2-heptyl-3-hydroxy-4-quinolone and Pseudomonas-Infections

Pseudomonas quinolone signal (PQS) is a quorum-sensing molecule associated with Pseudomonas aeruginosa that regulates quorum sensing, extracellular vesicle biogenesis, iron acquisition, and the secretion of virulence factors. PQS has been shown to have immunomodulatory effects on the host. It induces oxidative stress, modulates cytokine levels, and activates regulated cell death in the host. In this study, we investigated the effects of PQS (10 μM) on host organelle dynamics and dysfunction in human macrophages at the interphase of endoplasmic reticulum (ER), mitochondria, and lysosome. This study showed that PQS increases cytosolic Ca

Topics: Humans; Inflammation; Macrophages; Organelles; Pseudomonas aeruginosa; Pseudomonas Infections

During infections, fast identification of the microorganisms is critical to improve patient treatment and to better manage antibiotics use. Electrochemistry exhibits several advantages for rapid diagnostic: it enables easy, cheap and in situ analysis of redox molecules in most liquids. In this work, several culture supernatants of different Pseudomonas aeruginosa strains (including PAO1 and its isogenic mutants PAO1ΔpqsA, PA14, PAK and CHA) were analyzed by square wave voltammetry on glassy carbon electrode during the bacterial growth. The obtained voltamograms shown complex traces exhibiting numerous redox peaks with potential repartitions and current amplitudes depending on the studied bacterium and/or growth time. Among them, some peaks were clearly associated to the well-known redox toxin Pyocyanin (PYO) and the autoinducer Pseudomonas Quinolone Signal (PQS). Other peaks were observed that are not yet attributed to known secreted species. Each complex electrochemical response (number of peaks, peak potential and amplitude) can be interpreted as a fingerprint or "ID-card" of the studied strain that may be implemented for fast bacteria strain identification.

Topics: Electrochemical Techniques; Humans; Oxidation-Reduction; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Quinolones

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; China; Drosophila melanogaster; Drosophila Proteins; Female; Flavonoids; Models, Animal; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Rats; Rats, Sprague-Dawley; Transcription Factors; Type III Secretion Systems; Virulence; Virulence Factors

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) - a crucial transcriptional regulator serving major functions in PA virulence - can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC

Topics: Animals; Biofilms; Disease Models, Animal; Mice; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Tobramycin

Reduced efficacy of antibiotics in bacterial diseases is a global concern in clinical settings. Development of anti-virulence compounds which disarm bacterial virulence is an attractive therapeutic agent for complementary antibiotics usage. One potential target for anti-virulence compounds is quorum sensing (QS), the intercellular communication system in most pathogens, such as Pseudomonas aeruginosa. QS inhibitors (QSIs) can inhibit QS effectively, attenuate QS-mediated virulence, and improve host clearance of infections. While studies focused on developing homoserine-based las QSI, few targeted the quinolone-based pqs QS, which implicated host cytotoxicity and biofilm formation. It is imperative to develop novel anti-pqs-QS therapeutics for combinatorial antibiotic treatment of microbial diseases. We employed a gfp-based transcriptional pqs biosensor to screen a natural compounds library and identify vanillin (4-hydroxy-3-methoxybenzaldehyde), the primary phenolic aldehyde of vanilla bean. The vanillin inhibited pqs expression and its associated phenotypes, namely pyocyanin production and twitching motility in P. aeruginosa. Molecular docking results revealed that vanillin binds to the active site of PqsR, the PQS-binding response regulator. Combinatorial treatment of vanillin with antimicrobial peptide (colistin) inhibited biofilm growth in vitro and improved treatment in the in vivo C. elegans acute infection model. We demonstrated that vanillin could dampen pqs QS and associated virulence, thus providing novel therapeutic strategies against P. aeruginosa infections.

Topics: Animals; Benzaldehydes; Biofilms; Caenorhabditis elegans; Catalytic Domain; Colistin; Drug Therapy, Combination; Gene Expression; Models, Molecular; Molecular Docking Simulation; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Quinolones; Quorum Sensing; Virulence

Pseudomonas aeruginosa is an important opportunistic pathogen normally associated with increasing morbidity and mortality of immunocompromised hosts with respiratory infections. The phenotypic and genetic features of P. aeruginosa from patients with chronic obstructive pulmonary disease (COPD) remain poorly understood. By using the sputum samples of 25 hospitalized COPD patients from the affiliated hospital of Southwest Medical University (China), we identified a P. aeruginosa isolate, COP2, which showed multiple antibiotic resistance and enhanced Pseudomonas quinolone signal (PQS) production but decreased motility, biofilm formation and virulence compared with the model strain PAO1. Importantly, COP2 harbored a substantial amount of mutations that might influence the functions of 1771 genes in the genome and the evolutionary status of this isolate was clearly distinct from the PAO1 lineage. Accordingly, COP2 had a discrepant transcriptional pattern relating to flagellar assembly, antibiotic resistance, biofilm and PQS production, and can increase the capacities of compound degradation in response to resource/space stresses. Therefore, the identification of COP2 in this study provides preliminary information regarding the genetic features and survival strategy of P. aeruginosa in colonizing COPD lungs and lays the foundations for further understanding of the pathogenic mechanisms of pseudomonal infections.

Topics: Biofilms; China; Drug Resistance, Bacterial; Humans; Locomotion; Lung; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Disease, Chronic Obstructive; Quinolones; Sputum; Transcriptome; Virulence; Whole Genome Sequencing

There is a general lack of understanding about how communities of bacteria respond to exogenous toxins such as antibiotics. Most of our understanding of community-level stress responses comes from the study of stationary biofilm communities. Although several community behaviors and production of specific biomolecules affecting biofilm development and associated behavior have been described for

Topics: Anti-Bacterial Agents; Biofilms; Humans; Hydroxyquinolines; Mass Spectrometry; Microbial Viability; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Spectrum Analysis, Raman; Tobramycin

The small RNA ReaL of the opportunistic pathogen Pseudomonas aeruginosa has been characterized. Our results indicate that ReaL contributes to P. aeruginosa virulence. In the Galleria mellonella infection model, reaL gene deletion resulted in decreased virulence, while ReaL overexpression resulted in a hyper-virulent phenotype. We also demonstrate that ReaL is embedded in the P. aeruginosa quorum sensing (QS) with the role of linking las to pqs systems. We show that ReaL is negatively regulated by the las regulator LasR and impacts positively the synthesis of the pqs quinolone signal PQS by a positive post-transcriptional effect on the pqsC gene. Perturbations of ReaL levels affect pyocyanin synthesis, biofilm formation and swarming motility, processes that are known to be influenced by PQS synthesis. In addition to being regulated by LasR, ReaL is also responding to infection relevant cues that P. aeruginosa can experience in mammalian hosts such as temperature and oxygen availability. Furthermore, ReaL shows a growth phase-dependent pattern of expression, being up-regulated in stationary phase, due to the activity of the alternative σ factor RpoS. Together, these regulations of ReaL expression are expected to contribute to the fine co-modulation of PQS synthesis and, ultimately, virulence.

Topics: Animals; Bacterial Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Moths; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Quinolones; Quorum Sensing; RNA, Small Nuclear; Sigma Factor; Signal Transduction; Trans-Activators; Virulence

Quorum sensing inhibitors (QSIs) act as antivirulent agents since quorum sensing (QS) plays a vital role in regulating pathogenesis of Pseudomonas aeruginosa. However, application of single QSI may not be effective as pathogen is vulnerable to successful mutations. In such conditions, combination of QSIs can be exploited as there can be synergistic or adjuvant action. In the present study, we evaluated the antivirulence efficacy of combination of Vaccinium macrocarpon proanthocyanidin active fraction (PAF) and ciprofloxacin (CIP) at their sub-MICs using standard methods followed by analysis of their mode of action on QS using TLC and molecular docking. There was significant improvement in action of CIP when it was combined with PAF in reducing the QS controlled virulence factors (p < 0.05), motilities and biofilm of P. aeruginosa. TLC profiles of QS signals [(Acyl homoserine lactone (AHL) and Pseudomonas quinolone signal (PQS)] indicated that CIP in combination with PAF, besides showing inhibitory action on production of AHLs, also modulated production and inactivation of PQS. Docking scores also supported the observation. We therefore hypothesize that PAF-CIP combination, having improved anti-virulence property; can be exploited as a potent drug pairing against P. aeruginosa.

Topics: 4-Butyrolactone; Acyl-Butyrolactones; Adjuvants, Pharmaceutic; Anti-Bacterial Agents; Biofilms; Ciprofloxacin; Drug Synergism; Microbial Sensitivity Tests; Molecular Docking Simulation; Plant Extracts; Proanthocyanidins; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Vaccinium macrocarpon; Virulence

Cystic fibrosis (CF) lungs are filled with thick mucus that obstructs airways and facilitates chronic infections. Pseudomonas aeruginosa is a significant pathogen of this disease that produces a variety of toxic small molecules. We used molecular networking-based metabolomics to investigate the chemistry of CF sputa and assess how the microbial molecules detected reflect the microbiome and clinical culture history of the patients. Metabolites detected included xenobiotics, P. aeruginosa specialized metabolites and host sphingolipids. The clinical culture and microbiome profiles did not correspond to the detection of P. aeruginosa metabolites in the same samples. The P. aeruginosa molecules that were detected in sputum did not match those from laboratory cultures. The pseudomonas quinolone signal (PQS) was readily detectable from cultured strains, but absent from sputum, even when its precursor molecules were present. The lack of PQS production in vivo is potentially due to the chemical nature of the CF lung environment, indicating that culture-based studies of this pathogen may not explain its behavior in the lung. The most differentially abundant molecules between CF and non-CF sputum were sphingolipids, including sphingomyelins, ceramides and lactosylceramide. As these highly abundant molecules contain the inflammatory mediator ceramide, they may have a significant role in CF hyperinflammation. This study demonstrates that the chemical makeup of CF sputum is a complex milieu of microbial, host and xenobiotic molecules. Detection of a bacterium by clinical culturing and 16S rRNA gene profiling do not necessarily reflect the active production of metabolites from that bacterium in a sputum sample.

Topics: Adolescent; Ceramides; Cystic Fibrosis; Humans; Lung; Metabolome; Microbiota; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; RNA, Ribosomal, 16S; Sputum; Xenobiotics

Pseudomonas aeruginosa quorum-sensing (QS) is a sophisticated network of genome-wide regulation triggered in response to population density. A major component is the self-inducing pseudomonas quinolone signal (PQS) QS system that regulates the production of several nonvital virulence- and biofilm-related determinants. Hence, QS circuitry is an attractive target for antivirulence agents with lowered resistance development potential and a good model to study the concept of polypharmacology in autoloop-regulated systems per se. Based on the finding that a combination of PqsR antagonist and PqsD inhibitor synergistically lowers pyocyanin, we have developed a dual-inhibitor compound of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS). In vitro, this compound markedly reduced virulence factor production and biofilm formation accompanied by a diminished content of extracellular DNA (eDNA). Additionally, coadministration with ciprofloxacin increased susceptibility of PA14 to antibiotic treatment under biofilm conditions. Finally, disruption of pathogenicity mechanisms was also assessed in vivo, with significantly increased survival of challenged larvae in a Galleria mellonella infection model. Favorable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, the ability to address two targets of the PQS autoinduction cycle at the same time with a single compound holds great promise in achieving enhanced synergistic cellular effects while potentially lowering rates of resistance development.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Drug Discovery; Gene Expression Regulation, Bacterial; Humans; Lepidoptera; Oligopeptides; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Quinolones; Quorum Sensing; Virulence Factors

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 μM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

Topics: Administration, Intranasal; Animals; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cations, Divalent; Cell Line, Tumor; Drug Repositioning; Enzyme Inhibitors; Humans; Iron Chelating Agents; Male; Manganese; Metalloendopeptidases; Mice; Pentetic Acid; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Small Molecule Libraries; Virulence; Zinc

Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.

Topics: 4-Quinolones; Bacterial Proteins; Benzamides; Benzoates; Enzyme Inhibitors; Magnetic Resonance Spectroscopy; Molecular Docking Simulation; Protein Binding; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Structure-Activity Relationship; Sulfonamides; Surface Plasmon Resonance; Transcription Factors

Cis-2-dodecenoic acid (BDSF) is well known for its important functions in intraspecies signaling in Burkholderia cenocepacia. Previous work has also established an important role of BDSF in interspecies and inter-kingdom communications. It was identified that BDSF modulates virulence of Pseudomonas aeruginosa. However, how BDSF interferes with virulence of P. aeruginosa is still not clear.. We report here that BDSF mediates the cross-talk between B. cenocepacia and P. aeruginosa through interference with quorum sensing (QS) systems and type III secretion system (T3SS) of P. aeruginosa. Bioassay results revealed that exogenous addition of BDSF not only reduced the transcriptional expression of the regulator encoding gene of QS systems, i.e., lasR, pqsR, and rhlR, but also simultaneously decreased the production of QS signals including 3-oxo-C12-HSL, Pseudomonas quinolone signal (PQS) and C4-HSL, consequently resulting in the down-regulation of biofilm formation and virulence factor production of P. aeruginosa. Furthermore, BDSF and some of its derivatives are also capable of inhibiting T3SS of P. aeruginosa at a micromolar level. Treatment with BDSF obviously reduced the virulence of P. aeruginosa in both HeLa cell and zebrafish infection models.. These results depict that BDSF modulates virulence of P. aeruginosa through interference with QS systems and T3SS.

Topics: 4-Butyrolactone; Animals; Antibiosis; Bacterial Secretion Systems; Biofilms; Burkholderia cenocepacia; Disease Models, Animal; Epithelial Cells; Fatty Acids, Monounsaturated; Gene Expression Profiling; HeLa Cells; Homoserine; Humans; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Signal Transduction; Virulence; Virulence Factors; Zebrafish

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.

Topics: Animals; Bacterial Proteins; Bacterial Secretion Systems; Caco-2 Cells; Caenorhabditis elegans; Cichorium intybus; Gene Expression Regulation, Bacterial; Humans; Plant Leaves; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Reverse Transcriptase Polymerase Chain Reaction; Virulence; Virulence Factors

Pseudomonas aeruginosa, an opportunistic pathogen of clinical importance, causes chronic airway infections in patients with cystic fibrosis (CF). Current literature suggests that pockets with reduced oxygen tension exist in the CF airway mucus. However, virulence features of this opportunistic pathogen under such conditions are largely unknown. Cell-free supernatant of the standard laboratory P. aeruginosa strain PAO1 obtained from anaerobic culture, but not aerobic culture, failed to kill A549 human airway epithelial cells. Further investigation revealed that this reduced cytotoxicity upon anaerobiosis was due to the suppressed secretion of elastase, a virulence factor controlled by P. aeruginosa quorum sensing (QS). Both a lacZ-reporter fusion assay and quantitative real-time PCR (RT-PCR) analysis demonstrated that transcription of the elastase-encoding lasB gene was substantially decreased during anaerobic growth compared with aerobic growth. Moreover, transcription of other genes controlled by the LasI/R QS system, such as rhlR, vqsR, mvfR, and rsaL, was also repressed under the same anaerobic growth conditions. Importantly, synthesis of 3-oxo-C(12)-HSL (PAI-1), an autoinducer molecule that mediates induction of the LasI/R QS system, was >22-fold decreased during anaerobic growth while C(4)-HSL (PAI-2), which mediates RhlI/R QS, was nondetectable under the same growth conditions. Transcription of the lasB gene was restored by exogenous supplementation with autoinducers, with PAI-2 more effective than PAI-1 or Pseudomonas quinolone signal (PQS) at restoring transcription of the lasB gene. Together, these results suggest that anaerobiosis deprives P. aeruginosa of the ability to regulate its virulence via QS and this misregulation attenuates the pathogenic potential of this important pathogen.

Topics: Anaerobiosis; Bacterial Proteins; Blotting, Western; Cell Line, Tumor; Epithelial Cells; Gene Expression Regulation, Bacterial; Genes, Reporter; Humans; Metalloendopeptidases; Pancreatic Elastase; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Polymerase Chain Reaction; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Virulence Factors

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent post-translational control, specifically governing type III cytotoxin secretion, exists in this species.

Topics: Gene Expression Regulation, Bacterial; Humans; Leukocidins; Oligopeptides; Pancreatic Elastase; Pneumonia, Ventilator-Associated; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Quorum Sensing; Virulence Factors

Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection in cystic fibrosis (CF) lungs. The effects of subinhibitory concentrations of colistin on gene expression in P. aeruginosa were investigated by transcriptome and functional genomic approaches. Analysis revealed altered expression of 30 genes representing a variety of pathways associated with virulence and bacterial colonization in chronic infection. These included response to osmotic stress, motility, and biofilm formation, as well as genes associated with LPS modification and quorum sensing (QS). Most striking was the upregulation of Pseudomonas quinolone signal (PQS) biosynthesis genes, including pqsH, pqsB and pqsE, and the phenazine biosynthesis operon. Induction of this central component of the QS network following exposure to subinhibitory concentrations of colistin may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF lung.

Topics: Anti-Bacterial Agents; Colistin; DNA, Bacterial; Dose-Response Relationship, Drug; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Humans; Hydroxyquinolines; Oligonucleotide Array Sequence Analysis; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Quinolones; Quorum Sensing; Transcriptional Activation

MvfR (PqsR), a Pseudomonas aeruginosa LysR-type transcriptional regulator, plays a critical role in the virulence of this pathogen. MvfR modulates the expression of multiple quorum sensing (QS)-regulated virulence factors; and the expression of the phnAB and pqsA-E genes that encode functions mediating 4-hydroxy-2-alkylquinolines (HAQs) signalling compounds biosynthesis, including 3,4-dihydroxy-2heptylquinoline (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ). PQS enhances the in vitro DNA-binding affinity of MvfR to the pqsA-E promoter, to suggest it might function as the in vivo MvfR ligand. Here we identify a novel MvfR ligand, as we show that HHQ binds to the MvfR ligand-binding-domain and potentiates MvfR binding to the pqsA-E promoter leading to transcriptional activation of pqsA-E genes. We show that HHQ is highly produced in vivo, where it is not fully converted into PQS, and demonstrate that it is required for MvfR-dependent gene expression and pathogenicity; PQS is fully dispensable, as pqsH-mutant cells, which produce HHI but completely lack PQS, display normal MvfR-dependent gene expression and virulence. Conversely, PQS is required for full production of pyocyanin. These results uncover a novel biological role for HHQ; and provide novel insights on MvfR activation that may aid in the development of therapies that prevent or treat P. aeruginosa infections in humans.

Topics: Animals; Bacterial Proteins; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Ligands; Mice; Mutation; Promoter Regions, Genetic; Protein Binding; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolines; Quinolones; Signal Transduction; Survival Rate; Time Factors; Trans-Activators; Transcription Factors; Virulence

In Pseudomonas aeruginosa the production of multiple virulence factors depends on cell-to-cell communication through the integration of N-acylhomoserine lactone (AHL)- and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)- dependent signalling. Mutation of genes encoding the efflux protein MexI and the porin OpmD from the MexGHI-OpmD pump resulted in the inability to produce N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-c12-hsl) and pqs and a marked reduction in n-butanoyl-L-homoserine lactone levels. Both pump mutants were impaired in growth and exhibited enhanced rather than reduced antibiotic resistance. Provision of exogenous PQS improved growth and restored AHL and virulence factor production as well as antibiotic susceptibility, indicating that the pump mutants retained their capacity to respond to PQS. RT-PCR analysis indicated that expression of the PQS biosynthetic genes, phnA and pqsA, was inhibited when the mutants reached stationary phase, suggesting that the pleiotropic phenotype observed may be due to intracellular accumulation of a toxic PQS precursor. To explore this hypothesis, double mexI phnA (unable to produce anthranilate, the precursor of PQS) and mexI pqsA mutants were constructed; the improved growth of the former suggested that the toxic compound is likely to be anthranilate or a metabolite of it. Mutations in mexI and opmD also resulted in the attenuation of virulence in rat and plant infection models. In plants, addition of PQS restored the virulence of mexI and opmD mutants. Collectively, these results demonstrate an essential function for the MexGHI-OpmD pump in facilitating cell-to-cell communication, antibiotic susceptibility and promoting virulence and growth in P. aeruginosa.

Topics: 4-Butyrolactone; 4-Quinolones; Animals; Bacterial Outer Membrane Proteins; Base Sequence; DNA, Bacterial; Drug Resistance, Multiple, Bacterial; Female; Gene Expression; Genes, Bacterial; Lactuca; Membrane Transport Proteins; Mutation; Phenotype; Plant Diseases; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Rats; Rats, Inbred Lew; Signal Transduction; Virulence

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.

Topics: Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Humans; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones; Signal Transduction; Sputum