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.
