Bacterial adaptation is associated with tryptophan metabolism and persistence of nontypeable Haemophilus influenzae

Bacterial adaptation to the nutritional environment of the host is critical for infection. We hypothesize that responses to nutrient stress through microevolution enhance invasion, intracellular bacterial community (IBC) formation, and persistence of nontypeable Haemophilus influenzae (NTHI). We previously identified a mutant that arose during prolonged nutrient deprivation and exhibited in vitro persistence and increased IBC formation during experimental otitis media (OM). A single mutation in the gene encoding cyclic AMP phosphodiesterase reprioritizes NTHI metabolism during biofilm growth in this evolved strain with a significant increase in all proteins involved in tryptophan biosynthesis. Given the importance of tryptophan and tryptophan metabolites in host immunity and bacterial virulence, we evaluated the contribution of tryptophan biosynthesis and import on NTHI pathogenesis. Through mutational analyses, NTHI viability in vitro is dependent on tryptophan biosynthesis but independent of tryptophan import. We also observed decreased persistence of IBCs in the absence of tryptophan biosynthesis. In an experimental model of OM, both tryptophan biosynthesis and import mutant strains demonstrated increased fitness compared to the parental strain. The NTHI trp operon has a distinct organization from Escherichia coli and contains an ortholog of ydfG , an NADP-dependent 3-hydroxyl acid dehydrogenase, that contributes to oxidative stress resistance. This ydfG ortholog exhibits greater frequency in mucosal-associated NTHI strains, as compared with commensal or invasive strains. Understanding the underlying mechanisms that NTHI use to respond and survive within the host environment, particularly tryptophan biosynthesis, will inform the development of therapeutics to attenuate current NTHI infections and limit recurrent episodes of OM. IMPORTANCE Bacteria exquisitely adapt to environmental cues to survive and, in some cases, cause disease. We observed that bacteria starved for heme-iron evolve to increase the expression of tryptophan biosynthesis genes. We determined that loss of tryptophan biosynthesis decreased intracellular bacterial survival. In an experimental model of middle ear infection, both tryptophan biosynthesis and import mutant strains demonstrated increased fitness compared to the parental strain. We determined that the organization of tryptophan biosynthesis and transport genes is distinct and contains a gene that encodes a protein that contributes to oxidative stress resistance. This gene exhibits greater frequency in mucosal-associated NTHI strains, as compared with commensal or invasive strains. These data reveal coordination of tryptophan metabolism with oxidative stress resistance for bacterial persistence.