Pf4 Phage Variant Infection Reduces Virulence-Associated Traits in Pseudomonas aeruginosa.

Pf4 is a filamentous bacteriophage integrated as a prophage into the genome of Pseudomonas aeruginosa PAO1. Pf4 virions can be produced without killing P. aeruginosa. However, cell lysis can occur during superinfection when Pf virions successfully infect a host lysogenized by a Pf superinfective variant. We have previously shown that infection of P. aeruginosa PAO1 with a superinfective Pf4 variant abolished twitching motility and altered biofilm architecture. More precisely, most of the cells embedded into the biofilm were showing a filamentous morphology, suggesting the activation of the cell envelope stress response involving both AlgU and SigX extracytoplasmic function sigma factors. Here, we show that Pf4 variant infection results in a drastic dysregulation of 3,360 genes representing about 58% of P. aeruginosa genome; of these, 70% of the virulence factors encoding genes show a dysregulation. Accordingly, Pf4 variant infection (termed Pf4*) causes in vivo reduction of P. aeruginosa virulence and decreased production of N-acyl-homoserine lactones and 2-alkyl-4-quinolones quorum-sensing molecules and related virulence factors, such as pyocyanin, elastase, and pyoverdine. In addition, the expression of genes involved in metabolism, including energy generation and iron homeostasis, was affected, suggesting further relationships between virulence and central metabolism. Altogether, these data show that Pf4 phage variant infection results in complex network dysregulation, leading to reducing acute virulence in P. aeruginosa. This study contributes to the comprehension of the bacterial response to filamentous phage infection. IMPORTANCE Filamentous bacteriophages can become superinfective and infect P. aeruginosa, even though they are inserted in the genome as lysogens. Despite this productive infection, growth of the host is only mildly affected, allowing the study of the interaction between the phage and the host, which is not possible in the case of lytic phages killing rapidly their host. Here, we demonstrate by transcriptome and phenotypic analysis that the infection by a superinfective filamentous phage variant causes a massive disruption in gene expression, including those coding for virulence factors and metabolic pathways.

Bacterial Quorum-Sensing Signal Arrests Phytoplankton Cell Division and Impacts Virus-Induced Mortality.

Interactions between phytoplankton and heterotrophic bacteria fundamentally shape marine ecosystems by controlling primary production, structuring marine food webs, mediating carbon export, and influencing global climate. Phytoplankton-bacterium interactions are facilitated by secreted compounds; however, linking these chemical signals, their mechanisms of action, and their resultant ecological consequences remains a fundamental challenge. The bacterial quorum-sensing signal 2-heptyl-4-quinolone (HHQ) induces immediate, yet reversible, cellular stasis (no cell division or mortality) in the coccolithophore Emiliania huxleyi; however, the mechanism responsible remains unknown. Using transcriptomic and proteomic approaches in combination with diagnostic biochemical and fluorescent cell-based assays, we show that HHQ exposure leads to prolonged S-phase arrest in phytoplankton coincident with the accumulation of DNA damage and a lack of repair despite the induction of the DNA damage response (DDR). While this effect is reversible, HHQ-exposed phytoplankton were also protected from viral mortality, ascribing a new role of quorum-sensing signals in regulating multitrophic interactions. Furthermore, our data demonstrate that in situ measurements of HHQ coincide with areas of enhanced micro- and nanoplankton biomass. Our results suggest bacterial communication signals as emerging players that may be one of the contributing factors that help structure complex microbial communities throughout the ocean.IMPORTANCE Bacteria and phytoplankton form close associations in the ocean that are driven by the exchange of chemical compounds. The bacterial signal 2-heptyl-4-quinolone (HHQ) slows phytoplankton growth; however, the mechanism responsible remains unknown. Here, we show that HHQ exposure leads to the accumulation of DNA damage in phytoplankton and prevents its repair. While this effect is reversible, HHQ-exposed phytoplankton are also relieved of viral mortality, elevating the ecological consequences of this complex interaction. Further results indicate that HHQ may target phytoplankton proteins involved in nucleotide biosynthesis and DNA repair, both of which are crucial targets for viral success. Our results support microbial cues as emerging players in marine ecosystems, providing a new mechanistic framework for how bacterial communication signals mediate interspecies and interkingdom behaviors.

Production of the antimicrobial compound tetrabromopyrrole and the Pseudomonas quinolone system precursor, 2-heptyl-4-quinolone, by a novel marine species Pseudoalteromonas galatheae sp. nov.

Novel antimicrobials are urgently needed due to the rapid spread of antibiotic resistant bacteria. In a genome-wide analysis of Pseudoalteromonas strains, one strain (S4498) was noticed due to its potent antibiotic activity. It did not produce the yellow antimicrobial pigment bromoalterochromide, which was produced by several related type strains with which it shared less than 95% average nucleotide identity. Also, it produced a sweet-smelling volatile not observed from other strains. Mining the genome of strain S4498 using the secondary metabolite prediction tool antiSMASH led to eight biosynthetic gene clusters with no homology to known compounds, and synteny analyses revealed that the yellow pigment bromoalterochromide was likely lost during evolution. Metabolome profiling of strain S4498 using HPLC-HRMS analyses revealed marked differences to the type strains. In particular, a series of quinolones known as pseudanes were identified and verified by NMR. The characteristic odor of the strain was linked to the pseudanes. The highly halogenated compound tetrabromopyrrole was detected as the major antibacterial component by bioassay-guided fractionation. Taken together, the polyphasic analysis demonstrates that strain S4498 belongs to a novel species within the genus Pseudoalteromonas, and we propose the name Pseudoalteromonas galatheae sp. nov. (type strain S4498T = NCIMB 15250T = LMG 31599T).

Engineered Biosynthesis of Fungal 4-Quinolone Natural Products.

Quinolone-containing natural products are widely found in bacteria, fungi, and plants. The fungal quinolactacins, which are N-methyl-4-quinolones, display a wide spectrum of biological activities. Here we uncovered a concise nonribosomal peptide synthetase pathway involved in quinolactacin A biosynthesis from Penicillium by using heterologous reconstitution and in vitro enzymatic synthesis. The N-desmethyl analog of quinolactacin A was accessed through the construction of a hybrid bacterial and fungi pathway in the heterologous host.

A structure-function analysis of interspecies antagonism by the 2-heptyl-4-alkyl-quinolone signal molecule from Pseudomonas aeruginosa.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa, modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.

The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, P. aeruginosa transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation of antR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation of antR, demonstrating that the PrrF sRNAs interact with the antR 5' untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. The P. aeruginosa Hfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd [dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to the antR mRNA (0.3 µM), and PrrF was able to bind to antR mRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to the antR UTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, in P. aeruginosaIMPORTANCE The iron-responsive PrrF sRNAs play a central role in regulating P. aeruginosa iron homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and the antR mRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target in P. aeruginosa.

Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse tissue using LC-MS/MS; and its application to a pharmacodynamic evaluation of MvfR inhibition.

Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) are important quorum sensing signals, which play a mediatory role in the pathogenesis of acute and chronic Pseudomonas aeruginosa infection. A targeted approach inhibiting the bacterial 'multiple virulence factor regulon' (MvfR) protein complex, offers the possibility to block the synthesis of MvfR-dependant signal molecules. Here, a high throughput bioanalytical method was developed using LC-MS/MS detection for the selective determination of HHQ and PQS in mouse tissue homogenate, over a sensitive range of 1-5000 and 10-5000pg/mL, respectively. Chromatographic peak distortion of the iron chelator PQS was overcome with the applied use of a bidentate chelator mobile phase additive 2-Picolinic acid at 0.2mM concentration, giving an improved separation and response for the analyte, whilst maintaining overall MS system robustness. Following thigh infection with P. aeruginosa strain 2-PA14 in mice, the concentration and time course of HHQ and PQS (4-hydroxy-2-alkyl-quinolone (HAQ) biomarkers) residing in the biophase were evaluated, and exhibited a low level combined with a substantial inter-individual variability. Quantifiable levels could be obtained from approximately 15h post infection, to the study termination at 21-22h. A dose dependant reduction in HAQ tissue concentrations at selected time points were obtained following MvfR inhibitor administration versus drug vehicle (p<0.01, Kruskal-Wallis-one way ANOVA) and meta -analyses of several studies enabled an inhibitory concentration (IC50) of 80nM free drug to be determined. However, due to the experimental limitations a defined time profile for in-vivo HAQ production could not be characterised. Microsomal stability measurements demonstrated a rapid metabolic clearance of both alkyl quinolone biomarkers in the bacterial host, with a hepatic extraction ratio greater than 0.96 (the measurable assay limit). High clearance underpinned the low concentrations present in the well-perfused thigh tissue. Along with method development and validation details, this paper considers the kinetics of in-vivo HAQ bio-synthesis during Pseudomonas infection; and risks of biomarker over-estimation from samples which contain an exogenous population of bacteria.

Synthesis, alkaline phosphatase inhibition studies and molecular docking of novel derivatives of 4-quinolones.

New and convenient methods for the functionalization of the 4-quinolone scaffold at positions C-1, C-3 and C-6 were developed. The 4-quinolone derivatives were evaluated for their inhibitory potential on alkaline phosphatase isozymes. Most of the compounds exhibit excellent inhibitory activity and moderate selectivity. The IC50 values on tissue non-specific alkaline phosphatase (TNAP) were in the range of 1.34 ± 0.11 to 44.80 ± 2.34 µM, while the values on intestinal alkaline phosphatase (IAP) were in the range of 1.06 ± 0.32 to 192.10 ± 3.78 µM. The most active derivative exhibits a potent inhibition on IAP with a ≈14 fold higher selectivity as compared to TNAP. Furthermore, molecular docking calculations were performed for the most potent inhibitors to show their binding interactions within the active site of the respective enzymes.

Unravelling the Genome-Wide Contributions of Specific 2-Alkyl-4-Quinolones and PqsE to Quorum Sensing in Pseudomonas aeruginosa.

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl-4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response.

New P2X3 receptor antagonists. Part 2: Identification and SAR of quinazolinones.

Numerous potent P2X3 antagonists have been discovered and the therapeutic potential of P2X3 antagonism already comprises proof-of-concept data obtained in clinical trials with the most advanced compound. We have lately reported the discovery and optimization of thia-triaza-tricycle compounds with potent P2X3 antagonistic properties. This Letter describes the SAR of a back-up series containing a 4-oxo-quinazoline central ring. The discovery of the highly potent compounds 51 is presented.

PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal: CRYSTAL STRUCTURE, INHIBITION, AND REACTION MECHANISM.

Pseudomonas aeruginosaproduces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the ß-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used forin vitrobinding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.

PqsE of Pseudomonas aeruginosa Acts as Pathway-Specific Thioesterase in the Biosynthesis of Alkylquinolone Signaling Molecules.

Pseudomonas aeruginosa uses the alkylquinolones PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) and HHQ (2-heptyl-4(1H)-quinolone) as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. The biosynthesis of HHQ is generally accepted to require the pqsABCD gene products. We now reconstitute the biosynthetic pathway in vitro, and demonstrate that in addition to PqsABCD, PqsE has a role in HHQ synthesis. PqsE acts as thioesterase, hydrolyzing the biosynthetic intermediate 2-aminobenzoylacetyl-coenzyme A to form 2-aminobenzoylacetate, the precursor of HHQ and 2-aminoacetophenone. The role of PqsE can be taken over to some extent by the broad-specificity thioesterase TesB, explaining why the pqsE deletion mutant of P. aeruginosa still synthesizes HHQ. Interestingly, the pqsE mutant produces increased levels of 2,4-dihydroxyquinoline, resulting from intramolecular cyclization of 2-aminobenzoylacetyl-coenzyme A. Overall, our data suggest that PqsE promotes the efficiency of alkylquinolone signal molecule biosynthesis in P. aeruginosa and balances the levels of secondary metabolites deriving from the alkylquinolone biosynthetic pathway.

Role of Pseudomonas aeruginosa quorum sensing (QS) molecules on the viability and cytokine profile of human mesenchymal stem cells.

Pseudomonas aeruginosa infections represent one of the major threats for injured or transplanted lungs and for their healing. Considering that the mesenchymal stem cells (MSCs) are a major tool for the regenerative medicine, including therapy of lung damaging diseases, the aim of this paper was to investigate the effects of P. aeruginosa quorum sensing signaling molecules (QSSMs) on human MSCs death signaling pathways and cytokine profile. Our data revealed that N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), N-butanoyl-L-homoserine lactone (C4-HSL), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), and its precursor, 2-heptyl-4-quinolone (HHQ), significantly impact on several core signaling mechanisms of MSCs in a specific and time-dependent manner. Even if all tested autoinducers interfered with the MSCs apoptotic genes expression, only OdDHL and HHQ significantly promoted MSCs apoptosis, by 14- and 23-fold respectively, this aspect being confirmed by the flow cytometry assay. The tested QSSMs induced a heterogeneous cytokine profile of the treated MSCs. The level of IL-1ß was increased by OdDHL, IL-8 production was stimulated by all tested autoinducers, IL-6 was modulated mostly by PQS and IL-10 by HHQ. The significant influence of the purified bacterial autoinducers on the MSCs signaling pathways may suggest that the accumulation of these mediators could interfere with the normal function of these cells in the human body, and eventually, impair or abolish the success of the stem cells therapy during P. aeruginosa infections.

Structure optimization of 2-benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa infections and elucidation of binding mode by SPR, STD NMR, and molecular docking.

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.

Structure-function analysis of the C-3 position in analogues of microbial behavioural modulators HHQ and PQS.

2-Heptyl-3-hydroxy-4-quinolone (PQS) and its precursor 2-heptyl-4-quinolone (HHQ) are key signalling molecules of the important nosocomial pathogen Pseudomonas aeruginosa. We have recently reported an interkingdom dimension to these molecules, influencing key virulence traits in a broad spectrum of microbial species and in the human pathogenic yeast Candida albicans. For the first time, targeted chemical derivatisation of the C-3 position was undertaken to investigate the structural and molecular properties underpinning the biological activity of these compounds in P. aeruginosa, and using Bacillus subtilis as a suitable model system for investigating modulation of interspecies behaviour.

Pseudomonas aeruginosa Alkyl quinolones repress hypoxia-inducible factor 1 (HIF-1) signaling through HIF-1α degradation.

The transcription factor hypoxia-inducible factor 1 (HIF-1) has recently emerged to be a crucial regulator of the immune response following pathogen perception, including the response to the important human pathogen Pseudomonas aeruginosa. However, as mechanisms involved in HIF-1 activation by bacterial pathogens are not fully characterized, understanding how bacteria and bacterial compounds impact on HIF-1α stabilization remains a major challenge. In this context, we have focused on the effect of secreted factors of P. aeruginosa on HIF-1 regulation. Surprisingly, we found that P. aeruginosa cell-free supernatant significantly repressed HIF-1α protein levels. Further characterization revealed that HIF-1α downregulation was dependent on a subset of key secreted factors involved in P. aeruginosa pathogenesis, the 2-alkyl-4-quinolone (AQ) quorum sensing (QS) signaling molecules, and in particular the pseudomonas quinolone signal (PQS). Under hypoxic conditions, the AQ-dependent downregulation of HIF-1α was linked to the suppressed induction of the important HIF-1 target gene hexokinase II. Furthermore, we demonstrated that AQ molecules directly target HIF-1α protein degradation through the 26S-proteasome proteolytic pathway but independently of the prolyl hydroxylase domain (PHD). In conclusion, this is the first report showing that bacterial molecules can repress HIF-1α protein levels. Manipulation of HIF-1 signaling by P. aeruginosa AQs could have major consequences for the host response to infection and may facilitate the infective properties of this pathogen.

Biofilm formation by Pseudomonas aeruginosa in solid murine tumors - a novel model system.

The ability of opportunistic bacterial pathogens to grow in biofilms is decisive in the pathogenesis of chronic infectious diseases. Growth within biofilms does not only protect the bacteria against the host immune system but also from the killing by antimicrobial agents. Here, we introduce a mouse model in which intravenously administered planktonic Pseudomonas aeruginosa bacteria are enriched in transplantable subcutaneous mouse tumors. Electron microscopy images provide evidence that such bacteria reside in the tumor tissue within biofilm structures. Immunohistology furthermore demonstrated that infection of the tumor tissue elicits a host response characterized by strong neutrophilic influx. Interestingly, the biofilm defective PA14 pqsA transposon mutant formed less biofilm in vivo and was more susceptible to clearance by intravenous ciprofloxacin treatment as compared to the wild-type control. In conclusion, we have established an experimentally tractable model that may serve to identify novel bacterial and host factors important for in vivo biofilm formation and to re-evaluate bactericidal and anti-biofilm effects of currently used and novel antibacterial compounds.

2-Heptyl-4-quinolone, a precursor of the Pseudomonas quinolone signal molecule, modulates swarming motility in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors, including biofilm formation and swarming motility. These group behaviors are regulated by both the intracellular signaling molecule c-di-GMP and acylhomoserine lactone quorum-sensing systems. Here, we show that the Pseudomonas quinolone signal (PQS) system also contributes to the regulation of swarming motility. Specifically, our data indicate that 2-heptyl-4-quinolone (HHQ), a precursor of PQS, likely induces the production of the phenazine-1-carboxylic acid (PCA), which in turn acts via an as-yet-unknown downstream mechanism to repress swarming motility. We show that this HHQ- and PCA-dependent swarming repression is apparently independent of changes in global levels of c-di-GMP, suggesting complex regulation of this group behavior.

Investigations on the 4-quinolone-3-carboxylic acid motif. 4. Identification of new potent and selective ligands for the cannabinoid type 2 receptor with diverse substitution patterns and antihyperalgesic effects in mice.

Experimental evidence suggests that selective CB2 receptor modulators may provide access to antihyperalgesic agents devoid of psychotropic effects. Taking advantage of previous findings on structure-activity/selectivity relationships for a class of 4-quinolone-3-carboxamides, further structural modifications of the heterocyclic scaffold were explored, leading to the discovery of the 8-methoxy derivative 4a endowed with the highest affinity and selectivity ever reported for a CB2 ligand. The compound, evaluated in vivo in the formalin test, behaved as an inverse agonist by reducing at a dose of 6 mg/kg the second phase of the formalin-induced nocifensive response in mice.