Transcription Activation at the rhaBAD Operon

描述(申请人提供)：自20世纪40年代以来，抗生素的广泛应用大大减少了因细菌感染而死亡的人数，但随着抗生素耐药性的日益普遍，我们正在迅速失去相对于我们的细菌敌人的优势。我们必须研究抗菌剂的新靶点，以避免因细菌感染而再次出现常见的死亡。在细菌病原体中表达毒力因子需要许多转录激活剂家族的成员。在一些情况下，研究表明，缺失AraC/XylS激活剂可显著降低致病作用，表明该家族成员有可能成为抗菌药物的靶点。我们的目标是确定AraC/XylS家族激活剂RHAS的小分子抑制剂及其作用机制，部分是通过确定RHAS活性的分子机制。我们的中心假设是，可以确定干扰rHAs激活转录能力的活动的小分子抑制物。我们的理论基础是，这些抑制剂有可能被开发成治疗细菌疾病的新策略。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来确定rHAS转录激活的机制-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，我们将结合体内遗传和体外研究，确定rHAS用来联系RNA聚合酶并从而激活转录的详细分子相互作用。在第三个目标中，我们将使用高通量筛选大的小分子文库来识别RHAS活性的抑制剂及其作用机制。我们的筛选将在体内进行，以提高我们确定的化合物对细菌细胞有效的机会，并将采用一种对照菌株，使我们能够从考虑中排除不直接影响RHAS活性的化合物。在未来，我们将测试在这个筛选中鉴定的化合物对细菌病原体中毒力因子的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.