Genetic requirements for <i>Staphylococcus aureus</i> nitric oxide resistance and virulence

<div><p><i>Staphylococcus aureus</i> exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). <i>S</i>. <i>aureus</i> NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for <i>S</i>. <i>aureus</i> NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for <i>S</i>. <i>aureus</i> to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F₁F₀ ATPase, have not been previously reported to contribute to <i>S</i>. <i>aureus</i> NO· resistance. The most striking finding was that loss of any genes encoding components of the F₁F₀ ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in <i>S</i>. <i>aureus</i>, the F₁F₀ ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the Δ<i>atpG</i> mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the Δ<i>atpG</i> mutant cannot thrive. Thus, <i>S</i>. <i>aureus</i> NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F₁F₀ ATPase enzyme complex.</p></div

Optimization of an assay to select for mutant fitness during NO· stress.

<p>Known NO· sensitive mutants in <i>S</i>. <i>aureus</i> LAC were mixed with WT at a ratio of 1:100. Mixed cultures were serially passaged either <b>A.</b> aerobically or <b>B.</b> with 5mM DETA/NO. Every 12-hrs (for NO· exposed) or 5-hrs (for aerobic), cultures were diluted 1:100 into fresh media (with or without DETA/NO) and were plated on selective media for cfu enumeration. Asterisks indicate the generation at which the mutant becomes significantly underrepresented from the initial 1:100 ratio (n = 3 for each mutant, Student’s t-test).</p

NO· sensitive mutants identified by Tn-Seq are confirmed with clean deletion mutants.

<p>Representative growth curves are shown of <i>S</i>. <i>aureus</i> LAC WT and mutants grown in TSB 5g/L glucose either <b>A.</b> aerobically or <b>B.</b> with 10mM DETA/NO added at the time of inoculum (n = 3). <b>C.</b> Growth rates of mutants and a complemented Δ<i>atpG</i> relative to that of WT with/without NO· exposure at inoculum (10 mM DETA/NO).</p

Distribution of Tn-insertions along the <i>S</i>. <i>aureus</i> genome.

<p><b>A.</b> Insertions appear uniform all along the genome with no obvious gaps or hot spots. <b>B.</b> Distribution of insertions on each strand reveal no apparent strand bias. <b>C.</b> 2,132 Tn insertions were at identical TA dinucleotides but on opposite strands. The 1066 sites appear to be uniformly distributed across the entire genome.</p

Δ<i>atpG</i> exhibits both elevated ATP levels and membrane potential both before and after NO· exposure.

<p><i>S</i>. <i>aureus</i> WT and Δ<i>atpG</i> were grown in TSB 5g/L glucose and were exposed to NO· mix (10mM NOC12, 1mM DEANO) at OD₆₅₀ 0.25. <b>A.</b> Prior to NO· addition and at 1-hr post addition ATP levels were determined. <b>B.</b> Prior to NO· addition and 1-hr post addition, membrane potential was quantified. (n = 2 for ATP levels, n = 3 for membrane potential). Significance was determined with two-sided Student’s t tests (*, P ≤ .05; **, P ≤ .01).</p

Δ<i>atpG</i> is attenuated during non-respiratory growth and is also sensitive to peroxide and kanamycin.

<p><b>A.</b> and <b>B.</b> <i>S</i>. <i>aureus</i> LAC WT and Δ<i>atpG</i> were grown in TSB 5g/L glucose aerobically, with 10mM DETA/NO, with 2mM 2,2’-dipyridyl, anaerobically, or anaerobically with nitrate. <b>A.</b> Maximum absorbance (650nm) reached over a 24-hr growth curve and <b>B.</b> maximum growth rate reached over a 24-hr growth curve are shown for the Δ<i>atpG</i> mutant as a percentage of the WT maximums. <b>C.</b> Minimum inhibitory concentrations were measured for <i>S</i>. <i>aureus</i> WT and Δ<i>atpG</i> in TSB 5g/L glucose and are shown as a ratio to the WT MIC (n = 3).</p

The Δ<i>atpG</i> mutant is severely attenuated in a murine skin abscess model.

<p><b>A.</b> Abscess area over time; the Δ<i>atpG</i> mutant did not cause visible abscesses. <b>B.</b> Viable cfu within abscesses 3 days post inoculation.</p

Biphasic distribution of representation values (R) for all genes in <i>S</i>. <i>aureus</i>.

<p>R-values below 3 SD of the log transformed mean are considered essential whereas R-values between 2 and 3 SD below the mean are considered to be required for full fitness.</p