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

本项目主要以大肠杆菌为模型，研究细菌异源输出子的综合分析及其表达调控机制。我们先前建立了完整的大肠杆菌异源输出子的表达克隆文库。在文库的基础上，我们对四个典型的异源输出物进行了分析。其中，我们发现所有5个RND转运体、2个MFS转运体和1个ABC转运体都与相同的外膜通道TolC相连。在此之前，从序列分析来看，MFS和ABC出口蛋白没有估计到TolC相互作用所需的大的周质结构域。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，发现MACB和EMRB分别只有4个和5个跨膜螺旋，并且含有足够与TolC相互作用的大的周质环。至于…中主要的外源输出子AcrB(RND)更多的大肠杆菌，我们在2002年成功地确定了X射线晶体结构，这是第一个不仅作为异源输出体，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底物结合形式的结构。在外源基因的表达调控方面，我们首次发现外源基因的表达调控是由细菌环境反应系统中的双组分信号转导系统诱导的。我们用双组分系统揭示了外源基因表达的复杂网络。此外，我们还发现吲哚作为一种细胞间信号转导分子，诱导了一些外源基因的表达。此外，我们还发现，异源生物出口商不仅与异源生物的排泄有关，还与细菌的致病性有关。也就是说，虽然口服接种鼠伤寒沙门氏菌的小鼠在几天内死亡，但口服接种鼠伤寒沙门氏菌并不会导致死亡，因为所有主要的异源输出基因都被敲除了。其中，MacB基因的敲除导致了几乎无毒的细菌。MacB基因是唯一的大环内酯类特异性输出子。这些结果有力地表明，某些外来生物输出体除了具有外来生物出口外，还可能具有其内在的生理作用。这些发现表明，一种全新的抗菌药物靶点有可能在不杀死细菌的情况下消除致病性。与细菌共存有望大大减少耐药病原体的出现。较少

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