Regulation of P. aeruginosa PoxB Oxacillinase by AmpR and a Two-Component System

描述(申请人提供)：囊性纤维化(CF)患者慢性铜绿假单胞菌感染的两个关键特征是藻酸盐过度产生粘液菌落和产生抗生素耐药性。在CF患者中，由于体内选择产生过量AmpC酶的菌株，导致铜绿假单胞菌对抗生素的耐药性迅速出现，AmpC酶是一种分解内酰胺类抗生素的酶，导致发病率和死亡率。先前利用遗传学和分子生物学技术研究铜绿假单胞菌Amp基因调控的研究导致了第二种内酰胺酶的鉴定，即PoxB苯唑西林酶。我们发现AmpC和poxB的表达都受AMPR的控制，AMPR是LysR转录调节家族的成员。我们已经证明AMPR是一个全球转录调节因子，它调节许多毒力基因的表达，这些基因对于确定慢性感染是重要的。 我们的长期目标是建立存在于Amp和Pox基因之间的调节电路，以及它们在内酰胺类药物耐药性的形成和慢性感染建立中的作用。到目前为止，我们对控制痘操纵子表达的分子机制知之甚少。这项资助中提议的实验将扩大我们的理解，因为它们将集中在(1)表征poxAB操纵子并确定AMPR是否通过转录去抑制来调节，以及(2)确定poxAB操纵子的表达是否受以前未表征的双组分系统的调控。这些分析将为细菌对抗生素冲击的适应和免疫反应提供宝贵的见解。这可能会导致新的治疗方法来对抗持续的铜绿假单胞菌感染，最终导致CF患者的过早死亡。

DESCRIPTION (provided by applicant): Two critical hallmarks of chronic Pseudomonas aeruginosa infection in patients with cystic fibrosis (CF) are the appearance of alginate over-producing mucoid colonies and the development of antibiotic resistance. A rapid emergence of antibiotic resistance in P. aeruginosa isolates in CF patients due to in vivo selection of strains that overproduce AmpC ¿-lactamase, an enzyme that hydrolyzes the ¿-lactams, contributes to both morbidity and mortality. Previous studies investigating the regulation of the amp genes in P. aeruginosa using genetic and molecular biological techniques led to the identification of a second ¿-lactamase, PoxB oxacillinase. We showed that the expression of both ampC and poxB are under the control of AmpR, a member of the LysR family of transcriptional regulators. We have demonstrated that AmpR is a global transcriptional regulator which regulates expression of many virulent genes that are important for establishing chronic infections. Our long-term goal is to establish the regulatory circuit that exists between the amp and pox genes and their role in the development of resistance to ¿-lactams and the establishment of chronic infection. To date, we know very little about the molecular mechanisms that govern the expression of pox operon. The experiments proposed in this grant will extend our understanding as they will focus on (1) characterizing the poxAB operon and determining if AmpR regulation is by transcriptional derepression, and (2) determining if expression of the poxAB operon is regulated by a previously uncharacterized two-component system. These analyses will provide invaluable insight into the bacterial adaptation to the onslaught of antibiotics and the immune response. It will likely lead to novel therapeutic approaches in combating the persistent P. aeruginosa infection that ultimately leads to the untimely death of CF patients.