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

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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