2-heptyl-3-hydroxy-4-quinolone and pyoverdin

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

We synthesized a range of PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone) analogues and tested them for their ability to stimulate MvfR-dependent pqsA transcription, MvfR-independent pyoverdine production, and membrane vesicle production. The structure-activity profile of the PQS analogues was different for each of these phenotypes. Certain inactive PQS analogues were also found to strongly synergize PQS-dependent pyoverdine production.

Topics: Gene Expression Regulation, Bacterial; Molecular Structure; Oligopeptides; Pseudomonas; Quinolones; Structure-Activity Relationship

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

Summary Using flow chamber-grown Pseudomonas aeruginosa biofilms as model system, we show in the present study that formation of heterogeneous biofilms may occur through mechanisms that involve complex subpopulation interactions. One example of this phenomenon is expression of the iron-siderophore pyoverdine in one subpopulation being necessary for development of another subpopulation that does not itself express the pyoverdine synthesis genes. Another example is quorum sensing-controlled DNA release in one subpopulation being necessary for development of another subpopulation that does not itself express the quorum-sensing genes.

Topics: Biofilms; DNA, Bacterial; Iron; Oligopeptides; Pseudomonas aeruginosa; Quinolones; Symbiosis

Previously, we identified the uncharacterized predicted membrane protein PA2663 of Pseudomonas aeruginosa PAO1 as a virulence factor using a poplar tree model; PA2663 was induced in the poplar rhizosphere and, upon inactivation, it caused 20-fold lower biofilm formation (Attila et al., Microb Biotechnol, 2008). Here, we confirmed that PA2663 is related to biofilm formation by restoring the wild-type phenotype by complementing the PA2663 mutation in trans and investigated the genetic basis of its influence on biofilm formation through whole-transcriptome and -phenotype studies. Upon inactivating PA2663 by transposon insertion, the psl operon that encodes a galactose- and mannose-rich exopolysaccharide was highly repressed (verified by RT-PCR). The inactivation of PA2663 also repressed 13 pyoverdine genes, which eliminated the production of the virulence factor pyoverdine in P. aeruginosa. The inactivation of PA2663 also affected other quorum-sensing-related phenotypes in that it repressed the Pseudomonas quinolone signal (PQS) genes, which abolished PQS production, and repressed lasB, which decreased elastase activity sevenfold. Genes were also induced for motility and attachment (PA0499, PA0993, PA2130, and PA4549) and for small molecule transport (PA0326, PA1541, PA1632, PA1971, PA2214, PA2215, PA2678, and PA3407). Phenotype arrays also showed that PA2663 represses growth on D: -gluconic acid, D: -mannitol, and N-phthaloyl-L: -glutamic acid. Hence, the PA2663 gene product increases biofilm formation by increasing the psl-operon-derived exopolysaccharides and increases pyoverdine synthesis, PQS production, and elastase activity while reducing swarming and swimming motility. We speculate that PA2663 performs these myriad functions as a novel membrane sensor.

Topics: Bacterial Proteins; Biofilms; Biological Transport; DNA Transposable Elements; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Gluconates; Glutamates; Locomotion; Mannitol; Metalloendopeptidases; Mutagenesis, Insertional; Oligonucleotide Array Sequence Analysis; Oligopeptides; Operon; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Quinolones; Quorum Sensing; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial; RNA, Messenger; Virulence

In some Proteobacteria biogenesis of c-type cytochromes depends on the products of the ccmABCDEFG(H) genes, which encode inner-membrane proteins. Inactivation of some ccm genes, in particular ccmC, has an impact on other processes as well, including siderophore production and utilization. Non-polar insertions were generated in the Pseudomonas aeruginosa ccmA, ccmC, ccmE, ccmF and ccmH genes, and their impacts on different phenotypes were compared. Only in the case of the ccmC mutant was cytochrome c production totally abrogated. The ccmC mutant, and to a lesser extent the ccmF mutant, showed a range of other phenotypic changes. The production of the siderophore pyoverdine was very low and growth under the condition of iron limitation was severely restricted, but production of the second siderophore, pyochelin, was increased. Interestingly, other traits were also strongly affected by the ccmC mutation, including the production of pyocyanin, swarming and twitching motility, and rhamnolipid production. The production of N-acyl homoserine lactones or the Pseudomonas quinolone signal (PQS) was, however, not affected in the ccmC and ccmF mutants. The ccmC mutant was also found to accumulate porphyrins, and catalase production was undetectable, consistent with the increased sensitivity to hydrogen peroxide. Finally, reduction in the content of [Fe-S] clusters was evidenced in both ccmC and ccmF mutants. Wild-type phenotypes were restored by complementation with a ccmC gene from Pseudomonas fluorescens ATCC 17400. In conclusion, we have demonstrated that CcmC is a key determinant for cytochrome c biogenesis, pyoverdine maturation, and expression of some quorum sensing-regulated traits.

Topics: Acyl-Butyrolactones; Bacterial Outer Membrane Proteins; Bacterial Proteins; Catalase; Cytochromes c; Genetic Complementation Test; Glycolipids; Locomotion; Membrane Proteins; Mutagenesis, Insertional; Mutation; Oligopeptides; Phenols; Porphyrins; Pseudomonas aeruginosa; Pyocyanine; Quinolones; Thiazoles