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Post-Genomic Approach to Bacterial Xenobiotic Exporter Gene Resources and Investigation of Novel Drug Resistance Mechanisms of Bacteria

细菌异生素输出基因资源的后基因组方法和细菌新型耐药机制的研究

基本信息

批准号：
13854012
负责人：
YAMAGUCHI Akihito
金额：
$ 78.96万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

This project aimed to investigate comprehensive analysis of bacterial xenobiotic exporters including their expression regulation mechanisms using mainly Escherichia coli as a model. We previously established the complete expression-cloning library of E.coli putative xenobiotic exporters. On the basis of the library, we analyzed the four typical groups of xenobiotic exporters. Among them, we found that all five of the RND exporters, two of the MFS transporters and one of the ABC transporters couple with the same outer membrane channel TolC. Before that, MFS and ABC exporters had not estimated to have large periplasmic domain required for TolC interaction from their sequence analysis. So, we determined the topology of MacB (ABC) and EmrB (MFS) by site-directed chemical modification and revealed that MacB and EmrB have only four and five transmembrane helices, respectively, and contain large periplasmic loops enough to interact with TolC.As for the major xenobiotic exporter AcrB (RND) in … More E.coli, we succeeded to determine the X-ray crystallographic structure in 2002, which was the first structure not only as xenobiotic exporters but also as secondary transporters. Then, last year, we succeeded to elucidate the structure of the substrate-binding form of AcrB. As a result, we revealed that xenobiotic recognition is based on the membrane vacuum cleaner mechanism from outer leaflet of the inner membrane.As for the expression control of xenobiotic exporter genes, we first found that two-component signal transduction systems, which are the bacterial environmental response systems, induce xenobiotic exporter expression. We revealed the complex network of xenobiotic exporter gene expression by two-component systems. Besides, we found that indole acts as an intercellular signal transduction molecule and induce some xenobiotic exporter genes. Xenobiotic exporters were also controlled by the regulatory systems for ferrous homeostasis.In addition, we found that xenobiotic exporters confer not only to xenobiotic extrusion but also bacterial pathogenicity. That is, although mice which were perorally inoculated Salmonella typhimurium died within a few days, peroral inoculation of S.typhimurium of which all major xenobiotic exporter genes were knocked out did not cause death. Among them, the knock out of MacB gene, which is the only macrolide-specific exporter, resulted in almost nonpoisonous bacteria. These results strongly suggested that some xenobiotic exporters must have their intrinsic physiological roles other than xenobiotic export. These findings suggest the possibility of a completely novel antibacterial drug target which removes pathogenicity without killing bacteria. Coexistence with bacteria is expected to greatly decrease the emergence of drug resistant pathogens. Less

本课题主要以大肠杆菌为模型，对细菌异生物质输出体及其表达调控机制进行综合分析。我们以前建立了完整的表达克隆库的大肠杆菌推定外源性出口商。在此基础上，分析了四类典型的外源性物质出口者。其中，我们发现所有的五个RND输出者，两个MFS转运体和一个ABC转运体与相同的外膜通道TolC偶联。在此之前，MFS和ABC出口商没有估计有大的周质结构域所需的TolC相互作用从他们的序列分析。因此，我们通过定点化学修饰确定了MacB（ABC）和EmrB（MFS）的拓扑结构，并揭示MacB和EmrB分别只有四个和五个跨膜螺旋，并且含有足够与TolC相互作用的大周质环。 ...更多信息 2002年，我们成功地确定了大肠杆菌的X射线晶体结构，这是第一个不仅作为异生物质输出者而且作为二级转运体的结构。然后，去年，我们成功地阐明了AcrB的底物结合形式的结构。结果表明，外源性物质的识别是基于内膜外叶的膜真空吸尘器机制;外源性物质输出基因的表达调控，首次发现细菌环境响应系统中的双组分信号转导系统诱导外源性物质输出基因的表达。我们揭示了复杂的网络的外源性输出基因表达的双组分系统。此外，我们还发现吲哚作为细胞间的信号转导分子，诱导了一些外源性物质的输出基因。此外，我们还发现，外源性物质的输出不仅影响外源性物质的排出，而且还影响细菌的致病性。也就是说，虽然经口接种鼠伤寒沙门氏菌的小鼠在几天内死亡，但经口接种所有主要外源性输出基因被敲除的鼠伤寒沙门氏菌不会导致死亡。其中，MacB基因是唯一的大环内酯特异性输出基因，敲除该基因后，产生的细菌几乎无毒。这些结果有力地表明，某些外源性物质输出者除了输出外源性物质外，还具有其内在的生理功能。这些发现表明，一个全新的抗菌药物靶点的可能性，消除致病性，而不杀死细菌。与细菌共存有望大大减少耐药病原体的出现。少