A series of 3-oxo-C12-HSL, tetramic acid, and tetronic acid analogues were synthesized to gain insights into the structural requirements for quorum sensing inhibition in Staphylococcus aureus. Compounds active against agr were noncompetitive inhibitors of the autoinducing peptide (AIP) activated AgrC receptor, by altering the activation efficacy of the cognate AIP-1. They appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17, which reduced nasal cell colonization and arthritis in a murine infection model.

•Multi-antibiotic resistant bacteria pose a global healthcare threat.•Broad spectrum growth inhibitory antibiotics select for resistance.•Attenuating bacterial virulence offers an alternative approach.•Quorum sensing systems provide multiple targets for drugs which attenuate virulence.•Progress in the discovery and development of quorum sensing inhibitors is described.Almost a century on from the discovery of penicillin, the war against bacterial infection still rages compounded by the emergence of strains resistant to virtually every clinically approved antibiotic and the dearth of new antibacterial agents entering the clinic. Consequently there is renewed interest in drugs which attenuate virulence rather than bacterial growth. Since the metaphors of warfare are often used to describe the battle between pathogen and host, we will describe in such a context, the molecular communication (quorum sensing) mechanisms used by bacteria to co-ordinate virulence at the population level. Recent progress in exploiting this information through the design of anti-virulence deception strategies that disrupt quorum sensing through signal molecule inactivation, inhibition of signal molecule biosynthesis or the blockade of signal transduction and their advantages and disadvantages are considered.Graphical abstract

2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) is a quorum-sensing signal molecule used by Pseudomonas aeruginosa. The structural similarity between 3-hydroxy-2-methyl-4(1H)-quinolone, the natural substrate for the 2,4-dioxygenase, Hod, and PQS prompted us to investigate whether Hod quenched PQS signaling. Hod is capable of catalyzing the conversion of PQS to N-octanoylanthranilic acid and carbon monoxide. In P. aeruginosa PAO1 cultures, exogenously supplied Hod protein reduced expression of the PQS biosynthetic gene pqsA, expression of the PQS-regulated virulence determinants lectin A, pyocyanin, and rhamnolipids, and virulence in planta. However, the proteolytic cleavage of Hod by extracellular proteases, competitive inhibition by the PQS precursor 2-heptyl-4(1H)-quinolone, and PQS binding to rhamnolipids reduced the efficiency of Hod as a quorum-quenching agent. Nevertheless, these data indicate that enzyme-mediated PQS inactivation has potential as an antivirulence strategy against P. aeruginosa.

Communication through quorum sensing (QS) enables bacterial populations to coordinate their behavior. Recent work on N-acylhomoserine lactone-mediated QS has revealed that some soil bacteria exploit host-derived substrates to generate an alternative N-substituted homoserine lactone. New light has also been shed on the mechanism by which N-(3-oxo-dodecanoyl)-l-homoserine lactone modulates host inflammatory signaling pathways to promote bacterial survival.

Bacteria release a wide variety of small molecules including secondary metabolites such as antibiotics and siderophores (iron chelators), metabolic end products and cell-to-cell signaling molecules. The latter facilitate the coordination of gene expression within a bacterial population1, 2, 3, 4. Cell-to-cell communication in bacteria is referred to as “quorum sensing” (QS) and depends on the action of diffusible signal molecules or “autoinducers”. As a bacterial culture grows, signal molecules are released into the extracellular milieu and accumulate. Once a critical threshold concentration of the QS signal molecule (and consequently a specific population density) has been achieved, a coordinated change in bacterial behavior is initiated via a specific sensor kinase or response regulator, so facilitating the expression of QS-dependent target genes1, 2, 3, 4. Secondary metabolite production, bioluminescence, competence, plasmid transfer, biofilm development and pathogenicity are all known to be controlled by QS in many different Gram-positive and Gram-negative bacteria.QS signal molecules are chemically diverse, ranging from peptides and N-acylhomoserine lactones (AHLs) to furanones and AHQs1, 2, 3, 4. Given the vast number of extracellular metabolites, the chemical diversity among known QS signal molecules is likely to represent only the “tip of the iceberg”. Indeed, it has been suggested that the majority of low-molecular-weight organic compounds made and secreted by microbes are likely to function as QS signal molecules5.Studies of QS in diverse bacteria have greatly benefited from the availability of biosensor assays, which respond sensitively to specific QS signal molecule classes and do not require sophisticated instrumentation. Such assays can provide tentative information on the chemical identity, number and concentrations of the QS signal molecules present in bacterial culture supernatants. For example, AHLs can be readily detected and quantified using AHL biosensors based on lux 6, 7, gfp 8 or lacZ 9 reporter gene fusions or violacein pigment induction10 while autoinducer-2 is generally assayed using a bioluminescent Vibrio harveyi strain11.In the 1940s and 1950s, a number of antibacterial AHQ compounds were isolated and purified from P. aeruginosa culture supernatants4. Subsequently, AHQs were discovered to inhibit the growth of Gram-positive bacteria, algae and phytoplankton, to modulate host immune defences, chelate iron and act as QS signal molecules12, 13, 14, 15, 16, 17, 18. Other pseudomonads, Burkholderia and Alteromonas species also produce AHQs and it is highly likely that other Gram-negative bacteria such as Ralstonia species synthesize these compounds as they possess homologs of the P. aeruginosa and B. pseudomallei AHQ biosynthetic genes19.Although over 50 different AHQs have been identified in P. aeruginosa, most are present at low concentrations where they are unlikely to be physiologically active20, 21. AHQs exist in another tautomeric form and can also be termed 4-hydroxy-2-alkylquinolines (HAQs)17, 20. However, the neutral 4-quinolone rather than the 4-hydroxy-quinoline is the predominant species in the pH range 4–6 and hence under physiological conditions17; so here we use the AHQ rather than HAQ nomenclature. The major P. aeruginosa AHQ signal molecules are 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal (PQS)) and 2-heptyl-4-quinolone (HHQ, Fig. 1)16, 21, 22, 23. While HHQ is the immediate precursor of PQS, it can also function as a QS signal molecule not only in P. aeruginosa but also in Burkholderia pseudomallei 19 and probably in other AHQ-producing bacteria, although this remains to be confirmed.In P. aeruginosa, AHQ biosynthesis depends on the pqsABCDE operon and mutation of pqsA renders the organism AHQ-negative19, 21, 24. Both PQS and HHQ act as autoinducers as they are required to drive their own synthesis by binding to the LysR-type regulator, PqsR(MvfR), such that PqsR(MvfR) binding to the pqsA promoter is enhanced17, 23, 25. To construct an AHQ biosensor, we first deleted pqsA in strain PAO1 before introducing a luxCDABE-based pqsA promoter fusion onto the chromosome of the pqsA mutant17 (M.P.F., S.P.D., S. Crusz, S.R. Chhabra, M.C. and P.W., unpublished manuscript) using the mini-CTX luxCDABE plasmid system described by Becher and Schweizer26. The surrogate pqsA promoter was inserted into a non-coding site in the chromosome where it can be activated independently of the native gene promoter. The use of the entire luxCDABE operon dispenses with the need to add exogenously a long-chain fatty aldehyde substrate for the luciferase7. Figure 2 shows the mechanism by which an AHQ such as PQS activates the biosensor and induces bioluminescence. As the pqsA mutant does not produce any AHQs, it is dark and light is emitted only upon exposure to an exogenous AHQ source. Bioluminescence can be detected on TLC plate overlays using an X-ray film or a photon video camera or quantified by using a luminometer for liquid-based assays. As P. aeruginosa is not naturally bioluminescent, the biosensor background light level is very low and it responds to a wide dynamic range of both PQS and HHQ, from nanomolar to micromolar concentrations (M.P.F., S.P.D., S. Crusz, S.R. Chhabra, M.C. and P.W., unpublished manuscript). In addition, although less amenable to rapid quantification, the bioreporter produces the blue–green pigment pyocyanin in response to both PQS and HHQ, which is readily apparent on the TLC overlays. Furthermore, the AHQ biosensor is most sensitively activated by PQS and HHQ, both of which have C7 alkyl chains. Analogs of both compounds with alkyl side chains from C1 to C11 are capable of activating the reporter albeit at higher concentrations (M.P.F., S.P.D., S. Crusz, S.R. Chhabra, M.C. and P.W., unpublished manuscript).AHQs such as PQS and HHQ can be detected in crude cell-free culture supernatants using the lux-based AHQ biosensor in a liquid microtiter plate assay. If AHQ levels are low or where information on cell or vesicle-associated AHQs is required, they can simply be concentrated by extraction with solvents such as dichloromethane or ethyl acetate, as AHQs readily partition into the organic phase. After removal of the solvent by rotary evaporation, the residue can be rehydrated in an aqueous buffer before AHQ biosensor analysis. Although the P. aeruginosa AHQ bioreporter can detect HHQ and PQS at nanomolar concentrations (detection limit 12 nM for both AHQs), it responds in a dose-dependent manner to both compounds (M.P.F., S.P.D., S. Crusz, S.R. Chhabra, M.C. and P.W., unpublished manuscript). The AHQ biosensor is most sensitively activated by PQS and HHQ both of which have C7 alkyl chains. Analogs with C5, C9 and C11 are also capable of activating the biosensor albeit less sensitively (M.P.F., S.P.D., S. Crusz, S.R. Chhabra, M.C. and P.W., unpublished manuscript). Consequently, the light output from the bioreporter in response to a P. aeruginosa culture supernatant reflects the combined concentrations of HHQ and PQS as well the other AHQs listed above. However, the latter, at least in P. aeruginosa, are mostly present at substantially lower concentrations except for the corresponding C9 analogs, 2-nonyl-4-quinolone (HNQ) and 2-nonyl-3-hydroxy-4-quinolone (C9-PQS) (see ref. 27). The AHQ liquid bioassay therefore provides a positive indication for the presence of PQS, HHQ and closely related AHQs. However, it is only a semiquantitative indication of the total AHQs present in a given spent bacterial culture supernatant. In P. aeruginosa, for example, the data obtained will reflect predominantly the PQS and HHQ concentrations. The assay is however fully quantitative if used for bacteria, which make only a single AHQ and for synthetic standards. A typical dose–response curve of maximal light output from the AHQ biosensor in response to a range of PQS and HHQ concentrations is shown in Figure 3. The liquid assay is therefore highly appropriate where comparative overall, rather than absolute, AHQ concentrations for individual AHQs are required. The liquid assay cannot provide information on the concentrations of the individual AHQs present in a mixture as this would require the introduction of additional separation and purification stages by, for example, HPLC. An additional limitation of the biosensor is that it will not respond to all AHQ analogs although this could be considered as a major advantage with respect to HHQ and PQS analysis.To determine whether both PQS and HHQ are present together with other AHQs, cell-free supernatant, cell or vesicle extracts can be extracted with ethyl acetate and subjected to TLC. After chromatography, the TLC plate is overlaid with a thin nutrient agar containing the bioreporter strain, which is subsequently monitored for bioluminescence and pyocyanin production (Fig. 4). Tentative identification of positive spots can be made by comparison of their relative migration (R f) values with synthetic standards. Unequivocal chemical identification of any positive compounds can be made by MS and/or NMR analysis. The method can also be made more quantitative by spotting a range of concentrations of a synthetic AHQ(s) on the TLC plate.Preparing bacterial cultures for AHQ extraction, Steps 1–4: 2 daysAHQ extraction of bacterial cultures, Steps 5–9: 5 hPreparation of TLC plates and running of samples, Steps 10–14: 3 h. Steps 15 and 16 can be carried out in advance of Steps 10–14.Overlay of TLC plates with the AHQ reporter, Steps 15–20: 2 days (to view light output) or 3 days (to view pyocyanin production)Remember that a substantial proportion of the quoted times for both assays involve 'dormant' periods, for example, for growth of cultures, running of TLCs, etc. The actual workload involved is much less than that implied by the total time frame.Troubleshooting advice can be found in Table 1.

Kynurenine formamidase (KynB) forms part of the kynurenine pathway which metabolises tryptophan to anthranilate. This metabolite can be used for downstream production of 2-alkyl-4-quinolone (AQ) signalling molecules that control virulence in Pseudomonas aeruginosa. Here we investigate the role of kynB in the production of AQs and virulence-associated phenotypes of Burkholderia pseudomallei K96243, the causative agent of melioidosis. Deletion of kynB resulted in reduced AQ production, increased biofilm formation, decreased swarming and increased tolerance to ciprofloxacin. Addition of exogenous anthranilic acid restored the biofilm phenotype, but not the persister phenotype. This study suggests the kynurenine pathway is a critical source of anthranilate and signalling molecules that may regulate B. pseudomallei virulence.

•Phenotypic complementation identifies a CsrA/RsmA family member from P. aeruginosa•Crystallography reveals a dimeric fold for RsmN•The RsmN complex was solved with a hairpin motif from the noncoding sRNA RsmZ-2•Details of binding affinity and specificity with target RNA 5′-ANGGAN motifs are revealedIn bacteria, the highly conserved RsmA/CsrA family of RNA-binding proteins functions as global posttranscriptional regulators acting on mRNA translation and stability. Through phenotypic complementation of an rsmA mutant in Pseudomonas aeruginosa, we discovered a family member, termed RsmN. Elucidation of the RsmN crystal structure and that of the complex with a hairpin from the sRNA, RsmZ, reveals a uniquely inserted α helix, which redirects the polypeptide chain to form a distinctly different protein fold to the domain-swapped dimeric structure of RsmA homologs. The overall β sheet structure required for RNA recognition is, however, preserved with compensatory sequence and structure differences, allowing the RsmN dimer to target binding motifs in both structured hairpin loops and flexible disordered RNAs. Phylogenetic analysis indicates that, although RsmN appears unique to P. aeruginosa, homologous proteins with the inserted α helix are more widespread and arose as a consequence of a gene duplication event.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the α-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.