Quorum sensing is the intercellular communication used by a bacterial population, once it reaches a certain threshold, to collectively synchronize the expression of pathogenic traits, such as biofilm formation, swarming motility, and the production of virulence factors. This coordinated behaviour is mediated via small molecules called auto-inducers. In quorum sensing research, the aim is to inhibit quorum sensing molecular mechanisms, which could provide an alternative to the conventional antibiotic control of infections. Several natural (such as coumarin, curcumin, and garlic) and synthetic compounds have been suggested as quorum sensing inhibitors.
Less than two decades ago, at the end of the 20th century, humanity started seeing the comeback of several lethal infectious diseases. Since then, it has become apparent that bacteria can adapt to the selective pressure of antibiotics. One of today’s global concerns is that we are entering a post‐antibiotic era with no knowledge on how to fight microbes.1 The World Health Organization anticipates that antibiotic resistance will be one of the biggest problems, and if the existing trajectory is sustained, it could kill up to 1.3 million people in Europe by 2050. Humanity is being threatened by antibiotic-resistant bacteria and we need to do something about it fast. Other antimicrobial strategies are required, and quorum sensing might be one road to discover them.
What is Quorum Sensing (QS)?
It would be of little value for just a few bacterial cells to produce a gene product such as an extracellular enzyme or virulence protein, because the concentration of the protein would be too low to be effective. QS is a type of regulatory process that ensures that there is sufficient cell density (the quorum) before a particular gene product is made. Such a process allows bacteria to increase in numbers before starting to produce a specific gene product. Each species that uses QS produces a small signal molecule, called an autoinducer.
Autoinduction was discovered around 50 years ago in the Gram-negative bacterium Vibrio fischeri. V. fischeri is a bioluminescent symbiont of the Hawaiian bobtail squid, whose rich nutrients allow the bacteria to proliferate. Bioluminescence genes are expressed when the density of the bacteria is significantly high. Interestingly, the light produced serves as an anti-predatory response, stopping the squid from producing a shadow under the moonlight.
There are several molecular mechanisms for intercellular signalling.2
- QS based on AHL (Acyl Homoserine Lactone) or ‘autoinducer-1’ (AI-1) system
In Gram-negative bacteria, the signal molecule is acyl homoserine lactone, or AHL. When the AHL signal molecule reaches a threshold concentration, it binds to and activates a regulatory protein which then binds to a specific site on the DNA. The binding of this regulatory protein transcription activator results in the production of the specific quorum-dependent protein, as well as more enzymes to make the AHL.
- QS based on autoinducing peptides (AIPs)
In Gram-positive bacteria, QS involves a different type of signal molecule. First, a precursor oligopeptide is cleaved into functional signal molecules of 10 to 20 amino acids. These molecules are transported out of the cell through a special transporter protein. When the signal oligopeptides reach a threshold concentration on the outside of the cell, they are detected by a sensor protein on the surface of the cell. When the oligopeptides react with the sensor protein, the protein becomes phosphorylated on the inside of the cell membrane. The phosphate is then transferred to a response regulator protein which allows it to bind to a specific site on the DNA. This binding results in alteration in the transcription of target genes. Finally, quorum-dependent proteins such as specific virulence factors are produced.
- QS based on the ‘autoinducer-2’ (AI-2) system
Based on the molecule furanosyl borate diester, the AI-2 system is found in both Gram-negative and Gram-positive bacteria. For instance, it is found in pathogenic bacteria such as Salmonella typhimurium, Streptococcus gordonii and Vibrio harveyi.3 Depending on the cell density and threshold concentration, the Al-2 molecule changes between the inter- and intra-species signal, and is used to serve bacteria in their surrounding environment, including signal transduction.
- Other QS systems
Over the last decades, other molecules have been identified; these include 4-quinolones, fatty acids, and the A-factor from Streptomyces.3 Pyrones have also been recognised as QS signals in Photorhabdus luminescens.4 In all probability, this only represents a small percentage of the metabolites involved in QS.
Interfering with QS Signalling
QS signalling has provided important benefits to bacteria in their adaptation to changing environments, defence against competitors, formation of biofilms, and host colonisation. Several QS-controlled activities are involved in the pathogenic and virulence potential of bacteria. Indeed, understanding and targeting QS molecular mechanisms could provide an alternative to the conventional antibiotic control of infections. However, while bacterial QS inhibitors (QSIs) have been studied for their QS interfering capabilities, none have been applied in the clinical setting so far. In general, this is attributed to the co-existence of multiple AHL QS systems in individual bacterial species.
In principle, QSIs should inhibit the genes that are QS-controlled. Specifically, QSIs should target the QS regulator without detrimental effects on the bacteria. It is important that the essential life processes that aid bacterial growth are not disrupted by QSIs, because then the selective pressure to develop resistance is abated,5 which is a key problem with many antibiotics.
In QSI research, one of the most commonly studied bacterial species is Pseudomonas aeruginosa. P. aeruginosa is a Gram-negative bacterium with three QS systems, namely Las, Pqs, and Rhl.6 It is estimated that ~5% of the total number of genes in P. aeruginosa is controlled by QS. Critically, P. aeruginosa is responsible for a large number of nosocomial infections, and is particularly damaging for patients with weakened immune systems.7 Therefore, intensive research efforts are focused on P. aeruginosa in particular, on both natural and synthetic QSIs.
Both natural and synthetic compounds have been identified as QSIs.5 In recent years, however, the focus may have shifted towards medicinal natural plants since they offer a phytochemical repertoire with microbial disease-controlling potential.8 In part, this is due to the range of secondary metabolites found in extracts, which include alkaloids, flavonoids, phenolics, polyacetylenes, quinones, and terpenoids. Most of these compounds inhibited QS in screens that used AHL-dependent biosensor strains.
The Coumarin family of natural plant-derived compounds, for instance, possesses bioactive molecules that have been investigated for their efficacy as QSIs. Coumarin has been demonstrated to have anti-biofilm activity in P. aeruginosa, particularly in hydroxylated coumarins.9 It has also been shown to reduce swarming motility.10 Zhang et al. (2018)11 have recently shown that in coumarin-treated P. aeruginosa, integral genes involved in QS are downregulated, further demonstrating the potential application of coumarin as a QSI.
Curcumin (a bright yellow compound found in turmeric) also has potential as a QSI.12 Bahari et al. (2017) showed that P. aeruginosa QS is inhibited by sub-inhibitory concentrations of curcumin with azithromycin and gentamicin. Promisingly, curcumin has similar effects in the bacteria Aeromonas sobria13 and Streptococcus mutans.14 In keeping with this, in 2014 Packiavathy demonstrated that curcumin enhanced the susceptibility of a marker strain and uropathogens to conventional antibiotics.15 On a side note, the antibiotic azithromycin has been shown to prevent P. aeruginosa ventilator-associated pneumonia by inhibition of QS.16
- aeruginosa is also known to form biofilms in the cystic fibrosis lung. QS controls biofilm maturation. In a pilot trial, Smyth et al. (2010)17 randomised over 30 patients to garlic or olive oil capsules (both 656 mg daily) to ascertain the QS inhibitory activity of garlic. In general, the clinical trial showed that garlic inhibits P. aeruginosa QS in cystic fibrosis – a result which should be further investigated in a larger trial. Intriguingly, Jakobsen et al. (2012)18 determined that it is the sulfur-rich molecule ajoene in garlic that inhibits genes controlled by QS.
In another investigation, Rajkumari et al. (2018)19 studied the pentacyclic triterpenes betulin and betulinic acid. Betulin, found in the bark of birch trees, can be converted to betulinic acid, which is a more active compound than betulin itself. In this study, the researchers reported that these two triterpenes, at sub-lethal concentrations, attenuated biofilm formation and the production of QS-regulated virulence factors in P. aeruginosa.
Lee et al. (2011)20 showed that, at low concentrations (0.5% v/v), acacia and multifloral Korean honeys reduce biofilm formation in an Escherichia coli strain. Truchado et al. (2009)21 also showed that chestnut honey and its aqueous extract inhibit QS in the bacteria Aeromonas hydrophila, Erwinia carotovora, and Yersinia enterocolitica. Specifically, these compounds degraded AHLs and inhibited AHL production by the bacterial strains.
Adonizio et al. (2008)22 assessed six south Florida medicinal plants for their anti-QS activities against P. aeruginosa. Interestingly, each plant had a different effect on the las and rhl QS genes and their corresponding signals. In addition, all extracts inhibited QS genes and QS-controlled factors, with negligible effects on bacterial growth.
Furthermore, the essential oils of rosemary and tea tree, as well as resveratrol and extracts of bee pollen, pomegranate, and propolis were tested for their QS inhibitory activities.8 Overall, the results revealed that the essential oils of rosemary and tea tree have the highest inhibitory activity, whereas pomegranate extract and resveratrol have the lowest anti-QS activity.
The list goes on and indeed, other sources are also being investigated like extracts from aquatic fungi.23 However, in addition to natural QSIs, numerous synthetic compounds are being developed to target and disrupt genes vital to QS systems. For example, Qiu et al. (2019)24 have synthesised compounds derived from quinoline that inhibit biofilm formation and virulence in P. aeruginosa, and disrupt rhl expression. In a different investigation, Welsh et al. (2015)6 screened synthetic N-acyl l-homoserine lactones and identified compounds that can change the production of two P. Aeruginosa virulence factors: pyocyanin and rhamnolipid. Overall, their results suggest that designing chemical agents to disrupt QS signalling could be a functional strategy to combat this common opportunistic pathogen.
Although new synthetic and natural compounds are continually being tested for their QSI efficacy, existing drugs and compounds may also serve as prospective QSIs. Yang et al. (2009)25 used a computer-aided screening method - on a database comprised of both natural compounds and existing approved drugs that share similar structural properties to known QSIs - to detect previously unidentified potential QSIs. Many QSIs have been recognised in recent years, and the number of QSI-related patent applications is rapidly increasing.26 However, the potential of QSIs as future therapeutic strategies will rest upon the results of clinical trials in humans; this is the next step in QSI research but is to date effectively unexplored.
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- Brachmann AO, Brameyer S, Kresovic D, et al. Pyrones as bacterial signaling molecules. Nature Chemical Biology 2013; 9:573.
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- Welsh MA, Eibergen NR, Moore JD, et al. Small molecule disruption of quorum sensing cross-regulation in Pseudomonas aeruginosa causes major and unexpected alterations to virulence phenotypes. J Am Chem Soc 2015; 137:1510-9.
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- D'Almeida RE, Molina RDI, Viola CM, et al. Comparison of seven structurally related coumarins on the inhibition of quorum sensing of Pseudomonas aeruginosa and Chromobacterium violaceum. Bioorg Chem 2017; 73:37-42.
- Gutierrez-Barranquero JA, Reen FJ, McCarthy RR, et al. Deciphering the role of coumarin as a novel quorum sensing inhibitor suppressing virulence phenotypes in bacterial pathogens. Appl Microbiol Biotechnol 2015; 99:3303-16.
- Zhang Y, Sass A, Van Acker H, et al. Coumarin Reduces Virulence and Biofilm Formation in Pseudomonas aeruginosa by Affecting Quorum Sensing, Type III Secretion and C-di-GMP Levels. Front Microbiol 2018; 9:1952.
- Bahari S, Zeighami H, Mirshahabi H, et al. Inhibition of Pseudomonas aeruginosa quorum sensing by subinhibitory concentrations of curcumin with gentamicin and azithromycin. Journal of Global Antimicrobial Resistance 2017; 10:21-28.
- Ding T, Li T, Wang Z, et al. Curcumin liposomes interfere with quorum sensing system of Aeromonas sobria and in silico analysis. Scientific Reports 2017; 7:8612.
- Li B, Li X, Lin H, et al. Curcumin as a Promising Antibacterial Agent: Effects on Metabolism and Biofilm Formation in S. mutans. Biomed Res Int 2018; 2018:1-11.
- van Delden C, Köhler T, Brunner-Ferber F, et al. Azithromycin to prevent Pseudomonas aeruginosa ventilator-associated pneumonia by inhibition of quorum sensing: a randomized controlled trial. Intensive Care Med 2012; 38:1118-25.
- Packiavathy IA, Priya S, Pandian SK, et al. Inhibition of biofilm development of uropathogens by curcumin - an anti-quorum sensing agent from Curcuma longa. Food Chem 2014; 148:453-60.
- Smyth AR, Cifelli PM, Ortori CA, et al. Garlic as an inhibitor of Pseudomonas aeruginosa quorum sensing in cystic fibrosis - a pilot randomized controlled trial. Pediatr Pulmonol 2010; 45:356-62.
- Jakobsen TH, van Gennip M, Phipps RK, et al. Ajoene, a Sulfur-Rich Molecule from Garlic, Inhibits Genes Controlled by Quorum Sensing. Antimicrob Agents Chemother 2012; 56:2314-2325.
- Rajkumari J, Borkotoky S, Murali A, et al. Attenuation of quorum sensing controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by pentacyclic triterpenes, betulin and betulinic acid. Microb Pathog 2018; 118: 48-60.
- Lee JH, Park JH, Kim JA, et al. Low concentrations of honey reduce biofilm formation, quorum sensing, and virulence in Escherichia coli O157:H7. Biofouling 2011; 27:1095-104.
- Truchado P, López-Gálvez F, Gil MI, et al. Quorum sensing inhibitory and antimicrobial activities of honeys and the relationship with individual phenolics. Food Chemistry 2009; 115:1337-1344.
- Adonizio A, Kong KF, Mathee K. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrob Agents Chemother 2008; 52:198-203.
- Martín-Rodríguez AJ, Reyes F, Martín J, et al. Inhibition of bacterial quorum sensing by extracts from aquatic fungi: first report from marine endophytes. Mar Drugs 2014; 12:5503-26.
- Qiu MN, Wang F, Chen SY, et al. Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa virulence and biofilm formation. Bioorg Med Chem Lett 2019;29(5):749-754.
- Yang L, Rybtke MT, Jakobsen TH, et al. Computer-aided identification of recognized drugs as Pseudomonas aeruginosa quorum-sensing inhibitors. Antimicrob Agents Chemother 2009; 53:2432-43.
- Chen X, Zhang L, Zhang M, et al. Quorum sensing inhibitors: a patent review (2014-2018). Expert Opin Ther Pat 2018; 1-17.