PROJECT SUMMARY Antibiotics are critical therapeutics for modern medicine, but their frequent misuse has led to the emergence of multidrug-resistant organisms and can perturb the endogenous microbiome, providing opportunities for secondary infection by opportunistic pathogens. The long-term goal of this work is to develop novel therapeutics that are “microbiome safe” by determining how antibiotics and other bioactive secondary metabolites (SMs) mediate interspecies interactions between commensal bacteria and pathogens in the human microbiome. The overall objectives in this application are to (i) assess how co-culture with niche-specific pathogens shapes secondary metabolism in commensal bacteria from the human aerodigestive tract (ADT) and (ii) isolate and identify SMs with antibiotic activity against pathogens. The central hypothesis is that bacteria in the ADT modulate their secondary metabolism in response to invading pathogens and produce antibiotics that benefit the host and microbiome. The rationale for this project is that metabolites produced by symbiotic bacteria are likely to have been evolutionarily selected for narrow-spectrum activity and to not exert toxic effects on the host or microbiome. Such metabolites will become leads for the development of new therapeutics. The central hypothesis will be tested by pursuing two specific aims: 1) Assess how pathogen exposure changes secondary metabolism in commensal ADT bacteria; and 2) Isolate and characterize bioactive SMs produced by ADT bacteria using genetics and genomics. Under Specific Aim 1, select bacterial genera that have been previously shown to be rich in biosynthetic gene clusters (BGCs) for SM production will be cultured from nasal and oral specimens. Co-culture bioassays and comparative metabolomics will be used to assess how exposure to either ecologically relevant or general pathogens changes SM production in these bacteria. For Specific Aim 2, the genomes of ADT bacteria that inhibit pathogens in vitro will be sequenced to identify their encoded BGCs. Then, BGCs associated with bioactivity against pathogens will be identified using comparative genomics and systematically disrupted. After comparing the metabolite production of ADT bacteria and their isogenic BGC mutants, molecular features responsible for antibiotic activity will be identified and purified. Mass spectrometry and nuclear magnetic resonance spectroscopy will be used for structural elucidation of these compounds. The research proposed in this application is innovative because it departs from current approaches to drug discovery and instead focuses on ecologically relevant interactions to reveal fundamental biology and chemistry of human ADT microbiomes. The proposed research is significant because it could lead to the discovery of new SMs that can be developed into narrow-spectrum antibiotics that specifically eliminate pathogens. Ultimately, ecologically inspired screening of the human microbiome may represent ...