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.Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of causing infection in the immunocompromised host. The types of infection caused by P. aeruginosa include otitis media (19), infection of burn wounds (37), and lung infection in cystic fibrosis (CF) patients (27). In many instances, infection by P. aeruginosa assumes the form of a biofilm, which is highly resistant to antibiotics and to attack by immune effector cells (20).P. aeruginosa growth in biofilms is characterized by its ability to grow in nutrient-and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through a denitrification enzyme pathway that catalyzes a four-step sequential reduction of nitrate to nitrogen gas, with nitrite, nitric oxide, and nitrous oxide, respectively, as intermediates. Two different nitrate reductase complexes mediate nitrate reduction to nitrite in P. aeruginosa (4, 68), a plasma membrane-bound nitrate reductase complex encoded by the narK1K2GHJI operon and a periplasmic nitrate reductase encoded by napEFDABC. Reduction of either nitrate or nitrite substrates provides energy for P. aeruginosa anaerobic growth, with nitrate reduction to nitrite via nitrate reductase contributing more significantly to proton motive force and hence energy production (4, 68). A well-described environment for P. aeruginosa growth under anoxic conditions is as a biofilm within the mucus of the CF lung (66). Nitrate and nitrite levels in CF mucus, generated in part by the host inflammatory response to infection, are sufficient to support anaerobic metabolism of P. aeruginosa (27).Biofilm formation and organism dispersal leading to spread of infection by P. aeruginosa are dependent on motility. ...
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 (⌬narGH) and nitrite reductase (⌬nirS) 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 ⌬nirS 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.The ubiquitous gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for both acute and chronic infections. P. aeruginosa is commonly an etiologic agent in ear infections (8), infections of burn wounds (27), corneal keratitis (21, 30), and pulmonary infections in patients with cystic fibrosis (16) and ventilator-associated pneumonia (5). P. aeruginosa is frequently resistant to conventional antibiotic therapy and the antimicrobial effector mechanisms of phagocytes (9), particularly in the biofilm mode of growth (15).The establishment of P. aeruginosa infection is accompanied by the synthesis of a diverse array of virulence factors composed of various exoproteins, such as elastase and exotoxin A, as well as the mucoid exopolysaccharide alginate (20). The type III secretion system (T3SS), a mechanism whereby cytotoxic effector proteins are directly secreted into the host cell cytoplasm following contact of the bacterium with a target cell (11), has also been identified as a virulence determinant of P. aeruginosa that contributes significantly to the pathogenesis of acute infection (45, 46). The P. aeruginosa T3SS plays a major role in triggering cell death in phagocytes and epithelial cells (17) and is composed of at least 20 proteins, including (i) a secretory apparatus, (ii) machinery devoted to the direct translocation of effectors into the host cell cytoplasm, and (iii) four effector proteins, ExoS, ExoT, ExoU, and ExoY, that contribute to host cell toxicity (19,46). Regulation of virulence factor expression in P. aeruginosa is hierarchical and highly complex, involving quorum sensing (37, 44) and responses to environmental signals, including stresses applied via host defense mechanisms (46). P. aeruginosa is capable of growin...
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.
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