<div><p>Nontypeable <i>Haemophilus influenzae</i> (NTHi) is an opportunistic pathogen that is a common cause of acute and recurrent mucosal infections. One uncharacterized NTHi toxin-antitoxin (TA) module, NTHI1912-1913, is a <b><i><u>h</u></i></b>ost <b><i><u>i</u></i></b>nhibition of <b><i><u>g</u></i></b>rowth (<i>higBA</i>) homologue. We hypothesized that this locus, which we designated <i>toxAvapA</i>, contributed to NTHi survival during infection. We deleted <i>toxAvapA</i> and determined that growth of the mutant in defined media was not different from the parent strain. We tested the mutant for persistence during long-term <i>in vitro</i> co-culture with primary human respiratory tissues, which revealed that the Δ<i>toxAvapA</i> mutant was attenuated for survival. We then performed challenge studies using the chinchilla model of otitis media and determined that mutant survival was also reduced <i>in vivo</i>. Following purification, the toxin exhibited ribonuclease activity on RNA <i>in vitro</i>, while the antitoxin did not. A microarray comparison of the transcriptome revealed that the tryptophan biosynthetic regulon was significantly repressed in the mutant compared to the parent strain. HPLC studies of conditioned medium confirmed that there was no significant difference in the concentration of tryptophan remaining in the supernatant, indicating that the uptake of tryptophan by the mutant was not affected. We conclude that the role of the NTHi <i>toxAvapA</i> TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways.</p></div
