<em>Pseudomonas aeruginosa</em> PA1006, Which Plays a Role in Molybdenum Homeostasis, Is Required for Nitrate Utilization, Biofilm Formation, and Virulence

<div><p><em>Pseudomonas aeruginosa (Pae)</em> is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that Δ<em>PA1006</em> cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of Δ<em>PA1006</em> aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of <em>PA1006</em> confers extensive changes in <em>Pae</em> metabolism. Based upon homology of PA1006 to the <em>E. coli</em> YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the Δ<em>PA1006</em> mutant.</p> </div

<i>PA1006</i> is necessary for virulence.

<p>A/B) Mouse thermal injury. A) Mice were scalded as described in Materials and Methods and a total of 1×10³ CFU of the <i>Pae</i> strain to be tested was injected subcutaneously in the burn eschar immediately after burning. Mortality was observed for 5 days post-burn/infection. Three separate experiments were conducted with each strain. The average percent mortality values are shown (** = p<0.01, n = 15/strain tested). (•) WT; (○)Δ<i>PA1006</i>; (▾)Δ<i>PA1006:attb:PA1006</i>. B) <i>PA1006</i> is required for full dissemination in the mouse thermal injury model. Quantitation of bacteria recovered from the livers of burned and infected mice. The number of CFU was calculated per gram of tissue. p = 0.04 (between PAO1 and PA1006), and p = 0.0002 (between PA1006 and the complemented strain), via student t-test. There were 10 mice total for each group. C) Effect of Δ<i>PA1006</i> on inflammation in a rat lung model of infection. ^aMean ± SD. ANOVA, Bonferroni multiple comparisons test indicated: P<0.001 for PAO1 vs <i>ΔPA1006</i>, P>0.05 for PAO1 vs Δ<i>PA1006</i>:<i>attb:PA1006</i>), and P<0.001 for Δ<i>PA1006</i> vs Δ<i>PA1006</i>:<i>attb:PA1006</i>).</p

<i>PA1006</i> is critical for nitrate reductase activity.

<p>A/B) <i>PA1006</i> does not appear to affect aerobic growth in rich media but is required for anaerobic growth with nitrate. (•) WT; (○)Δ<i>PA1006</i>; (▾)Δ<i>PA1006:attb:PA1006</i>. Growth curves were performed in duplicate as indicated in the Methods average values are plotted. Data showed excellent agreement. C) Δ<i>PA1006</i> whole cell suspensions lack periplasmic and membrane nitrate reductase activity. D) Western blot with α-NarGH antisera of whole cell extract of wild-type (wt) and Δ<i>PA1006</i> (Δ) cells indicates that the membrane nitrate reductase is present but inactive. E) Summary of nitrate and nitrite reductases in <i>Pae</i>, their cofactors, and what is known about functionality in the Δ<i>PA1006</i> mutant.</p

<i>PA1006</i> affects PQS production.

<p>PQS production by <i>Pae</i> strains. PQS samples extracted from 24 h cultures were analyzed by TLC. The arrowhead indicates the position of PQS.</p

Notable nitrate metabolism and virulence genes whose expression levels are altered in the ΔPA1006 mutant in the absence of nitrate.

<p>Notable nitrate metabolism and virulence genes whose expression levels are altered in the ΔPA1006 mutant in the absence of nitrate.</p

Activation and inhibition of PqsR in <i>P. aeruginosa</i> by the 2-alkyl-4(1<i>H</i>)-quinolones.

<p>*EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsA</i> CTX::<i>pqsA'-lux</i> strain; **EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsAH</i> CTX::<i>pqsA'</i>-lux strain; – no activity.</p

Crystallographic data collection and refinement statistics.

*<p>Values in parentheses are for highest-resolution shell.</p>a<p>R_merge = Σh Σi|<i>I</i>i(h)−<<i>I</i>(h)>/|Σh Σi <i>I</i>i(h), where <i>I</i> is the observed intensity and <<i>I</i>> is the average intensity of multiple observations from symmetry-related reflections calculated with XDS.</p>b<p>Correlation co-efficient value calculated using XDS to determine the resolution cutoff.</p>c<p>All values calculated using REFMAC. R_work = Σh||Fo|h−|Fc|h|/Σh|Fo|h, where Fo and Fc are the observed and calculated structure factors, respectively. R_free computed as in R_work, but only for (5%) randomly selected reflections, which were omitted in refinement.</p

3-NH₂-7Cl-C9-QZN is a competitive inhibitor of PqsR.

<p>(<b>A</b>) PqsR activity as reflected by the maximum bioluminescence produced by a miniCTX::<i>pqsA'-lux</i> fusion in a Δ<i>pqsA</i> mutant in the presence of 100 µM QZN (inset) and increasing concentrations of PQS; (<b>B</b>) Topology diagram of the PqsR^CBD ligand-binding site occupied by the QZN shown in stick (orange), with hydrogen bonds coloured purple; (<b>C</b>) Orientation of 4-quinolone ring (right panel) and 7Cl-substituted QZN ring (left panel) within the PqsR ligand-binding pocket. The 7Cl is accommodated within a crevice forming a hydrogen bond (dotted line) with Thr265 (left panel); (<b>D</b>) Superposed PqsR^CBD-3-NH₂-7Cl-C9-QZN and PqsR^CBD-NHQ structures with residues shown as stick (orange and yellow respectively).</p

Response of PqsR^CBD ligand binding site mutants to AQs.

<p>(<b>A</b>) PqsR-6His is functional in <i>P. aeruginosa</i>. The <i>pqsR</i> gene with or without a 6His tag on pME6032 was introduced into a <i>P. aeruginosa pqsR</i> deletion mutant (Δ<i>pqsR</i>) containing a chromosomal miniCTX::<i>pqsA'-lux</i> reporter gene fusion. Relative PqsR activity was determined as a % of the maximum bioluminescence produced by the miniCTX::<i>pqsA'-lux</i> reporter fusion in a wild type <i>P. aeruginosa</i> PAO1 background carrying the empty pME6032 vector; (<b>B</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with one of each of the 13 site-specific mutations introduced into the PqsR^CBD ligand-binding pocket. Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to PAO1 Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> expressing the PqsR-6His protein (WT); (<b>C</b>) Western blot analysis confirming expression of each of the PqsR-6His mutant proteins; (<b>D</b>) Light output from the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain transformed with either the gene coding for PqsR-6His or one of the 13 site-specific mutants and supplemented with either HHQ or PQS (40 µM). Bioluminescence is presented as % of <i>pqsA</i> promoter activity with respect to the <i>P. aeruginosa</i> Δ<i>pqsA</i> Δ<i>pqsH</i> Δ<i>pqsR</i> miniCTX::<i>pqsA'-lux</i> strain expressing PqsR-6His.</p

Activation and inhibition of PqsR in <i>P. aeruginosa</i> by the 2-alkyl-4(3<i>H</i>)-quinazolinones.

<p>*EC₅₀ determined in a <i>P. aeruginosa</i> Δ<i>pqsA</i> miniCTX::<i>pqsA'-lux</i> strain; **IC₅₀ determined in a <i>P. aeruginosa</i> wild type strain incorporating a miniCTX::<i>pqsA'-lux</i> fusion; – no activity; <b>^‡</b>compounds exhibited growth inhibition; X compounds did not dissolve in MeOH at a workable concentration; 3-NH₂-7Cl-PhOBn-QZN (46) is barely soluble.</p