This application addresses broad Challenge Area (03) Biomarker Discovery and Validation and specific Challenge Topic, 03-AI-101: Identification, characterization and evaluation of novel pathogen or host targets that may lead to the development of antimicrobials with broad spectrum activity. A major challenge in the treatment of bacterial infections is the emergence of antibiotic resistance. Widely distributed, conserved families of bacterial virulence proteins are potentially ideal targets for novel antimicrobial agents. Inhibition of virulence proteins results in bacteria that are attenuated and more easily cleared by the human immune system. We have characterized a bacterial pathway that is required for function of a large family of virulence proteins. These proteins, the autotransporters, are widely distributed among gram-negative bacterial pathogens. They are secreted at the bacterial pole, a process that is required for their proper secretion and function;consequently, the polar targeting pathway presents an ideal target for the development of a novel category of antibiotics that utilize a unique mechanism of action. We have developed a reporter assay that enables us to determine whether the model autotransporter IcsA is localized or not to the bacterial pole. Our goal in this application is to use this assay to identify molecules that inhibit this family of virulence proteins and to then optimize these molecules for the inhibition of diseases caused by these bacterial pathogens. Our specific aims are as follows: Aim 1. Identify small molecules that inhibit autotransporter localization to the bacterial pole;and Aim 2. Optimize compounds and investigate their mechanisms of activity. Our long-term goal is to develop a novel category of antibiotics that utilize a unique mechanism of action. Since autotransporter proteins are not essential to bacterial survival, antibiotics that target this pathway are likely to minimally select for the development of microbial resistance. Moreover, since autotransporters are absent in eukaryotes, at least some of the compounds that are found to be active against autotransporters will likely have no adverse effects on mammalian cells. Finally, short of their development into antibiotics, the small molecules identified and characterized in this proposal would constitute valuable pharmacological tools for investigations into the fundamental mechanism of localization of autotransporters, and perhaps other polar proteins, to the bacterial pole. PUBLIC HEALTH RELEVANCE: A major challenge in the treatment of bacterial infections is the emergence of antibiotic resistance;our long-term goal is to develop a novel category of antibiotics that utilize a unique mechanism of action. We have characterized a bacterial pathway that is required for the function of a large family of virulence proteins. Our goal in this application is to identify molecules that inhibit this family of virulence proteins and to optimize these molecules for the inhibition of diseases caused by these bacterial pathogens.