Transcription Activation at the rhaBAD Operon

描述（由申请人提供）：自20世纪40年代以来，抗生素的广泛使用大大减少了因细菌感染导致的死亡人数，然而，随着抗生素耐药性日益普遍，我们正在迅速失去对细菌敌人的优势。我们必须研究抗菌剂的新靶点，以避免细菌感染导致的常见死亡再次发生。许多非常大的AraC/XylS转录激活因子家族的成员是细菌病原体中毒力因子表达所必需的。在一些病例中，研究表明，AraC/XylS激活剂的缺失可显著减少发病机制，这表明该家族成员具有作为抗菌剂靶点的潜力。我们的目标是鉴定AraC/XylS家族激活剂RhaS的小分子抑制剂及其作用机制，部分是通过鉴定RhaS活性的分子机制。我们的中心假设是，可以确定干扰RhaS激活转录能力的活性的小分子抑制剂。我们的基本原理是，这些抑制剂有潜力开发成治疗细菌性疾病的新策略。我们的目标将通过三个目标来实现。在第一个目标中，我们将使用各种遗传和生化方法确定RhaS功能的机制，包括二聚化，鼠李糖结合，对鼠李糖的反应和自动调节。在第二个目标中，我们将使用体内遗传和体外研究相结合的方法，确定RhaS接触RNA聚合酶从而激活转录的详细分子相互作用。在第三个目标中，我们将使用一个大的小分子文库的高通量筛选来鉴定RhaS活性的抑制剂及其作用机制。我们的筛选将在体内进行，以提高我们鉴定的化合物对细菌细胞有效的机会，并将采用一种控制菌株，使我们能够从考虑中消除不直接影响RhaS活性的化合物。在未来，我们将测试在该筛选中鉴定的化合物对致病菌毒力因子的AraC/XylS激活剂的作用。

DESCRIPTION (provided by applicant): The widespread availability of antibiotics since the 1940's has dramatically reduced the number of deaths due to bacterial infections, however we are rapidly losing our advantage over our bacterial foes as antibiotic resistance becomes increasingly prevalent. We must investigate novel targets for antibacterial agents to avoid the return of commonplace deaths due to bacterial infections. Many members of the very large AraC/XylS family of transcription activators are required for the expression of virulence factors in bacterial pathogens. In several cases it has been shown that deletion of an AraC/XylS activator drastically reduces pathogenesis, indicating that members of this family have potential as targets for antibacterial agents. Our objective is to identify small molecule inhibitors of the AraC/XylS family activator RhaS and their mechanism of action, in part by identifying the molecular mechanisms of RhaS activity. Our central hypothesis is that small molecule inhibitors can be identified that interfere with the activities that underlie the ability of RhaS to activate transcription. Our rationale is that these inhibitors have potential to be developed into novel strategies for treatment of bacterial diseases. Our objective will be accomplished via 3 aims. In the first aim we will identify the mechanisms of RhaS functions that underlie transcription activation - including dimerization, rhamnose binding, the response to rhamnose, and autoregulation - using a variety of genetic and biochemical methods. In the second aim we will identify the detailed molecular interactions used by RhaS to contact RNA polymerase, and thereby activate transcription, using a combination of in vivo genetic and in vitro studies. In the third aim, we will use high throughput screening of a large library of small molecules to identify inhibitors of RhaS activity and their mechanism of action. Our screen will be performed in vivo to improve the chances that compounds we identify could be effective against bacterial cells and will employ a control strain that will allow us to eliminate compounds from consideration that do not directly influence RhaS activity. In the future, we will test the compounds identified in this screen against AraC/XylS activators of virulence factors in bacterial pathogens.