Matthew P. Fletcher

Quinolones: from antibiotics to autoinducers

Since quinine was first isolated, animals, plants and microorganisms producing a wide variety of quinolone compounds have been discovered, several of which possess medicinally interesting properties ranging from antiallergenic and anticancer to antimicrobial activities. Over the years, these have served in the development of many synthetic drugs, including the successful fluoroquinolone antibiotics. Pseudomonas aeruginosa and related bacteria produce a number of 2-alkyl-4(1H)-quinolones, some of which exhibit antimicrobial activity. However, quinolones such as the Pseudomonas quinolone signal and 2-heptyl-4-hydroxyquinoline act as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. Here, we review selectively this extensive family of bicyclic compounds, from natural and synthetic antimicrobials to signalling molecules, with a special emphasis on the biology of P. aeruginosa. In particular, we review their nomenclature and biochemistry, their multiple properties as membrane-interacting compounds, inhibitors of the cytochrome bc1 complex and iron chelators, as well as the regulation of their biosynthesis and their integration into the intricate quorum-sensing regulatory networks governing virulence and secondary metabolite gene expression.

Transcriptomic analysis reveals a global alkyl-quinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal hosts.

The quorum sensing (QS) system of Pseudomonas aeruginosa constitutes a sophisticated genome-wide gene regulatory network employing both N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. AQ signalling utilizes 2-heptyl-3-hydroxy-4-quinolone (PQS) and its immediate precursor, 2-heptyl-4-quinolone (HHQ). AQ biosynthesis requires the first four genes of the pqsABCDE operon and while the biochemical function of pqsE is not known, it is required for the production of secondary metabolites such as pyocyanin. To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant. Of 158 genes exhibiting altered expression in the pqsA mutant, 51% were also affected in the pqsE mutant. Following induction of pqsE, 237 genes were differentially expressed compared with the wild-type strain. In the pqsEind strain, pqsA was highly expressed but following induction both pqsA expression and AQ biosynthesis were repressed, revealing a negative autoregulatory role for PqsE. Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE. Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.

Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules

2-Alkyl-4-quinolones (AHQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) are quorum sensing signal molecules. Here, we describe methods for AHQ detection, tentative identification and quantification, which employ a lux-based Pseudomonas aeruginosa AHQ biosensor strain. The protocol describes both thin-layer chromatography (TLC) and microtiter plate assays, which use bioluminescence or the green color of pyocyanin as detection end points. Organic solvent extracts of bacterial cells or cell-free culture supernatants are chromatographed on TLC plates, which are dried and overlaid with the AHQ biosensor. AHQs appear as both luminescent and green spots. For the microtiter assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AHQ biosensor. Light output is proportional to the AHQ content of the sample. The assays described take approximately 2 days to complete, are simple to perform, do not require sophisticated instrumentation and are highly amenable to screening large numbers of bacterial samples. However, apart from PQS and HHQ in P. aeruginosa, definitive AHQ identification will require additional MS and NMR analyses.

Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

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.

Detection of 2-Alkyl-4-Quinolones Using Biosensors

2-alkyl-4-quinolones (AQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-quinolone (HHQ) are quorum sensing signal molecules. Here we describe two methods for AQ detection and quantification that employ thin layer chromatography (TLC) and microtitre plate assays in combination with a lux-based Pseudomonas aeruginosa AQ biosensor strain. For TLC detection, organic solvent extracts of bacterial cells or spent culture supernatants are chromatographed on TLC plates, which are then dried and overlaid with the AQ biosensor. After detection by the bioreporter, AQs appear as both luminescent and green (pyocyanin) spots. For the microtitre assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AQ biosensor. Light output by the bioreporter is proportional to the AQ content of the sample. The assays described are simple to perform, do not require sophisticated instrumentation, and are highly amenable to screening large numbers of bacterial samples.

Biosensors for qualitative and semiquantitative analysis of quorum sensing signal molecules.

Biosensors are biological tools that can be used to assay bacterial cultures for quorum sensing signal molecules (QSSMs) both qualitatively and semiquantitatively. QSSMs can be extracted from Pseudomonas aeruginosa cultures using organic solvents and tentatively identified via thin layer chromatography in combination with biosensor overlays. Alternatively, QSSMs can be quantified in spent culture supernatants or solvent extracts using biosensor-based spectrophotometric, luminescence, or fluorescence assays.

2-Alkyl-4(1H)-Quinolone Signalling in Pseudomonas aeruginosa

In addition to the las and rhl qorum sensing (QS) systems, Pseudomonas aeruginosa utilises a non-acyl homoserine lactone (AHL)-based systems which relies on the production of 2-alkyl-4(1H)-quinolone (AQ) molecules. Although this organism produces a large range of AQ derivatives, the two main AQ signal molecules described are 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), and its immediate precursor, 2-heptyl-3-hydroxy-4(1H)-quinolone (HHQ). AQ biosynthesis and signalling in this organism has proven extremely complex with many questions still remaining unanswered. The production of AQs is strongly influenced by the las and rhl systems indication a close relationship between these three regulatory networks. Originally discovered as potential antibiotic compounds, AQs are highly versatile molecules playing a central role in the biology of P.aeruginosa. PQS has multiple functions including the ability to trigger iron depletion-mediated responses due to its ability to sequester iron. In addtion, AQs play a central role in the virulence of this organism although there are still many gaps in the knowledge of the exact molecular mechanisms underlying this control. AQ production is not exclusive to P. aeruginosa and these molecules have also been found in the other bacteria, mainly other Pseudomonads and Burkholderiaceae, however, their roles in these organisms have not yet been very well defined. The relevance of AQs in the biology of P.aeruginosa places AQ signalling systems as a potential target for the development of novel antimicrobials which can be used to treat infections caused by AQ producing bacterial pathogens.