PROJECT SUMMARY / ABSTRACT Despite extensive studies of cystic fibrosis (CF) bacterial pathogens, the efficacy of antibiotics achieved in vitro seldom translates to successful clinical outcomes. This limitation is partially driven by our lack of understanding of the host environments to which bacteria adapt, and the metabolic strategies that sustain community growth in vivo. Studies that generate greater insight into what CF pathogens are doing within the cystic fibrosis airways are necessary to inform more effective therapeutic strategies. This proposal addresses how CF pathogens obtain nutrients within the lung. Despite the presence of bacterial cells at high densities, the nutrient source(s) that sustains their growth is not apparent; the predominant carbon reservoir, mucins, are recalcitrant to degradation by the canonical pathogen Pseudomonas aeruginosa. Given their abundance in the lung and their ability to degrade salivary mucins, we propose that anaerobes typically associated with the oral cavity, facilitate carbon acquisition by Pseudomonas, which in turn drives disease. More specifically, commensal bacteria (e.g. Prevotella, Veillonella) are able to thrive off respiratory mucin secretions, releasing mixed fermentation byproducts. We hypothesize that if allowed to accumulate (due to impaired mucus clearance), fermentation-derived metabolites can support the growth of other organisms. We have recently shown that saliva-derived bacterial communities stimulate the growth of P. aeruginosa when provided mucin as the sole carbon source. This suggests that mucin-degrading anaerobes may support pathogens during infection. Based on these data, this project will use a multi-disciplinary approach to (1) identify the anaerobic bacteria and the mucin-derived metabolites they generate that can support P. aeruginosa growth, (2) Use a stable isotope labeling approach to track the flow of mucin-derived carbon throughout CF sputum, (3) Use a mucin-overproducing cell line to assess Pseudomonas-epithelium interactions in the presence of mucin-degrading anaerobes, and (4) use a murine chronic lung infection model to assess the growth and pathogenicity of Pseudomonas in vivo when co-infected with oral-derived anaerobes. At the completion of this study, we will have defined a pathogenic role for oral-associated bacteria in the lower airways and provide a detailed characterization of carbon flow within the CF lung. Altogether, these studies will have a meaningful impact on our understanding of the functional role of the CF microbiota and the potential development of therapeutic strategies. While the CF microbiome will serve as a starting point to investigate mucin-derived cross-feeding interactions, our approach is equally applicable to the prevention of other respiratory diseases – COPD, chronic sinusitis, and ventilator- associated pneumonias – where a viscous mucus environment harbors complex bacterial infections.