 第四， 设计和制造出的微流控装置根据白假丝酵母菌的大小分别将3μm、4μm、5μm、6μm直径的流道依次间隔排。不仅能保证白假丝酵母菌单个、单层、贴壁通过流道。通过我们设计制作的这个装置，结合将FLO8基因绿色荧光蛋白的融合子，发现该基因的表达与滞留菌的产生有相关性。. 本部分研究最大的创新点在于，突破传统抗生素研发模式，提出了筛选抗真菌小分子化合物，来尝试清除白假丝酵母菌生物膜新思路。最终我们希望将有效的小分子化合物与现有的抗真菌药物联用，研发出新型抗真菌药物，既能降低药物的毒性，提高原药物疗效。对控制由生物膜产生的慢性真菌感染提供新路径。