Undermining aminoglycoside resistance in Pseudomonas aeruginosa

描述(由申请人提供)：拟议研究的目标是将一种双组分调节剂(EnvZ-OmpR)表征为铜绿假单胞菌潜在的联合治疗抗生素靶点。我们发现，使调节器失活的突变会导致一些分离株对氨基糖苷(妥布霉素)敏感性的增强，有效地破坏对抗生素的内在耐药性。因此，对EnvZ-OmpR功能的药理抑制也应增强对药物的敏感性。这项拟议的工作将通过鉴定在envZ-ompR突变体中表达改变的基因，帮助定义EnvZ-OmpR在内源性氨基糖苷类耐药性中的需求。我们推测，一个或多个受调控的基因产物介导了固有的氨基糖苷类耐药性。我们还将确定EnvZ和OmpR是否都是耐药所必需的，以及是否需要通过磷酸化激活OmpR。其他实验将测试EnvZ-OmpR在氨基糖苷类耐药性中的作用模型，在该模型中，该调节器帮助中和氨基糖苷类主动外排引起的细胞内pH/质子动力应激。最后，将构建适合于筛选EnvZ-OmpR特异性抑制剂小分子文库的菌株。总体而言，这项工作应该有助于表征氨基糖苷类固有耐药性中对两种成分调节剂的要求，并为小分子筛查将其作为潜在的药物靶点奠定基础。公共卫生相关性拟议的研究将探索细菌病原体铜绿假单胞菌的特定调节者是否是联合抗菌治疗的有前途的药物靶点。该调节器的遗传失活使细胞对氨基糖苷类抗生素高度敏感，针对该调节器的药物治疗应该会使氨基糖苷类药物更有效地对抗细菌。这项研究将有助于阐明该调节剂是如何工作的，并将产生细菌菌株，这些菌株可用于识别抑制该调节剂的化合物。

DESCRIPTION (provided by applicant): The goal of the proposed research is to characterize a two component regulator (EnvZ- OmpR) as a potential combination therapy antibiotic target in P. aeruginosa. We have found that mutations inactivating the regulator lead to enhanced aminoglycoside (tobramycin) sensitivity in a number of isolates, effectively undermining intrinsic resistance to the antibiotic. Thus, pharmacological inhibition of EnvZ-OmpR function should also enhance sensitivity to the drugs. The proposed work will help define the requirement for EnvZ-OmpR in intrinsic aminoglycoside resistance by identifying genes whose expression is altered in envZ-ompR mutants. We assume that one or more of the regulated gene products mediates intrinsic aminoglycoside resistance. We will also determine whether both EnvZ and OmpR are required for resistance, and whether activation of OmpR by phosphorylation is necessary. Other experiments will test a model for the role of EnvZ-OmpR in aminoglycoside resistance in which the regulator helps counteract intracellular pH/proton motive force stress caused by active efflux of aminoglycosides. Finally, strains suitable for screening small molecule libraries for specific inhibitors of EnvZ-OmpR will be constructed. Overall, the work should help characterize the requirement for the two component regulator in intrinsic aminoglycoside resistance and set the stage for small molecule screens to pursue it as a potential drug target. PUBLIC HEALTH RELEVANCE The proposed research will explore whether a particular regulator of the bacterial pathogen Pseudomonas aeruginosa is a promising drug target for combination antibacterial therapy. Genetic inactivation of the regulator makes cells hypersensitive to aminoglycoside antibiotics and treatment with drugs targeting the regulator should make aminoglycosides more effective against the bacteria. The research will help elucidate how the regulator works and will generate bacterial strains which may be used to identify chemical compounds inhibiting the regulator.