The spectacular rise of bacterial antibiotic resistance is a serious problem, particularly among Gram-negative pathogens, increasing morbidity, mortality and healthcare costs. Despite over a century of intense effort focused on killing pathogenic bacteria, these organisms have developed and shared increasingly inventive ways to overcome the best drugs we have been able to produce. There is growing evidence that a different approach, aimed at inhibiting virulence without inhibiting growth per se, can prevent or cure disease without inevitably leading to resistance. Our long term goal is to develop approaches that will selectively prevent the onset of virulence in bacterial pathogens. Our objective for this application is to develop selective inhibitors that block virulence and inhibit biofilm formation in Gram-negative human pathogens without otherwise interfering with their growth. Our central hypothesis is that biosynthesis of the quorum sensing (QS) molecules that trigger a virulence response can be selectively inhibited, without affecting the essential aspects of bacterial metabolism, by altering the common precursor for these QS molecules. Because these organisms will remain viable, but functionally avirulent, there will be much weaker selective pressure for resistance to QS inhibition. We plan to accomplish our project goals through the following complementary initial specific aims: (1) design and test alternative substrates and inhibitors for target enzymes that selectively block the production of QS molecules; and (2) use a new screening method to identify QS inhibitors on a broader scale. Once these aims have been achieved we will move to Phase II of this project, which involves: (1) focusing our quorum sensing inhibitor development towards specific pathogenic species; and (2) testing the ability of these inhibitors to synergistically increase the effectiveness of currently used antibiotics. The contribution of our proposed research will be the development of an approach that leads to new drugs while minimizing selection for bacterial resistance. This work is significant because it is designed to validate an alternative approach that does not rely on microbial death to overcome bacterial infections. PUBLIC HEALTH RELEVANCE: Mortality from infections is the second leading cause of death worldwide, and is still the third leading cause in industrialized countries. The major threat comes from bacterial pathogens that have evolved resistance to most classes of antibiotics. An approach that can block virulence in Gram-negative bacteria while minimizing selection for resistance will lead to a new paradigm in the treatment of infections.