The ToxAvapA Toxin-Antitoxin Locus Contributes to the Survival of Nontypeable Haemophilus influenzae during Infection

Nontypeable Haemophilus influenzae (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 host inhibition of growth (higBA) homologue. We hypothesized that this locus, which we designated toxAvapA, contributed to NTHi survival during infection. We deleted toxAvapA 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 in vitro co-culture with primary human respiratory tissues, which revealed that the DeltatoxAvapA 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 in vivo. Following purification, the toxin exhibited ribonuclease activity on RNA in vitro, 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 toxAvapA TA module in persistence following stress is multifactorial and includes effects on essential metabolic pathways

HPLC results of the relative amounts of tryptophan in derivatized samples of conditioned medium from the wild-type or mutant NTHi strains.

<p>HPLC results of the relative amounts of tryptophan in derivatized samples of conditioned medium from the wild-type or mutant NTHi strains.</p

The <i>ToxAvapA</i> Toxin-Antitoxin Locus Contributes to the Survival of Nontypeable <i>Haemophilus influenzae</i> during Infection

<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

Number of viable gentamicin-resistant internalized bacteria that survived over time in the EpiAirway human tissue model (n = 6).

<p>Number of viable gentamicin-resistant internalized bacteria that survived over time in the EpiAirway human tissue model (n = 6).</p

Fold decrease in transcription of the tryptophan biosynthesis regulon in the NTHi Δ<i>toxAvapA</i> mutant compared to the wild-type strain.

<p>Fold decrease in transcription of the tryptophan biosynthesis regulon in the NTHi Δ<i>toxAvapA</i> mutant compared to the wild-type strain.</p

H&E-stained sections of chinchilla middle ears (400× magnification).

<p>A. Middle ear mucosa of an uninfected control animal. B. The middle ear of an animal infected with the Δ<i>toxAvapA</i> mutant for 4 days. Note the characteristic goblet cell hyperplasia associated with a productive NTHi infection. C. The middle ear of an animal infected with the wild type parent strain 86-028NP. The middle ear lumen is at the top of each micrograph.</p

Growth curve of the Δ<i>toxAvapA</i> mutant and parent strain in defined media.

<p>No significant difference was found between the growth of the parent and mutant strains (n = 3 in duodecuplicate).</p

HPLC results of the relative amounts of tryptophan in derivatized samples of conditioned medium from the wild-type or mutant NTHi strains.

<p>HPLC results of the relative amounts of tryptophan in derivatized samples of conditioned medium from the wild-type or mutant NTHi strains.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Gross otoscopic view of the tympanic membrane of a chinchilla.

<p>The same animal imaged (A) before infection; and (B) 4 days after inoculation with the Δ<i>toxAvapA</i> mutant. Edema and redness indicate a productive infection.</p