Nitrate Sensing and Metabolism Modulate Motility, Biofilm Formation, and Virulence in Pseudomonas aeruginosa▿

Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa▿

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to ΔnirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14

Pseudomonas aeruginosa PA1006 is a persulfide-modified protein that is critical for molybdenum homeostasis.

A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) "top-down" analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein's persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant

<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> 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

<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

Purification and properties of His₆-PA1006 from <i>Pae</i>.

<p>(A) SDS PAGE analysis and (B) absorption spectrum of His₆-PA1006 purified to near homogeneity from <i>Pae</i>. Analytical gel-filtration chromatography (C) shows that pure His₆-PA1006 is monodisperse and approximates the size of a monomer. (D) FT-ICR-MS analysis of His₆- PA1006 purified from <i>Pae</i> shows at least two major species of m/z 1003 and m/z 1009.</p

His₆-PA1006 purified from <i>Pae</i> contains a DTT-labile species.

<p>FT-ICR-MS analysis of His₆-PA1006 purified from <i>Pae</i> before (A) and after (B) treatment with 20 mM DTT for 20 min. Species m/z 1009 (C) and m/z 1003 (D) were isolated and fragmented by CID showed an identical fragmentation pattern indicated that they are derived from the same initial peptide.</p