Systematic analysis of multidrug efflux pumps in multidrug resistant bacteria and development of pump inhibitor

耐甲氧西林金黄色葡萄球菌（MRSA）、耐万古霉素肠球菌（VRE）、铜绿假单胞菌、肺炎克雷伯菌等多重耐药细菌感染是严重问题。多药外排泵在多药耐药菌的多药耐药过程中起着重要作用。我们系统地研究了这种细菌的多药物外排泵。基于MRSA N315的基因组序列，克隆了37个候选多药外排泵基因，并对多药外排泵进行了鉴定。我们还研究了37个基因在MRSA N315中的表达，明确了哪些基因在N315细胞中真正表达，并真正参与了多药耐药。由此可见，MRSA N315多药外排泵的全貌清晰可见。rnd型多药外排泵在铜绿假单胞菌多药耐药过程中起重要作用。我们克隆了铜绿假单胞菌PAO1中rnd型多药外排泵的11个可能基因，并获得了它们的基因组序列。我们描述了所有迄今为止尚未描述的泵。从而清晰了rnd型多药外排泵的全貌。我们分离了铜绿假单胞菌突变体，对多种抗菌药物的耐药性升高。我们从突变体的染色体上克隆了多药耐药基因，表达了这些基因并对泵进行了表征。我们确定了多药外排泵的每个基因的突变部分。从肺炎克雷伯菌、VRE、副溶血性弧菌和非01型霍乱弧菌的染色体中克隆出多药外排泵基因。我们描述了许多多药物外排泵。我们构建了多药外排泵缺乏基因的突变体，并研究了多药外排泵的生理作用。我们发现了多药外排泵的抑制剂，黄芩苷和黄芩苷。

Infections by multidrug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Pseudomonas aeruginosa, Klebsiella pneumoniae are serious problems. Multidrug efflux pumps are most importantly involved in the multidrug resistance in multidrug resistant bacteria. We systematically investigated the multidrug efflux pumps of such bacteria. Based on genome sequence of MRSA N315, we cloned all of the 37 genes for tentative multidrug efflux pumps, and characterized the pumps. Also we investigated expression of the 37 genes in MRSA N315, and made clear which ones are really expressed in N315 cells and really contributing to the multidrug resistance. Thus, total picture of multidrug efflux pumps was made clear in MRSA N315. RND-type multidrug efflux pumps play major role in multidrug resistance in P aeruginosa. We cloned all of the 11 possible genes for RND-type multidrug efflux pumps in P aeruginosa PAO1 with which genome sequence is available. We characterized all of the hitherto uncharacterized pumps. Thus, total picture of RND-type multidrug efflux pumps was made clear. We isolated mutants of P.aeruginosa with elevated resistance to multiple antimicrobial agents. We cloned genes responsible for the multidrug resistance from chromosome of the mutants, expressed the genes and characterized the pumps. We identified the mutated portion of each gene for multidrug efflux pump. Genes for multidrug efflux pumps were also cloned from chromosome of K.pneumoniae, VRE, Vibrio parahaemolyticus and V.cholerae non-O1. We characterized many multidrug efflux pumps. We constructed mutants lacking genes for the multidrug efflux pumps, and investigated physiological roles of the pumps.We found inhibitors for the multidrug efflux pumps, skyrin and baicalein.