Abstract Infections caused by nontypeable Haemophilus influenzae (NTHI) strains represent a major cause of otitis media (OM), exacerbations of chronic obstructive pulmonary disease (COPD), and bacterial sinusitis. Furthermore, NTHI causes chronic, treatment-refractory diseases, in part by forming intracellular bacterial communities (IBCs) within epithelial cells that provide a refuge from both the host immune system, and from clinical interventions. The bacterial and host elements that impact NTHI persistence and recurrence of OM are not completely understood. Therefore, the delineation of host-pathogen interactions and metabolic influences that lead to OM- derived sequelae will inform novel therapeutic approaches. Our prior studies revealed a significant increase in all tryptophan biosynthetic pathway proteins during biofilm growth of a persistent isolate of NTHI. This isolate also demonstrated a significant increase in IBC formation during experimental OM. Critical to host-pathogen interactions is modulation of tryptophan levels. Tryptophan biosynthesis promotes intracellular survival of other pathogens and degradation of host tryptophan limits bacterial growth. In this study, we will delineate the contribution of tryptophan metabolism to NTHI pathogenesis during OM. Our central hypothesis is that NTHI adaptation to nutrient stress enhances invasion, IBC formation, persistence and recurrence. Our published studies indicate that multiple aspects of metabolism contribute to NTHI IBC formation and persistence. Our approach is to Determine the contribution of tryptophan metabolism in NTHI persistence and IBC formation (Aim 1). We hypothesize that bacterial adaptation to nutrient limitation modulates NTHI tryptophan biosynthesis that directly impacts pathogenesis and persistence. We will also evaluate the efficacy of interference with tryptophan biosynthesis as a novel therapeutic target (Aim 2). We hypothesize that prevention of invasion of NTHI into epithelial cells will promote bacterial clearance and thereby reduce the potential for recurrence of OM. Our studies will reveal mechanisms of tryptophan acquisition on NTHI behaviors that correlate with host responses during OM. Exploitation of these pathways to mitigate IBC development will provide a platform to continue to develop combinatorial treatments that target both the extracellular and intracellular populations of NTHI as an advanced therapeutic option to treat clinical disease.