Using Genomics to Identify Antibiotic Sensitivity Genes

描述（由申请人提供）： 抗生素耐药性是一个日益严重的问题，影响到几乎所有传染病的治疗。仅在美国，治疗抗生素耐药性感染的费用就接近300亿美元/年。鉴定耐药表型的基因和机制是当前药物发现努力的焦点，其中目标是开发靶向细菌存活所必需的过程并同时使耐药机制无效的组合疗法。在实现这一目标的过程中，我们认为有必要了解耐药病原体再次对抗生素敏感的机制，然后在联合治疗的发展中利用这些机制。本研究的目的是开发一种新的基于基因组学的方法，用于有效鉴定其过表达恢复耐药病原体对抗生素敏感性的基因，并应用该方法。在氨基糖苷类耐药铜绿假单胞菌（发病率和死亡率的重要原因，特别是在囊性纤维化患者中）的情况下，除了研究铜绿假单胞菌的天然存在的耐药临床分离株之外，将两种临床相关的耐药机制工程化到铜绿假单胞菌的敏感菌株PAO 1中。这将使用表达酶6”-N-氨基糖苷乙酰转移酶7（其修饰氨基糖苷类）或表达phoP基因（其降低细菌对氨基糖苷类的渗透性）的质粒完成。用基因组文库转化这些抗性菌株将允许基于DNA微阵列的方法来鉴定表达基因组插入物的克隆，所述基因组插入物在氨基糖苷类存在下生长的细菌群体中不存在。正是这些插入片段可能恢复了对这些耐药菌株的敏感表型，导致细菌死亡和群体中这些插入片段的缺失。将进行验证性研究，然后进行机制研究，以进一步阐明所发现的敏感性表型背后的机制。

DESCRIPTION (provided by applicant): Antibiotic resistance is a growing problem that affects the treatment of almost all infectious diseases. The cost of treating antibiotic resistant infections is close to $30 billion/year in the United States alone. The identification of genes and mechanisms underlying resistant phenotypes is a focus of current drug discovery efforts wherein the goal is to develop combination therapies that target a process essential for bacterial survival and simultaneously render resistance mechanisms ineffective. On the way to this goal we believe it is necessary to understand the mechanisms by which resistant pathogens can once again be made sensitive to antibiotics and then exploit these mechanisms in the development of combination therapy. The objective of this study is to develop a new genomics based approach for the efficient identification of genes whose overexpression restores sensitivity to antibiotics in resistant pathogens and apply that approach. In the case of aminoglycoside resistant Pseudomonas aeruginosa, a significant cause of morbidity and mortality, particularly in cystic fibrosis patients, in addition to studying naturally occurring resistant clinical isolates of P. aeruginosa, two clinically relevant mechanisms of resistance with be engineered into PAO1 a sensitive strain of P. aeruginosa. This will be done with a plasmid either expressing the enzyme 6"-N-aminoglycoside acetyltransferase7, which modifies aminoglycosides or expressing the phoP gene, which reduces bacterial permeability to aminoglycosideds. Transforming these resistant strains with a genomic library will then allow for a DNA microarray based method to identify clones expressing genomic inserts which are absent in a bacterial population grown in the presence of aminoglycosides. It is these inserts which presumably restore a sensitive phenotype to these resistant strains, causing bacterial death and an absence of these inserts in the population. Confirmatory and then mechanistic studies will be carried out to further elucidate the mechanisms behind the sensitivity phenotypes discovered.