The ability of bacterial cells to synchronize their behaviour through quorum sensing (QS) regulatory networks enables bacterial populations to mount co-operative responses against competing micro-organisms and host immune defences and to adapt to environmental challenges. Since QS controls the ability of many pathogenic bacteria to cause disease, it is an attractive target for novel antibacterial agents that control infection through inhibition of virulence and by rendering biofilms more susceptible to conventional antibiotics and host clearance pathways. QS systems provide multiple druggable molecular targets for inhibitors (QSIs) that include the enzymes involved in QS signal molecule biosynthesis and the receptors involved in signal transduction. Considerable advances in our understanding of the chemical biology of QS systems and their inhibition have been made, some promising QS targets structurally characterized, QSI screens devised and inhibitors identified. However, much more work is required before any QSI ‘hits’ with the appropriate pharmacological and pharmacokinetic properties can enter human clinical trials. Indeed, the relative efficacy of QSIs alone or as prophylactics or therapeutics or as adjuvants in combination with conventional antibiotics still needs to be extensively evaluated in vivo. Particular attention must be given to the measurement of successful QSI therapy outcomes with respect to bacterial clearance, immune response and pathophysiology. Currently, our understanding of the potential of QS as a promising antibacterial target suggests that it is likely to be of value with respect to a limited number of major pathogens.

Introduction

The emergence, rapid spread and persistence of antibiotic-resistant bacteria poses a serious threat to human and animal health worldwide [1]. Antimicrobial resistance threatens the outcomes of even simple infections and common medical interventions that until recently were considered low-risk. Novel antibacterial agents with unique modes of action, capable of curing chronic, biofilm-centred infections that are refractive to conventional antibiotic-resistance mechanisms and do not damage the host microbiota, are required. In turn, the discovery of such agents depends on a thorough understanding of the basic molecular, chemical and structural biology of bacterial pathogens and their interactions with the host together with access to chemical diversity for probing putative targets and developing new therapeutic agents.

Conventional antibiotics were primarily developed against rapidly dividing planktonic bacterial cells and are either bacteriostatic or bactericidal as they target essential biochemical processes including protein, nucleic acid and cell wall biosynthesis [1,2]. This exerts enormous selective pressures that drive the emergence of antibiotic resistance rapidly eroding the clinical antibiotic arsenal, a problem further compounded by the dearth of new antibiotics reaching the clinic and the evolution of multiresistant strains [1]. Consequently, alternative therapeutic approaches are required to prevent or treat bacterial infections. In this context, the attenuation of bacterial virulence and biofilm formation have been viewed as attractive targets for the development of ‘anti-virulence’ or ‘anti-pathogenic’ agents that could be used individually or as antibiotic ‘adjuvants’ via combination therapy [26]. Although antivirulence agents by definition lack in vitro bacterial growth inhibitory activities, they may well affect on pathogen growth and viability in vivo by preventing colonization or by interfering with bacterial metabolism or the activities of tissue-damaging exotoxins and exoenzymes that facilitate bacterial nutrient acquisition and prevent phagocytic clearance. Such agents have been suggested to offer potential advantages that include expanding the number of bacterial drug targets, preserving the host commensal microbiota and reducing the probability of selecting for resistance [24].

Quorum sensing

For most pathogens, virulence is multifactorial and host specific. It relies on the expression of diverse cell-associated and secreted virulence determinants and the ability to adopt an intracellular lifestyle or form surface-associated antibiotic and host defence tolerant biofilms [24]. Virulence determinants and their regulatory systems both offer potential therapeutic targets for novel anti-infective agents [24]. Targeting global regulatory systems, such as quorum sensing (QS), offers opportunities to simultaneously inhibit the expression of multiple virulence factor genes. As a means of cell-to-cell communication, QS integrates information at the bacterial population level, co-ordinating the metabolic status of the cells with environmental cues [2,3,7]. QS enables the cells in a population to synchronize the expression (or repression) of specific subsets of genes through the production and sensing of self-generated signal molecules that accumulate extracellularly [2,3].

Once a QS signal molecule has reached a threshold concentration (at which point the population is considered to be ‘quorate’), it binds to and activates a cognate receptor protein that controls expression of the target genes [3]. These often include the signal synthase gene(s) establishing an autoinduction circuit for rapidly increasing signal molecule production. Consequently, QS signal molecules are often called autoinducers [2]. Once a QS system has been activated, a co-ordinated physiological response is triggered re-programming gene expression to enable the population to accomplish tasks difficult to achieve by single cells acting alone. However, it should be noted that the size of the ‘quorum’ is not fixed but will depend on the relative rates of production and loss of the QS signal molecule. A chemically diverse range of small molecules [from N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs) to peptides and fatty acids] are employed as QS signal molecules that together with the cognate signal transduction machineries (that incorporate signal molecule receptors, e.g. sensor kinases and response regulators) form interdependent networks of regulatory units that govern bacterial population behaviour [3]. In different bacterial species, QS regulates diverse activities that range from secondary metabolite (including antibiotic) biosynthesis, antibiotic resistance, biofilm development, plasmid transfer, primary metabolism motility and virulence [3].

The potential of QS as an antibacterial target

QS has potential as an antibacterial target in pathogens where strains carrying mutations in key QS genes exhibit attenuated virulence in animal infection models [8]. However, it should be noted that an inhibitor will confront an established infection, whereas the in vivo evaluation of a QS mutant will generally assess the ability of the pathogen to initiate an infection. Support that QS systems in pathogens such as Pseudomonas aeruginosa are active during human infections has arisen from clinical studies where QS signal molecules have been detected in patient body fluids or tissues [9,10]. Since QS by definition depends on small-molecule ligand/receptor interactions, it offers a direct pharmacological pathway to inhibitor development since the steric requirements for optimal ligand/receptor interactions mean that antagonists can be readily obtained through structural modification of native agonists [11]. Indeed, this occurs naturally among Staphylococcus aureus strains that can divided into four QS groups based on their respective autoinducing peptides that self-activate but cross-group inhibit [6]. While signal molecule biosynthetic pathways constitute a further target for pharmacological inhibition, QS can also be blocked by preventing the pathogen population becoming quorate through enzymatic inactivation or antibody-based sequestration of the QS signal molecule [3]. Figure 1 summarizes the potential targets within a generalized QS system.

Potential targets within a generalized QS system.

Figure 1.
Potential targets within a generalized QS system.

Drug-like QSIs may target signal molecule biosynthesis or inhibit QS signal receptor interactions or QS may be inhibited through sequestration of the QS signal molecule by antibodies or polymers or via enzymatic degradation (adapted from ref. [3]).

Figure 1.
Potential targets within a generalized QS system.

Drug-like QSIs may target signal molecule biosynthesis or inhibit QS signal receptor interactions or QS may be inhibited through sequestration of the QS signal molecule by antibodies or polymers or via enzymatic degradation (adapted from ref. [3]).

Currently, our understanding of the potential of QS as an antibacterial target suggests that it is only likely to be of value with respect to a limited number of pathogens where it is clear that disruption of QS has a major impact on virulence, i.e. QS inhibitors (QSIs) in common with the broader class of the antivirulence agents [4] are likely to be narrow rather than broad-spectrum antibacterial agents.

In this context, success in developing clinically useful QSIs is likely to be restricted to bacterial pathogens such as S. aureus [6] and P. aeruginosa [5]. The latter has probably the most intensively investigated Gram-negative QS regulatory network controlling multiple virulence determinants and biofilm maturation [11,12]. Some promising P. aeruginosa QS targets have been structurally characterized [11], QSI screens devised [11,13,14] and inhibitors identified [5,15]. Interestingly, antibiotics, including azithromycin, ciprofloxacin and ceftazidime, have been reported to act as QSIs at sub-growth inhibitory concentrations via an unknown mode of action [16]. Table 1 compares some of the potential advantages and disadvantages of QSIs.

Table 1
Some potential advantages and disadvantages of QSIs
Advantages Disadvantages 
Novel targets New diagnostics required 
Not susceptible to most conventional antibiotic-resistance mechanisms Potentially susceptible to efflux 
Reduced selective pressure for the emergence of resistance Uncertain efficacy in immunocompromised individuals 
Render biofilms susceptible to antimicrobials and host defences Active as prophylactics but not possibly not as therapeutics 
Non-lytic mode of action — minimize endotoxin release Not bactericidal 
Preserve commensal microbiota Narrow spectrum of activity 
Advantages Disadvantages 
Novel targets New diagnostics required 
Not susceptible to most conventional antibiotic-resistance mechanisms Potentially susceptible to efflux 
Reduced selective pressure for the emergence of resistance Uncertain efficacy in immunocompromised individuals 
Render biofilms susceptible to antimicrobials and host defences Active as prophylactics but not possibly not as therapeutics 
Non-lytic mode of action — minimize endotoxin release Not bactericidal 
Preserve commensal microbiota Narrow spectrum of activity 

Discovering QS inhibitors

The discovery of drug-like QSI compounds depends initially on a suitable high-throughput bacterial cell-based screen and access to chemical diversity. Since QS acts at the transcriptional level, bacterial cell-based assays originally designed as biosensors for detecting QS signal molecules have frequently been exploited for inhibitor screening [3,10,12,13,17]. These usually incorporate reporter genes, such as gfp− or lux−, fused to a QS-dependent promoter such that the assay read-out will be reduced in the presence of a putative inhibitor. Such reporter screens are best constructed in the target pathogen to account for any problems that may relate to uptake, efflux, metabolic modification or toxicity. These assays also require appropriate control experiments to account for the general effects of putative QSI compounds on bacterial growth and the reporter gene product itself [18]. Alternatively, fusing a gene coding for a lethal protein to a QS-controlled promoter offers an QSI screen where the reporter strain is unable to grow in the presence of the QS signal molecule unless a non-toxic QSI molecule is present at a sufficiently high concentration [12].

QSI screens can readily be refined to confirm the QSI target, mechanism of action and to establish whether or not a QSI is a competitive or non-competitive inhibitor or has an undesirable partial agonist activity [15]. While chemical diversity can be sampled through the use of drug-like compound libraries and natural products, access to protein target structural information can reduce the numbers of compounds that require laboratory evaluation by employing structure-based computational screening to identify candidate QSI compounds [19]. This in silico approach also offers significant advantages with respect to downstream medicinal chemistry optimization. Furthermore, it is imperative that any ‘hit’ compounds are subjected to PAINS (pan-assay interference compound) filters, i.e. many QSI ‘hits’ will be artefacts, whose activity does not depend on a specific, drug-like interaction between the compound and protein target [20,21]. Most PAINS function as reactive chemicals rather than discriminating drug molecules and show false-positive activity in multiple assays. Polyphenols, such as curcumin for example, have been reported to be QSIs [22] but are good examples of PAINs and so not useful as drug candidates [20,21]. Once a promising chemical scaffold has been identified as a ‘hit’ QSI compound, further work will be required to synthesize a suitable potent (with activity in the nanomolar range), low toxicity lead compound with the appropriate physico-chemical, pharmacokinetic and drug metabolism properties [23]. To date, although several QSI compounds have been tested in animal infection models [8,14], none have advanced sufficiently successfully to be evaluated in clinical trials given the costs and long time-frames involved. However, one potential short-cut is the ‘repurposing’ of drugs already approved for human use for alternative indications as this option reduces the risks and regulatory hurdles lowering the costs of drug development. For example, niclosamide, an anthelmintic drug, inhibits AHL-dependent QS in P. aeruginosa although the mechanism of action has yet to be elucidated [24].

The P. aeruginosa QS regulatory hierarchy

The multi-signal molecule QS regulatory network of P. aeruginosa controls both virulence and biofilm development and hence is an appropriate target on which to focus efforts to develop QSIs. In P. aeruginosa, a flexible hierarchical QS cascade incorporating the overlapping las, rhl and pqs regulons employs AHLs (las and rhl) and AQs (pqs), respectively, as signal molecules [3,12]. All three QS systems contain autoinduction loops whereby activation of a dedicated transcriptional regulator by the cognate QS signal molecule induces expression of the target synthase such that QS signal molecule production can be rapidly amplified to promote co-ordination of gene expression at the population level [2,3,12].

The LasRI and RhlRI QS systems consist of two pairs of LuxRI homologues, where LasR and RhlR are LuxR-type transcriptional regulators activated by N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone that are, respectively, synthesized by LasI and RhlI [12]. P. aeruginosa strains with mutations in las, rhl or both exhibit attenuated virulence in some but not all infection models [8,14,25,26]. The latter may be a consequence of secondary mutations acquired during mutagenesis [26], differences between wild-type strains or because QS is not activated in all infection sites. QSIs for AHL-dependent QS in P. aeruginosa have been described and recently. Moore et al. [15] presented a comprehensive comparative analysis of synthetic LasR inhibitors, their mechanism of action and susceptibility to active efflux.

Transcriptomics and virulence factor profiling of wild-type and pqs mutants have revealed that the pqs regulon substantially overlaps with both las and rhl regulons with respect to many virulence factors, secondary metabolites and biofilm development [27]. P. aeruginosa mutants defective in AQ biosynthesis or perception are severely attenuated in experimental plant and animal infection models [14,28].

P. aeruginosa produces a variety of AQ and AQ N-oxide congeners, of which 2-heptyl-3-hydroxy-4-quinolone (PQS, the Pseudomonas Quinolone Signal) and its immediate precursor 2-heptyl-4-hydroxyquinoline (HHQ) are most closely associated with QS (Figure 2) [3,27]. Most of the genes required for AQ biosynthesis (pqsABCDE) and response (pqsR) are located at the same genetic locus although pqsH and pqsL are distally located [27]. PqsA catalyses the formation of anthraniloyl-CoA that is condensed with malonyl-CoA by PqsD to form 2-aminobenzoylacetyl-CoA (2-ABA-CoA). The latter is converted into 2-aminobenzoylacetate (2-ABA) via the thioesterase activity of PqsE [29]. However, although PqsE is not essential for AQ biosynthesis, it is required for the AQ-independent production of several factors that contribute to biofilm maturation including pyocyanin, rhamnolipids and lectin A [27,28]. However, the AQ-independent, thioesterase-independent mechanism by which PqsE acts has yet to be elucidated. HHQ is generated from the PqsBC heterodimer-mediated reaction of 2-ABA with octanoyl-CoA [30] and PQS via PqsH-mediated oxidation of HHQ. The formation of the AQ N-oxides requires PqsL, an alternative mono-oxygenase [27].

Diagrammatic representation of the AQ-dependent QS network in P. aeruginosa and the potential QSI targets.

Figure 2.
Diagrammatic representation of the AQ-dependent QS network in P. aeruginosa and the potential QSI targets.

The PqsABCDE proteins synthesize HHQ, which is converted into PQS by PqsH. Autoinduction occurs when either HHQ or PQS binds to PqsR and amplifies expression of the pqsABCDE operon. PqsE is a thioesterase that contributes to AQ biosynthesis and also an effector of virulence genes, secondary metabolites and biofilm development via an unknown mechanism. The conversion of HHQ into PQS confers additional functionalities since PQS is an iron chelator and functions via PqsR-dependent and PqsR-independent regulatory pathways [11,27]. QSI targets include AQ biosynthesis, PqsE, PqsR and PQS itself. Adapted from [11].

Figure 2.
Diagrammatic representation of the AQ-dependent QS network in P. aeruginosa and the potential QSI targets.

The PqsABCDE proteins synthesize HHQ, which is converted into PQS by PqsH. Autoinduction occurs when either HHQ or PQS binds to PqsR and amplifies expression of the pqsABCDE operon. PqsE is a thioesterase that contributes to AQ biosynthesis and also an effector of virulence genes, secondary metabolites and biofilm development via an unknown mechanism. The conversion of HHQ into PQS confers additional functionalities since PQS is an iron chelator and functions via PqsR-dependent and PqsR-independent regulatory pathways [11,27]. QSI targets include AQ biosynthesis, PqsE, PqsR and PQS itself. Adapted from [11].

The pqs system is subject to positive autoregulation (Figure 2), since the LysR-type transcriptional regulator PqsR (MvfR) binds to the promoter region of pqsABCDE (PpqsA) triggering transcription once activated by HHQ or PQS or their C9 congeners [11,14,27]. Therefore, by analogy with other QS systems, these AQs act as autoinducers by generating a positive feedback loop that accelerates their biosynthesis. In contrast with HHQ which only regulates the pqsABCDE operon [27], PQS is a ferric iron chelator that not only drives AQ biosynthesis but also the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways [27]. PQS also induces membrane vesicle formation via a direct physical interaction with outer membrane lipopolysaccharides [31].

AQ-dependent QS in P. aeruginosa as an exemplar QSI target

Rapid progress in our understanding of the chemical and structural biology of AQ-dependent QS and the loss of virulence accompanying mutation of key pqs genes in animal infection models has highlighted the promise of this particular P. aeruginosa QS system as a target for novel anti-pseudomonal agents [5,11,14]. The detection of AQs in the sputum, serum and urine of individuals with cystic fibrosis whose lungs are chronically colonized with P. aeruginosa indicates that the pqs system is expressed during human infections. Indeed, the presence of AQs, such as 2-nonyl-4-hydroxyquinoline (NHQ), has been correlated with clinical status in that NHQ levels were elevated at the start of a pulmonary exacerbation and decreased significantly following treatment [9].

Within the pqs system, many targets can be identified (Figure 2). AQ production and consequently virulence gene expression can be abrogated by mutation of pqsA, pqsB, pqsC, pqsD or pqsR since the loss of any one of these pqs genes results in the complete loss of AQ biosynthesis [27]. Since the substrates for each gene product are known and as crystal structures for several of the proteins involved have been determined [11,30,32], experimental screening, ligand- and fragment-based design, biophysical and in silico approaches have been used to identify inhibitors of AQ biosynthesis [5]. Not surprisingly, some of the first compounds identified were anthranilate analogues given that the first step in AQ biosynthesis is driven by the anthranilate ligase, PqsA [5]. Although as yet no inhibitors of the PqsBC heterodimer have been reported, many effective inhibitors of the FabH-like PqsD enzyme have been described [5]. Many of these compounds have IC50 activities in the low μM range although none have yet been extensively tested in rodent P. aeruginosa infection models. Interestingly, chemically blocking the intracellular supply of anthranilate by inhibiting the kynureninase KynU with S-phenyl-l-cysteine sulfoxide, a compound with structural similarity to the anthranilate precursor kynurenine, reduces PQS production and hence virulence factor production [33].

The loss of pqs system signalling has also been achieved through enzymatic cleavage of PQS. In particular, the heterocyclic ring-cleaving Arthrobacter enzyme ‘Hod’ (1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase) is capable of cleaving PQS to release carbon monoxide (an antibacterial) and N-octanoylanthranilic acid [34]. The addition of exogenous Hod significantly reduced the expression of several key P. aeruginosa virulence factors in planktonic cultures and reduced growth and tissue damage in a plant leaf infection model [34]. However, the efficacy of exogenous Hod is reduced by LasB-dependent proteolytic cleavage [34,35]. In biofilm assays, Hod enhanced the biomass of a P. aeruginosa lasB mutant probably as a consequence of increasing iron bioavailability through increased PQS degradation, a finding that suggests that Hod would be contra-indicated for biofilm-centred P. aeruginosa infections [35].

Inhibition of pqsABCDE operon expression and, hence, AQ biosynthesis by PqsR can also be achieved with a receptor-mediated antagonist such as the quinazolinone (QZN), 3-NH2-7-Cl-C9-QZN [11]. The QZN chemical scaffold was evolved through ‘ligand-based design’ in combination with two whole cell assays — a P. aeruginosa wild-type and a pqsA mutant both carrying a chromosomal pqsA::lux reporter gene fusion. These assays revealed that a simple isosteric replacement was sufficient to switch a QZN from potent agonist to potent competitive antagonist capable of inhibiting virulence factor production and reducing biofilm development [11]. Confirmation that 3-NH2-7-Cl-C9-QZN acted as a competitive antagonist was obtained by determination of the complex crystal structure of the QZN antagonist bound to the PqsR ligand-binding domain. This revealed a similar orientation within the ligand-binding pocket as the native agonist NHQ [11]. Other PqsR antagonists of varying potencies have been reported ranging from quinolones and 2-amino-oxadiazoles to benzamide–benzimidazoles. The latter have shown significant potency in both murine wound and lung infections [14]. Recently, Thomann et al. [36] have taken an interesting new approach by developing dual PqsR/PqsD inhibitors that reduced P. aeruginosa pathogenicity in a Galleria mellonella larvae infection model and enhanced the efficacy of ciprofloxacin towards P. aeruginosa biofilms [36].

Perspectives and conclusions

While considerable advances in our understanding of the chemical biology of QS regulatory systems and their inhibition have been made, much more work is needed before any QSIs are likely to enter human clinical trials. The relative efficacy of QSIs alone or as prophylactics or therapeutics or as adjuvants in combination with conventional antibiotics needs to be extensively evaluated. Preclinical testing of high potency, low toxicity QSIs in relevant acute and chronic animal infection (including immunocompromised) models will be required with particular attention being paid to the measurement of successful outcomes with respect to bacterial clearance, immune response and pathophysiology. Determining when and where within host tissues during infection QS systems are expressed will be important given that environmental conditions significantly have an impact on QS. PQS, for example, is not produced under anaerobic conditions [37]. While QSIs may well exert less selective pressure driving the emergence of resistance, QS (e.g. lasR) mutants are known to frequently arise in chronic human P. aeruginosa infections [38]. In addition, QSIs may well be rendered ineffective if, for example, they are substrates for multi-drug efflux pumps [15,39]. Whether QSI resistance readily develops and spreads is likely to depend on the costs and benefits of virulence factor expression during infection [40]. Since QSIs are likely to be narrow-spectrum agents, appropriate new diagnostics will be needed as traditional MIC testing is not relevant and confirmation that the infecting organism is expressing a specific QS system will be required. Despite these challenges, QSIs offer some attractive opportunities for developing new antibacterial agents in the age of personalized medicine and the emergence of pan-antibiotic-resistant pathogens.

Summary
  • Bacterial populations synchronize gene expression through cell-to-cell communication via the production and sensing of small diffusible signal molecules. Termed ‘quorum sensing’, this strategy enables bacteria to mount co-operative responses against competing micro-organisms and higher organisms and adapt to environmental challenges.

  • Quorum sensing regulates bacterial pathogenicity and is an attractive target for novel antibacterial agents that control infection through the inhibition of virulence factor production and by rendering biofilms more susceptible to conventional antibiotics and host immune clearance pathways.

  • Quorum-sensing systems offer multiple druggable molecular targets for inhibition that include the enzymes involved in signal molecule biosynthesis and the receptors involved in signal transduction.

  • A number of quorum-sensing targets have been structurally characterized, screens devised and inhibitors identified. However, the relative efficacies of quorum sensing inhibitors alone or as prophylactics or therapeutics or as adjuvants in combination with conventional antibiotics needs to be extensively evaluated in relevant in vivo animal infection models.

  • Since quorum-sensing inhibitors by definition will not inhibit bacterial growth in vitro, the measurement of successful therapy outcomes will revolve around bacterial clearance, immune response and pathophysiology. New diagnostics will be required.

  • As no universal bacterial quorum-sensing systems controlling virulence have been identified, quorum-sensing inhibitors will be narrow and targeted rather than broad spectrum antibacterial agents.

Abbreviations

     
  • 2-ABA

    2-aminobenzoylacetate

  •  
  • AHLs

    N-acylhomoserine lactones

  •  
  • AQs

    2-alkyl-4-quinolones

  •  
  • HHQ

    2-heptyl-4-hydroxyquinoline

  •  
  • Hod

    1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase

  •  
  • MIC

    minimum inhibitory concentration

  •  
  • NHQ

    2-nonyl-4-hydroxyquinoline

  •  
  • PAINS

    pan-assay interference compounds

  •  
  • PQS

    2-heptyl-3-hydroxy-4-quinolone

  •  
  • QS

    quorum sensing

  •  
  • QSI

    quorum sensing inhibitor

  •  
  • QZN

    quinazolinone

Acknowledgments

The contributions to quorum sensing and quorum sensing inhibition research of many colleagues, students and collaborators and research grant providers over the past 25 years are gratefully acknowledged.

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

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