Nonencapsulated (nontypeable) Haemophilus influenzae (NTHi) are human-adapted commensal organisms that can also cause chronic mucosal infections, particularly otitis media. Since NTHi are not susceptible to the routinely-administered Hib vaccine, these infections are usually treated with antibiotics. Many children exhibit recurrent otitis media caused by NTHi despite antibiotic treatment, and these episodes can lead to deafness and language development delays in early life. Although the ability to form biofilms has been implicated in persistent infections, the exact molecular mechanism by which NTHi causes chronic disease is not fully understood. NTHi contain a set of four highly conserved gene pairs termed toxin-antitoxin (TA) loci. Our preliminary data shows that the deletion of two of these gene pairs significantly reduces the ability of the organism to cause long-term infections in a primary human tissue model. Our hypothesis is that these four TA loci are involved in protease-mediated metabolic regulation that allows the organism to enter a state of growth arrest precipitated by stress (such as antibiotic therapy or nutrient limitation), and this enhances their ability to survive under these suboptimal conditions. Once conditions improve (e.g. cessation of antibiotic treatment, or nutrient upshift), bacterial growth resumes. Since most antibiotics target essential biosynthetic pathways that are not utilized during growth arrest, this mechanism could explain the observed clinical failure in children with recurrent otitis media, and result in new targets for the treatment of chronic infections. To investigate this, we aim to a) progressively delete all four TA loci from two different NTHi strains; b) assess the ability of these mutants to survive in long-term infections of a primary human tissue model grown at the air-liquid interface, and c) analyze the mutants that were found to be significantly attenuated for persistence in the chinchilla model of otitis media. Because TA loci appear in hundreds of different bacterial species, in addition to discovering novel targets for immunological or pharmacological intervention for those patients that exhibit chronic disease, these studies have implications for the treatment of many other organisms that persist within a host or in the environment. PUBLIC HEALTH RELEVANCE: antibiotic treatment failure in children that results in recurrent middle ear infections (otitis media). These episodes can lead to deafness as well as language development delays. Our research could lead to the identification of new targets for vaccines or pharmaceuticals that would synergize with existing treatments for those patients that exhibit chronic otitis media.