Probing autoinducer-2 based quorum sensing: the biological consequences of molecules unable to traverse equilibrium states

Bacteria have developed a cell-to-cell communication system, termed quorum sensing (QS), which allows for the population-dependent coordination of their behavior via the exchange of chemical signals. Autoinducer-2 (AI-2), a class of QS signals derived from 4,5-dihydroxy-2,3-pentandione (DPD), has been revealed as a universal signaling molecule in a variety of bacterial species. In spite of considerable interest, the study of putative AI-2 based QS systems remains a challenging topic in part due to the rapid interconversion between the linear and cyclic forms of DPD. Herein, we report the design and development of efficient syntheses of carbocyclic analogues of DPD, which are locked in the cyclic form. The synthetic analogues were evaluated for the modulation of AI-2-based QS in Vibrio harveyi and Salmonella typhimurium. No agonists were uncovered in either V. harveyi or S. typhimurium assay, whereas weak to moderate antagonists were found against V. harveyi. On the basis of NMR analyses and DFT calculations, the heterocyclic oxygen atom within DPD appears necessary to promote hydration at the C3 position of cyclic DPD to afford the active tetrahydroxy species. These results also shed light on the interaction between the heterocyclic oxygen atom and receptor proteins as well as the importance of the linear form and dynamic equilibrium of DPD as crucial requirements for activation of AI-2 based QS circuits.     

An unexpected switch in the modulation of AI-2-based quorum sensing discovered through synthetic 4,5-dihydroxy-2,3-pentanedione analogues

Quorum sensing (QS) has traditionally referred to a mechanism of communication within a species of bacteria. However, emerging research implicates QS in interspecies communication and competition, and such systems have been proposed in a wide variety of bacteria. The AI-2-based QS system represents the most studied of these proposed interspecies systems, and has been proposed to regulate diverse functions such as bioluminescence, expression of virulence factors, and biofilm formation. As such, the development of modulatory compounds, both agonists and antagonists, is of great interest for the treatment of bacterial infections and the study of unknown AI-2-based QS systems. Toward this end, we have designed and synthesized a panel of 4,5-dihydroxy-2,3-pentanedione/AI-2 analogues and evaluated their effects on the AI-2 QS of various bacteria. The panel of compounds exhibited differential effects in the bacterial cell lines examined, providing a platform for the development of broad-spectrum modulators of AI-2-based QS.     

Deconvoluting interspecies bacterial communication

The universal bacterial signal molecule autoinducer-2 (AI-2) is derived from 4,5-dihydroxy-2,3-pentanedione (DPD). DPD exists in a complex equilibrium between multiple forms, and NMR spectroscopy has now been used to establish that the extent of the structural diversity displayed by DPD over a broad pH range is even greater than previously posited.     

Boron binding with the quorum sensing signal AI-2 and analogues

[reaction: see text] The unstable bacterial metabolic product, DPD, and the related natural product, laurencione, are shown to have a high affinity for borate complexation, through the hydrated analogue. The boron complex of DPD is Vibrio harveyi AI-2, an interspecies quorum sensing signal in bacteria, and an affinity column with a borate resin is effective in providing the first method for concentrating and purifying V. harveyi AI-2 from the biosynthetic product.     

An Efficient Synthesis of Optically Active [4-13C] Labelled Quorum Sensing Signal Autoinducer-2

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-¹³C]-AI-2 starting from [1-¹³C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-¹³C]-AI-2.     

Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing

Many bacterial pathogens coordinate their virulence factor expression in a cell density-dependent manner. This population-dependent coordination of gene expression in bacteria has been termed "quorum sensing" (QS). N-Acyl homoserine lactones (AHLs) are used by over 70 Gram-negative bacterial species as autoinducers. Inhibition of QS signaling might represent a new target for antimicrobial therapy. Here we report the hapten design, synthesis, generation of monoclonal antibodies (mAbs) against AHLs, and the evaluation of these mAbs for their ability to blunt QS signaling and inhibit virulence factor expression in P. aeruginosa. The mAbs can be envisioned as a tool for future investigations into AHL-based QS, which may aid in gaining new insights into the pathogenesis of P. aeruginosa and may ultimately lead to the development of new strategies to combat bacterial diseases.     

Natural Product Rottlerin Derivatives Targeting Quorum Sensing

Rottlerin is a natural product consisting of chalcone and flavonoid scaffolds, both of which have previously shown quorum sensing (QS) inhibition in various bacteria. Therefore, the unique rottlerin scaffold highlights great potential in inhibiting the QS system of Pseudomonas aeruginosa. Rottlerin analogues were synthesised by modifications at its chalcone- and methylene-bridged acetophenone moieties. The synthesis of analogues was achieved using an established five-step synthetic strategy for chalcone derivatives and utilising the Mannich reaction at C6 of the chromene to construct morpholine analogues. Several pyranochromene chalcone derivatives were also generated using aldol conditions. All the synthetic rottlerin derivatives were screened for QS inhibition and growth inhibition against the related LasR QS system. The pyranochromene chalcone structures displayed high QS inhibitory activity with the most potent compounds, 8b and 8d, achieving QS inhibition of 49.4% and 40.6% and no effect on bacterial growth inhibition at 31 µM, respectively. Both compounds also displayed moderate biofilm inhibitory activity and reduced the production of pyocyanin.     

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C₄-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

Short-chain reactive probes can be used as tools to shed new light on virulence mechanisms in bacterial pathogens.     

Convergence of hormones and autoinducers at the host/pathogen interface

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed “quorum sensing” (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host’s immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others’ soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.     

A Baylis-Hillman/ozonolysis route towards (±) 4,5-dihydroxy-2,3-pentanedione (DPD) and analogues

The Baylis-Hillman reaction between 2-(tert-butyldimethylsilyloxy)ethanal and 3-buten-2-one followed by desilylation gave rise to the corresponding α-methylene-β,γ-dihydroxy ketone further converted by reductive ozonolysis of the carbon-carbon double bond into racemic 4,5-dihydroxy-2,3-pentanedione (DPD), a significant molecule in bacterial cell-cell communication systems. The same sequence applied to other substrates allowed the preparation of chain elongated analogues and 5-O-acylated derivatives of DPD.     

Glycation Reactivity of a Quorum‐Sensing Signaling Molecule

Reported herein is that (4S)‐4,5‐dihydroxy‐2,3‐pentanedione (DPD) can undergo a previously undocumented non‐enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein‐labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum‐sensing signaling molecule.     

Controlling the activity of quorum sensing autoinducers with light

Bacteria use a communication system, called quorum sensing (QS), to organize into communities and synchronize gene expression to promote virulence and secure survival. Here we report on a proof-of-principle for externally interfering with this bacterial communication system, using light. By employing photoswitchable small molecules, we were able to photocontrol the QS-related bioluminescence in an Escherichia coli reporter strain, and the expression of target QS genes and pyocyanin production in Pseudomonas aeruginosa.     

Recent progress in the chemistry and chemical biology of microbial signaling molecules: quorum-sensing pheromones and microbial hormones

Communication among microorganisms is mediated by secretion and detection of microbial signaling molecules such as quorum-sensing pheromones and microbial hormones. The molecules elicit the regulation of important genes necessary for microbial survival and often play important roles in interspecies or even inter-kingdom communication. Recent progress in the study of the signaling molecules has enabled us to eavesdrop on microbial conversations to gain insight on their intercellular communication system. This review summarizes the recent advances in the chemistry and chemical biology of these important microbial signaling molecules: acyl-homoserine lactones (AHLs), AI-2, CAI-1 related α-hydroxy ketones (AHKs), ComX pheromones, diffusible signal factors (DSFs), diffusible extracellular factor (DF), and Phytophthora mating hormones.     

Pyranosylmagellanicus a novel structural class of polyhalogenated acetogenins from Ptilonia magellanica

Three naturally occurring pyranosyl-like polyhalogenated metabolites 1-3 as well as their likely biogenetic precursor, the linear compound 4, have been isolated from the red alga Ptilonia magellanica. They are the first compounds within the genus that incorporate chlorine in their network. Compound 3 have structural features reminiscent of the universal chemical signal AI-2 (autoinducer-2) for bacterial communication.     

A multivalent probe for AI-2 quorum-sensing receptors

Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum-sensing receptors. From these studies, we have discovered a chemical probe specific for Lsr-type AI-2 quorum-sensing receptors with the potential for enabling the identification of new bacterial species that utilize AI-2 as a quorum-sensing signaling molecule.     

Infection control by antibody disruption of bacterial quorum sensing signaling

Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.     

Coumarin’s Anti-Quorum Sensing Activity Can Be Enhanced When Combined with Other Plant-Derived Small Molecules

Coumarins are class of natural aromatic compounds based on benzopyrones (2H-1-benzopyran-2-ones). They are identified as secondary metabolites in about 150 different plant species. The ability of coumarins to inhibit cell-to-cell communication in bacterial communities (quorum sensing; QS) has been previously described. Coumarin and its derivatives in plant extracts are often found together with other small molecules that show anti-QS properties too. The aim of this study was to find the most effective combinations of coumarins and small plant-derived molecules identified in various plants extracts that inhibit QS in Chromobacterium violaceum ATCC 31532 violacein production bioassay. The coumarin and its derivatives: 7-hydroxycoumarin, 7.8-dihydroxy-4-methylcoumarin, were included in the study. Combinations of coumarins with gamma-octalactone, 4-hexyl-1.3-benzenediol, 3.4.5-trimethoxyphenol and vanillin, previously identified in oak bark (Quercus cortex), and eucalyptus leaves (Eucalyptus viminalis) extracts, were analyzed in a bioassay. When testing two-component compositions, it was shown that 7.8-dihydroxy-4-methylcoumarin, 4-hexyl-1.3-benzendiol, and gamma-octalactone showed a supra-additive anti-QS effect. Combinations of all three molecules resulted in a three- to five-fold reduction in the concentration of each compound needed to achieve EC₅₀ (half maximal effective concentration) against QS in C. violaceum ATCC 31532.     

Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative

Quorum sensing (QS) is a cell-to-cell communication process that controls bacterial collective behaviors. The QS network regulates and coordinates bacterial virulence factor expression, antibiotic resistance and biofilm formation. Therefore, inhibition of the QS system is an effective strategy to suppress the bacterial virulence. Herein, we identify a phosphate ester derivative of chrysin as a potent QS inhibitor of the human pathogen Pseudomonas aeruginosa (P. aeruginosa) using a designed luciferase reporter assay. In vitro biochemical analysis shows that the chrysin derivative binds to the bacterial QS regulator LasR and abrogates its DNA-binding capability. In particular, the derivative exhibits higher anti-virulence activity compared to the parent molecule. All the results reveal the potential application of flavone derivative as an anti-virulence compound to combat the infectious diseases caused by P. aeruginosa.     

Investigating the effect of antibiotics on quorum sensing with whole-cell biosensing systems

Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn’s disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS. We investigated the effect of ciprofloxacin, metronidazole, and tinidazole on quorum sensing. Several concentrations of individual antibiotics were allowed to interact with two different types of bacterial sensing cells, in both the presence and absence of a fixed concentration of N-acylhomoserine lactone (AHL) QS molecules. The antibiotic effect was then determined by monitoring the biosensor’s bioluminescence response. Ciprofloxacin, metronidazole, and tinidazole exhibited a dose-dependent augmentation in the response of both bacterial sensing systems, thus showing an AHL-like effect. Additionally, such an augmentation was observed, in both the presence and absence of AHL. The data obtained indicate that ciprofloxacin, metronidazole, and tinidazole may interfere with bacterial communication systems. The results suggest that these antibiotics, at the concentrations tested, may themselves act as bacterial signaling molecules. The beneficial effect of these antibiotics in the treatment of intestinal inflammation may be due, at least in part, to their effect on QS-related bacterial behavior in the gut.