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. 2024 Jan 25;7(2):533–543. doi: 10.1021/acsptsci.3c00354

Juglone as a Natural Quorum Sensing Inhibitor against Pseudomonas aeruginosa pqs-Mediated Virulence and Biofilms

Yeping Ma †, Wing Suet Tang †, Sylvia Yang Liu †, Bee Luan Khoo ‡,§,∥, Song Lin Chua †,⊥,#,∇,*
PMCID: PMC10863437  PMID: 38357290

Abstract

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Pseudomonas aeruginosa is a notorious opportunistic pathogen associated with chronic biofilm-related infections, posing a significant challenge to effective treatment strategies. Quorum sensing (QS) and biofilm formation are critical virulence factors employed by P. aeruginosa, contributing to its pathogenicity and antibiotic resistance. Other than the homoserine-based QS systems, P. aeruginosa also possesses the quinolone-based Pseudomonas quinolone signal (PQS) QS signaling. Synthesis of the PQS signaling molecule is achieved by the pqsABCDEH operon, whereas the PQS signaling response was mediated by the PqsR receptor. In this study, we report the discovery of a novel natural compound, Juglone, with potent inhibitory effects on pqs QS and biofilm formation in P. aeruginosa. Through an extensive screening of natural compounds from diverse sources, we identified Juglone, a natural compound from walnut, as a promising candidate. We showed that Juglone could inhibit PqsR and the molecular docking results revealed that Juglone could potentially bind to the PqsR active site. Furthermore, Juglone could inhibit pqs-regulated virulence factors, such as pyocyanin and the PQS QS signaling molecule. Juglone could also significantly reduce both the quantity and quality of P. aeruginosa biofilms. Notably, this compound exhibited minimal cytotoxicity toward mammalian cells, suggesting its potential safety for therapeutic applications. To explore the clinical relevance of Juglone, we investigated its combinatorial effects with colistin, a commonly used antibiotic against P. aeruginosa infections. The Juglone–colistin combinatorial treatment could eliminate biofilms formed by wild-type P. aeruginosa PAO1 and its clinical isolates collected from cystic fibrosis patients. The Juglone–colistin combinatorial therapy dramatically improved colistin efficacy and reduced inflammation in a wound infection model, indicating its potential for clinical utility. In conclusion, the discovery of Juglone provides insights into the development of innovative antivirulence therapeutic strategies to combat P. aeruginosa biofilm-associated infections.

Keywords: Pseudomonas aeruginosa, biofilm, quorum sensing inhibition, antivirulence agent, colistin

Antimicrobial resistance poses major threats to global health, economic stability, and food security, attributing to 1.27 million deaths annually with high burdens in low- and middle-income countries.1 The critical priority pathogens with antibiotic resistance listed by World Health Organization (WHO) and US Centers for Disease Control and Prevention (CDC) include Acinetobacter, Enterobacterales, Pseudomonas aeruginosa, Clostridioides difficile, and Neisseria gonorrheae.2 Other than antibiotic resistance, these pathogens can produce virulence factors and form biofilms. The sticky exopolymeric matrix in biofilms serves as physical protective barriers against immune system and antibiotics, allowing most pathogens to establish chronic and nosocomial infections, such as pneumonia, urinary tract infections, and implant-associated infections.3 Biofilms can also impede motility of the hosts and prevent host detection of bacteria.4

As a robust biofilm-forming pathogen, P. aeruginosa is a leading causative agent of microbial infections globally, where it causes infections in hospitalized patients, immunocompromised individuals, and elderly,5 leading to prolonged hospitalization and high mortality. Its resistance to many antibiotic classes limits therapeutic options, so polymyxins (polymyxin B and colistin) are often used as the last resort for treatment of extensively drug-resistant (XDR) P. aeruginosa infections. However, mcr-1 plasmid-based colistin resistance had emerged in P. aeruginosa.6 Other mechanisms of intrinsic resistance include modifications of lipopolysaccharide (LPS) structure and composition in response to colistin treatment to prevent effective binding by colistin.7 Another poorly understood mechanism is the colistin-induced quorum sensing (QS) activation and biofilm formation, where prior studies, including ours, showed that colistin induces pqs QS and biofilm formation in P. aeruginosa.8,9

The PQS synthesis clusters are pqsABCDE, phnAB, and pqsH which are involved in the synthesis of PQS signaling molecules.10,11 On the other hand, PqsR is a transcriptional response regulator that responds to PQS signaling molecules and controls the expression of downstream pqs-controlled genes, such as the synthesis of cytotoxic pyocyanin and release of extracellular DNA (eDNA) for biofilm formation.11 Pyocyanin is a toxic phenazine produced at high concentrations (up to 100 μM) in infected human lungs12 to impair mucus clearance by ciliary cells. Next, eDNA associates with other exopolysaccharides in P. aeruginosa biofilms,13 with concentrations in human sputum estimated to be 2–20 mg/mL,14 where it could protect biofilm cells from antibiotics. Furthermore, PQS signaling molecule can modulate immune signaling and kill host cells.15 This raises the rationale to target the pqs signaling system specifically, to improve colistin efficacy in treatment of P. aeruginosa biofilm infections.

Despite the public health threat, few drug candidates have been developed16 due to high research costs and rapid emergence of antibiotic resistance. Current drug research efforts have turned their attention to alternative antimicrobial strategies with targeting mechanisms differing from those of existing antibiotics. According to the 2019 report by World Health Organization (WHO), novel antimicrobial development strategies should include [1] limiting the development of bacterial pathogen resistance, [2] decreasing pathogen virulence or host harm, and [3] addressing the lack of target specificity in broad-spectrum antibiotics.17 Precision antimicrobial therapeutics belong to a recent concept which target key virulence factors of specific pathogens while leaving the host microbiota undisturbed,18 such as bacteriophages, quorum sensing inhibitors and antibiofilm agents.19 However, there are numerous hurdles impeding clinical development of precision antimicrobial therapeutics, including the complexity in synthesizing or modifying lead candidates and the toxicity of these compounds. Repurposing of known drugs or natural compounds for novel antimicrobial functions is an innovative approach to address antimicrobial resistance due to shorter development pipeline and lower risk of adverse effects.20

Naphthoquinones are naturally abundant phenolic compounds with great promise in drug development due to their diverse pharmacological properties. One of its most significant applications is as an anticancer agent, as it has been shown to inhibit tumor growth and induce cancer cell death.21 As direct addition of a substituent group can alter their properties, their derivatives have also been investigated for their potential use in treating a range of other diseases, including inflammation, neurodegeneration, and infectious diseases.22 One example is lawsone, a naturally occurring naphthoquinone derivative found in henna leaves, which has been shown to possess antiviral properties against herpes simplex virus.23 Additionally, naphthoquinone derivatives have been explored as potential antibiotics, with some showing promising results against drug-resistant strains of bacteria.24

Here, we show that Juglone (5-hydroxy-1,4-naphthalenedione), an active ingredient from walnut trees used in traditional herbal medicine and commercial coloring, could be repurposed to inhibit pqs signaling, resulting in the inhibition of pyocyanin production and biofilm formation in P. aeruginosa. Although a previous study had shown that Juglone could also inhibit P. aeruginosa growth via the production of reactive oxygen species,25 the concentration used in their study was more than 10-fold higher than those used in our study and was cytotoxic to human cells. In contrast, the Juglone concentration used in our study (2.5 μM) was noncytotoxic to human cell culture and Medaka fish models. This indicated that the inhibitory concentration used in our study was potentially safe for clinical use. Lastly, we showed that Juglone–colistin combinatorial therapy could effectively eliminate P. aeruginosa biofilm infections and reduce acute inflammation in a Medaka fish wound-based model. The combinatorial therapy could also eliminate biofilms formed by P. aeruginosa clinical isolates collected from cystic fibrosis patients, suggesting the clinical efficacy of Juglone. Hence, precision antivirulence therapeutics represent a promising approach in mitigating bacterial virulence and enabling the subsequent immune clearance of pathogens.

Materials and Methods

Bacterial Strains, Media, and Cultivation

The P. aeruginosa strains are listed in Table S1. Bacteria were cultivated in 2 mL of Luria–Bertani (LB) medium (Difco, Becton Dickinson) or ABT minimal media containing 2 g/L glucose (Sigma-Aldrich, Germany) and 2 g/L casamino acids (Difco, Becton Dickinson) (ABTGC)26 at 37 °C with shaking at 200 rpm (rpm) for 16 h. For marker selection in P. aeruginosa, 30 μg/mL gentamicin (Sigma-Aldrich, Germany), 100 μg/mL carbenicillin (Sigma-Aldrich, Germany), and 30 μg/mL tetracycline (Sigma-Aldrich, Germany) were used, as appropriate.

Compound Library Screening and Evaluation of Minimal Inhibitory Concentration (MIC) and Half-Maximal Inhibitory Concentrations (IC50)

To identify potential pqs inhibitors, the P. aeruginosa wild-type PAO1/ppqsA-gfp transcription fusion biosensor27 was used for screening of our lab’s in-house 500 natural compound library at 10 μg/mL. The biosensor measures the expression of pqs QS, as PqsA is an enzyme involved in the synthesis of the autoinducer PQS.28 After the identification and selection of Juglone for this study, the bacterial culture was cultivated in 200 μL of ABTGC media with varying concentrations of Juglone (0–10 μg/mL) in each well of a 96-well plate (SPL, Korea) at 37 °C for 16 h in a microplate reader (Tecan Infinite 200 Pro, Switzerland). The bacterial cultures were analyzed by a microplate reader for their OD600 every 15 min. The MIC and IC50 were calculated using the Graphpad Prism 6 software package (GraphPad Software Inc., CA).

Relative GFP Quantification of ppqsA-gfp Expression

As described in the previous section,9 we cultivated PAO1/ppqsA-gfp, ΔpqsA/pqsA-gfp, ΔpqsR/ppqsA-gfp, and ΔpqsR/plac-pqsABCDE/ppqsA-gfp in ABTGC media containing different Juglone concentrations in a 96-well plate. The bacterial samples were cultivated for 16 h in a microplate reader at 37 °C, with the analysis of OD600 and GFP fluorescence intensity (Ex: 495 nm; Em: 515 nm; Gain 60) conducted every 15 min. The relative GFP/OD600 was then derived from accounting for cell optical density.

Molecular Docking

As previously described,29 AutoDock Tool v.1.5.7 software was used for molecular docking of Juglone to the active site of PqsR (4JVC).30 The native ligand NHQ and water molecules were removed from the active site, followed by addition of polar hydrogens using PyMol by Schrödinger v. 2.5. We then generate a search space around the amino acid residues involved in the binding of NHQ to the PqsR active site, followed by detection of the torsion root of Juglone. In each simulation, nine runs were conducted with the predicted binding affinity (kilocalories per mole) per run. The BIOVIA Discovery Studio was used to detect and compare the docking sites of PqsR for ligands NHQ and Juglone. The 3D images were generated from PyMOL v.2.3.2.

Relative Quantification of PQS

Using triplicate wells of ΔpqsA/pqsA-gfp grown in ABTGC containing 0 to 20 uM PQS, we first developed a concentration–response curve to demonstrate the relationship between GFP expression of pqsA-gfp to PQS concentration. The ΔpqsA/pqsA-gfp mutant could not produce its own PQS signaling molecule, so it is dependent on exogenous PQS treatment to achieve GFP expression by pqsA-gfp.(31) The 96-well microplate was then incubated in the microplate reader at 37 °C, with the analysis of OD600 and GFP conducted every 15 min. The dose–response curve (PQS concentration to GFP/OD600) was then derived with GraphPad Prism.

To measure the PQS concentrations produced by bacteria treated with Juglone, we cultivated PAO1 treated with various Juglone concentrations in ABTGC at 37 °C and 200 rpm for 16 h, followed by collecting the supernatants from the cultures by centrifugation at 13 000g for 3 min. The supernatants were sterilized by filtering through the 0.2-μm syringe filter, and 100 μL of supernatant was transferred to a fresh well in a 96-well microplate. The ΔpqsA/pqsA-gfp mutant in ABTGC was then added to supernatant at equal volumes so that it could detect the presence of PQS in the supernatant and express GFP. The bacterial samples were cultivated for 16 h in a microplate reader at 37 °C, with the analysis of OD600 and GFP fluorescence intensity (Ex: 495 nm; Em: 515 nm; gain 60) conducted every 15 min. The relative GFP/OD600 values from the samples were compared to the dose–response curve (PQS concentration to GFP/OD600) to determine the absolute PQS concentration present in the bacterial supernatants.

Swarming Motility Assay

The swarming motility experiment was performed by placing 5 μL of PAO1 overnight culture directly on the top and middle of the LB agar plates containing 0.5% agar and various Juglone concentrations. The plates were incubated in the 37 °C incubator for 24 h. The diameter of the motility ring across the agar plate was measured with a ruler.

Relative Quantification of Pyocyanin

As previously described,32 PAO1 was cultured in 10 mL of ABTGC with various concentrations of Juglone in 37 °C, shaking at 200 rpm for 16 h. The cultures were centrifuged at 13 000g for 3 min, followed by transfer of supernatant to fresh 15 mL tubes (SPL, Korea). The 1 mL of chloroform was added to each supernatant and vortexed vigorously for 15 s. After allowing the aqueous and chloroform phases to separate, the bottom layer of chloroform was then carefully transferred to fresh 1.5 mL microcentrifuge tubes. The 100 μL aliquot of 0.2 M HCl was added to the chloroform phase and vortexed vigorously for 15 s. The HCl layer containing the extracted pyocyanin was then transferred to each well of the 96-well plate for measurement of OD520 in the microplate reader.

Biofilm Growth Inhibition Assay

The PAO1/plac-gfp with constitutive GFP expression was cultivated at 1:100× dilution in 300 μL of ABTGC with various treatments (no treatment as control, 1.25 μg/mL Juglone, 1 μg/mL colistin, and 1.25 μg/mL Juglone +1 μg/mL colistin) in an 8-well chamber slide (μSlide, ibiTreat, Ibidi, Germany) at 37 °C for 24 h. The spent media containing planktonic cells were removed, followed by washing of the bottom-adhering biofilms twice with 0.9% NaCl. Propidium iodide (PI) was added to all wells at a final concentration of 1 μM to stain dead bacteria.

Biofilm Elimination Assay

The PAO1/plac-gfp biofilm was cultivated in 300 μL of ABTGC in the 8-well chamber slide at 37 °C for 24 h. The spent media containing planktonic cells were removed, followed by washing of the bottom-adhering biofilms twice with 0.9% NaCl. The biofilms were then directly treated with 300 μL of ABTGC with various treatments (no treatment as control, 1.25 μg/mL Juglone, 1 μg/mL colistin, and 1.25 μg/mL Juglone + 1 μg/mL colistin) in an 8-well chamber slide (μSlide, ibiTreat, Ibidi, Germany) at 37 °C for an additional 24 h. The spent media containing with planktonic cells were removed, followed by washing of the bottom-adhering biofilms twice with 0.9% NaCl. The propidium iodide (PI) was added into all wells at a final concentration of 1 μM to stain dead bacteria.

Microscope Imaging

As previously described,33 all microscopic images of biofilms (GFP and PI) were acquired with Z-stack by confocal laser scanning microscopy (CLSM) (Leica TCS SP8Multiphoton/Confocal Microscope system, Germany) with the 63x oil objective. Experiments were performed in triplicate, where at least 3 images were captured for each replicate.

To analyze the biomass and live/dead ratio of biofilms, all images were processed by ImageJ software. To measure fluorescence density,34 the biofilm and background region of each image were selected and measured to provide the integrated density, mean fluorescence, and area values. The corrected total cell fluorescence (CTCF) was calculated with the following formula

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Quantification of Bacterial Numbers by CFU

As previously described,35 cell suspensions were serially diluted in 0.9% NaCl saline and transferred to LBA agar plates with 5 technical replicates. The agar plates were incubated at 37 °C for 12 h until colonies were observed. The colonies were enumerated and tabulated with the following formula

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Human Lung Fibroblasts Cytotoxicity Assay

As previously described,32 human lung fibroblasts (ATCC) were cultivated with Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (Gibco) until 70% confluency for 96 h at 37 °C, 5% CO2, and 99% humidity.

For experiment, 1 × 106 cells/mL were first cultivated in each well of a 96-well microplate for 24 h at 37 °C, 5% CO2 and 99% humidity. The cells were then washed once with phosphate-buffered saline (PBS) and treated with 200 μL of DMEM containing varying concentrations of Juglone (0, 0.625, 1.25, 2.5, and 5 μg/mL) for 24 h at 37 °C, 5% CO2, and 99% humidity. Alamar Blue cell viability reagent (Life Technologies) was added to the final concentration of 25 μM to each well, followed by incubation at 37 °C and 5% CO2 and 99% humidity for 3 h. To measure the cell viability, the Alamar Blue fluorescence intensity (Ex: 520 nm; Em: 590 nm) was measured with a microplate reader.

Medaka Fish Tail Wound Infection Model

As previously described,36 the Medaka fish experiments were carried out following the NACLAR Guidelines and Animal and Birds (Care and Use of Animals for Scientific Purposes) Rules by the Department of Health (Hong Kong SAR), with approval by the City University of Hong Kong animal research ethics committee (permit number ref No. A-0418).

Animals were reared collectively in treated freshwater with salinity 3 psu, pH 6.5 in room temperature at a ratio of 1 fish/1 L of water. The light rhythm was kept at a light/dark ratio of 14 h:10 h. The animals were fed with dry food (Marubeni Nisshin Feed, Japan) and Artemia nauplii three times daily (twice by dry food and once by Artemia).

To establish the wound infection model, the animals were first anesthetized with 0.015 mM anesthesia tricaine mesylate (MS-222, Sigma-Aldrich). The 1 cm skin of the tail was surgically cut with a syringe needle (26GX1 1/2″, 0.45 × 13 mm2, Terumo, Japan), followed by introduction of P. aeruginosa (1 × 107 cells/mL) onto the wound under direct observation with a light stereomicroscope. After 1 h post infection (h.p.i), the fish were washed with freshwater to remove residual anesthesia and unattached bacteria. The fish were placed in the fish tank containing freshwater for infection to establish over 72 h at room temperature. The infected animals were then treated with freshwater containing varying concentrations of Juglone (0, 0.625, 1.25, 2.5, and 5 μg/mL) for a further 24 h at room temperature.

At the end of the experiment, the fish were euthanized with an overdose of MS-222 (0.05 mM) for 30 min. For imaging, the fish tail was observed using CLSM with a 20x objective, where brightfield, GFP, and pyoverdine images were captured. For bacterial counts using the CFU assay, the tail tissue was surgically removed from the euthanized fish and ground manually by pellet pestle in 100 μL of 0.9% NaCl in 1.5 mL microcentrifuge tubes until homogenization. The homogenized samples were diluted serially in 0.9% NaCl and transferred on cetrimide agar (selective only for Pseudomonas growth) (Sigma-Aldrich) with 5 technical replicates. The agar plates were incubated at 37 °C for 12 h until colonies were observed, for CFU tabulation.

Quantification of Fish Cytokine Expression Using ELISA

Fish were assayed for innate immune cytokines (TNF-α and IL-6) using an ELISA kit (Bosun, Jiangsu, China) and measured at an OD of 450 mm by an enzyme marker (Tecan, China) Infinite M1000 Pro (Switzerland). Cytokine concentrations were normalized by protein concentrations, which were measured at an OD of 280 mm with a Nanodrop (Thermo Fisher, NanoDrop One, ND-One-W). CFUs were performed.

The tail tissues were first ground with a pellet pestle on ice to disrupt tissues, followed by sonication at 30% amplitude for 3 min with 10 s ON/10 s OFF output by an ultrasonication machine (SFX 550, SSE-1, Branson, Emerson) to release intracellular proteins. The fish innate immunity cytokines (TNF-α and IL-2) were quantified with the ELISA kit (Bo Shen, Jiangsu, China) and measured at OD450 by the microplate reader. The total protein concentration was measured by a Nanodrop (Thermo Fisher, NanoDrop One, ND-One-W) at OD280.

Statistical Analysis

Where applicable, one-way ANOVA and Student’s t test were used. All experiments were performed in triplicate, and results were shown as mean ± standard deviation.

Results

Inhibition Assay of Juglone on P. aeruginosa

Using the P. aeruginosa wild-type PAO1/ppqsA-gfp transcription fusion biosensor,27 we conducted a preliminary screening of an in-house natural compounds library to identify potential inhibitors of the pqs operon. The biosensor measures the expression of pqs QS, as PqsA is an enzyme involved in the synthesis of the autoinducer PQS.28 Our screening showed that Juglone (Figure 1a) acted as an inhibitor of the pqs operon. As the concentration of Juglone increased, we observed growth inhibition of PAO1, with a minimum inhibitory concentration (MIC) of 5 μg/mL (Figure 1b).

Figure 1.

Figure 1

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Juglone inhibits pqs QS signaling system. (a) Chemical structure of Juglone. (b) Cell density (OD600) of PAO1/ppqsA-ASV in varying concentrations of Juglone. (c) Inhibitory effect of various concentrations of Juglone on the GFP expression (normalized with OD600) of PAO1/ppqsA-gfp. (d) The half-maximal inhibitory concentration (IC50) of ppqsA-gfp (normalized with OD600) with different concentrations of Juglone. The slope of the curve was calculated based on its respective dose–response curves and plotted against the log concentration. The slope is indicative of the biosynthesis rate of GFP due to PQS induction. (e) Relative PQS concentration of PAO1 treated with Juglone, as quantified using ppqsA-gfp expression (normalized with OD600). Means and s.d. from triplicate experiments are shown. **P < 0.001, one-way ANOVA.

PAO1/ppqsA-gfp was exposed to Juglone, which resulted in dose-dependent inhibition of pqsA-gfp expression (Figure 1c). Thus, we determined that Juglone has a half-maximal inhibitory concentration (IC50) value of 0.3 μg/mL in inhibiting the expression of pqsA-gfp (Figure 1d), where bacterial growth was not affected. Further, Juglone could inhibit the production of the PQS signaling molecule, where we observed a lower PQS concentration in P. aeruginosa after Juglone treatment (Figure 1e).

Juglone Inhibits pqs Response but Not Biosynthesis

Since the pqs QS signaling is controlled by the biosynthesis of PQS by pqsABCD and pqsH and the response regulator PqsR,37 we next aim to determine if Juglone inhibits the synthesis or response genes. To test if Juglone inhibits PQS synthesis, we employed a ΔpqsA/ppqsA-gfp mutant, which was unable to synthesize its own PQS but still could respond via PqsR, and incubated it with exogenously added 10 μM PQS and increasing Juglone concentrations. In the absence of Juglone, ΔpqsA/ppqsA-gfp could express GFP and retain its response activity in the presence of exogenous PQS only (Figure 2a). However, addition of Juglone to ΔpqsA/ppqsA-gfp with exogenous PQS could reduce the expression of pqsA-gfp by around 40% (Figure 2a). This indicated that Juglone did not inhibit PQS biosynthesis.

Figure 2.

Figure 2

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Juglone inhibited pqs QS response but not pqs biosynthesis. (a) Relative GFP quantification (normalized with OD600) of ΔpqsA/ppqsA-gfp with exogenously added 10 μM PQS in varying concentrations of Juglone. (b) Relative GFP quantification (normalized with OD600) of PAO1, ΔpqsR/ppqsA-gfp, ΔpqsR/plac-pqsABCDE/ppqsA-gfp in varying concentrations of Juglone. Means and s.d. from triplicate experiments are shown. **P < 0.001, n.s: not significant. One-way ANOVA.

Next, to show if Juglone inhibited pqs response instead, we tested the ΔpqsR mutant which could not respond to PQS, and the ΔpqsR/plac-pqsABCDE mutant which possessed overexpression of pqs biosynthesis operon but could not respond to PQS. For simplicity sake, we showed the peak ppqsA-gfp expression values of each strain treated with different Juglone concentrations at 300 min (Figure 2b). Although there was lower GFP expression, the ΔpqsR/ppqsA-gfp mutant did not display a significant reduction in pqsA expression, indicating that it did not respond to increasing levels of Juglone. Moreover, even with overproduction of PQS in the ΔpqsR/plac-pqsABCDE/ppqsA-gfp strain, Juglone still could not inhibit pqs signaling (Figure 2b), indicating that Juglone could inhibit pqs QS via the PqsR response.

Molecular Docking of Juglone to PqsR

Since we had shown in the earlier section that Juglone could inhibit pqs signaling via PqsR response, we used molecular docking with Autodock Vina to evaluate if Juglone could potentially bind to the PqsR active site. A previous study had shown that the PqsR ligand, 2-nonyl-4-hydroxyquinoline (NHQ), binds to PqsR via several active site residues, such as L207 and L208.38 We showed that NHQ (yellow) and Juglone (green) could potentially bind to the PqsR active site (gray) in the same space (Figure 3a, superimposed). The NHQ could bind to the PqsR active site with a binding affinity of −6.9 kcal/mol (Figure 3b), while Juglone could potentially bind to PqsR at both the L207 and L208 sites (Figure 3c) with a binding affinity of −6.6 kcal/mol. This implied that Juglone might inhibit pqs signaling via PqsR.

Figure 3.

Figure 3

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Molecular docking of Juglone to PqsR. (a) Superimposed comparison of NHQ native ligand and Juglone binding to PqsR. (b) NHQ binding to PqsR, with a binding affinity of −6.9 kcal/mol at amino acid residues L207 and L208. (c) Juglone binding to PqsR, with a binding affinity of −6.6 kcal/mol at amino acid residues L207 and L208.

Juglone Could Inhibit PQS-Controlled Virulence Factors

Since Juglone could inhibit pqs signaling, we next evaluated if Juglone could inhibit the downstream pqs-controlled virulence factors. As swarming motility is controlled by PqsR,39 we performed the swarming assay where the distance that P. aeruginosa migrated across agar under Juglone inhibition was measured. Juglone could inhibit the swarming motility of P. aeruginosa, where there was reduced migration of P. aeruginosa across the agar plate (Figure 4a,b).

Figure 4.

Figure 4

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Inhibition of pqs signaling-controlled virulence factors by Juglone. (a) Representative images of swarming motility by P. aeruginosa with Juglone treatment. (b) Diameter of swarming area by P. aeruginosa with Juglone treatment. (c) Representative images of green pyocyanin pigment produced by P. aeruginosa with Juglone treatment. (d) Relative pyocyanin concentration of PAO1 after Juglone treatment. Means and s.d. from triplicate experiments are shown. ***P < 0.001, one-way ANOVA.

Next, as the pqs QS signaling controlled the expression of the highly cytotoxic pyocyanin production,40 we employed the chloroform–acid assay to extract and quantify pyocyanin extracted from P. aeruginosa treated with Juglone. We showed that Juglone could inhibit pyocyanin production via direct observation of green pyocyanin pigment (Figure 4c) and relative quantification of pyocyanin (Figure 4d).

Colistin–Juglone Combinatorial Therapy Could Eliminate P. aeruginosa Biofilms

Since colistin could promote pqs signaling and biofilm formation in P. aeruginosa,8,9 we next evaluate if addition of Juglone could enhance the efficacy of colistin efficacy in inhibiting biofilm formation or eliminating preformed biofilms. For the biofilm growth inhibition assay, we employed CLSM to observe that monotherapies of 1.25 μg/mL Juglone or 1 μg/mL colistin could not completely inhibit biofilm formation by P. aeruginosa, whereas 1.25 μg/mL Juglone–1 μg/mL colistin combinatorial therapy could completely inhibit formation of biofilms and kill existing biofilms (Figure 5a,b). Our observations were supported by the quantification of bacterial numbers via the CFU assay (Figure 5c).

Figure 5.

Figure 5

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Juglone–colistin combinatorial treatment could inhibit and eliminate P. aeruginosa PAO1 biofilms. (a) Representative CLSM images showing that Juglone–colistin combinatorial therapy could inhibit biofilm growth. Scale bar: 5 μm. (b) Biomass and (c) CFU/mL of biofilm cells treated with 0 and 1.25 μg/mL Juglone with various concentrations of colistin in the biofilm growth inhibition assay. (d) Representative CLSM images showing that Juglone–colistin combinatorial therapy could eliminate pre-established biofilms. Scale bar: 5 μm. (e) Biomass, (f) live/dead ratio, and (g) CFU/mL of biofilm cells treated with 0 and 1.25 μg/mL Juglone with various concentrations of colistin in the biofilm elimination assay. Means and s.d. from triplicate experiments are shown. ***P < 0.001, one-way ANOVA.

To evaluate if the Juglone–colistin combinatorial therapy could eliminate pre-established biofilms, we cultivated biofilms in 8-well chamber followed by chemical treatment for CLSM observation. We showed that 1.25 μg/mL Juglone or 1 μg/mL colistin monotherapy could not effectively eliminate biofilms, while the 1.25 μg/mL Juglone–1 μg/mL colistin combinatorial therapy could nearly eliminate the established biofilms (Figure 5d–f). Similarly, our CFU data also showed that Juglone–colistin combinatorial therapy was the most effective at reducing bacterial numbers (Figure 5g). Since P. aeruginosa was previously known to tolerate higher than 10 μg/mL colistin,41 Juglone–colistin combinatorial therapy could help to reduce the concentration of colistin used for eliminating biofilms.

Colistin–Juglone Combinatorial Therapy Could Eliminate Biofilms Formed by Clinical Isolates

We next expanded our antibiofilm analysis of the Juglone–colistin combinatorial therapy to 2 clinical bacterial isolates (CF173-2005 and CF273-2002).42 Both clinical isolates were pro-biofilm-forming rough and small colony variants (RSCVs) collected from cystic fibrosis patients.43 The Juglone–colistin combinatorial therapy was more effective than monotherapies in eliminating biofilms established by both clinical isolates (Figure 6a–c). Lower CFU was also observed in biofilms treated with combinatorial therapy (Figure 6d), implying that combinatorial therapy had the potential for clinical applications.

Figure 6.

Figure 6

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Juglone–colistin combinatorial treatment could inhibit and eliminate biofilms formed P. aeruginosa clinical isolates. (a) Representative CLSM images showing that Juglone–colistin combinatorial therapy could eliminate pre-established biofilms formed by CF173-2002. Scale bar: 5 μm. (b) Biomass, (c) live/dead ratio, and (d) CFU/mL of biofilm cells treated with 0 and 1.25 μg/mL Juglone with various concentrations of colistin in the biofilm elimination assay. (e) Representative CLSM images showing that Juglone–colistin combinatorial therapy could eliminate pre-established biofilms formed by CF273-2005. Scale bar: 5 μm. (f) Biomass, (g) live/dead ratio, and (h) CFU/mL of biofilm cells treated with 0 and 1.25 μg/mL Juglone with various concentrations of colistin in the biofilm elimination assay. Means and s.d. from triplicate experiments are shown. ***P < 0.001, one-way ANOVA.

In Vivo Efficacy of Juglone–Colistin Combinatorial Therapy against P. aeruginosa Biofilm Infections

Lastly, we aim to confirm that our in vitro results are translatable to in vivo infection models. Before drug testing in animals, we first showed that Juglone was not cytotoxic to human lung fibroblasts at concentrations relevant to our study (Figure S1). It is important to note that Juglone is cytotoxic to human cells at nearly 10-fold higher concentration (∼80 μM),44 so the concentration used in this study was significantly lower than the toxic concentration.

We then evaluated the efficacy of Juglone–colistin combinatorial therapy in our previously established Medaka fish tail wound-based biofilm infection model.45 The fish is a simple vertebral model for studying host–pathogen interactions with relevance to human immunity.46 Using CLSM, we showed that the Juglone–colistin combinatorial therapy could eliminate gfp-tagged P. aeruginosa biofilms more effectively than the Juglone and colistin monotherapies (Figure 7a). Furthermore, we also observed that the Juglone–colistin combinatorial therapy could also inhibit the self-fluorescent iron-scavenging pyoverdine that is controlled by pqs QS signaling47 and plays crucial role in biofilm formation48 (Figure 7a). We measured the GFP and pyoverdine fluorescence levels in the biofilms (Figure 7b,c), where the Juglone–colistin combinatorial therapy could eliminate biofilms and inhibit pyoverdine production. Our observations were also supported by the CFU assay (Figure 7d), indicating that there were significantly lower levels of live bacteria after combinatorial therapy than in nontreated control.

Figure 7.

Figure 7

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Juglone–colistin combinatorial therapy could eliminate biofilm and alleviate host inflammatory response in Medaka fish tail wound infection. (a) Representative CLSM images of gfp-tagged P. aeruginosa colonization on tail wounds. Scale bar: 50 μm. (b) Biomass, (c) pyoverdine, and (d) CFU/mL of biofilm cells treated with 0 and 1.25 μg/mL Juglone with various concentrations of colistin on tail wounds. ELISA assay of selected pro-inflammatory cytokines and (e) Tnf-α and (f) IL-2 in Medaka fish with and without P. aeruginosa PAO1 infection. Means and s.d. from triplicate experiments are shown. ***P < 0.001, **P < 0.01, one-way ANOVA.

Other than bacterial analysis, we also analyzed if the Juglone–colistin combinatorial therapy could reduce inflammation by the host animals. No mortality of the fish was observed, and we showed that Juglone–colistin combinatorial therapy could significantly reduce expressions of inflammatory cytokines (TNF-α and IL-6) (Figure 7e,f). This indicated that the combinatorial therapy had alleviated the inflammation in the fish.

Discussion

There is an increasing demand for novel precision antimicrobial therapeutics that mitigate pathogen virulence and enhance the efficacy of antibiotic treatment. While many studies have developed QSIs that inhibit homoserine-based QS systems, few QSIs target the pqs signaling system.30 However, most were synthetic and were not used in clinical trials. Natural phytocompounds offer several advantages over synthetic drugs, including food availability, high safety levels, and lesser side effects.49 We previously showed that vanillin from vanilla plant was a PqsR inhibitor that mitigated biofilm formation and eliminated infections.9 With an immense library of phytocompounds, potential QSIs that can have activity against the pqs signaling system have been poorly explored. Even as a natural product, Juglone had similar IC50 to other published synthetic compounds in inhibiting PQS-controlled virulence factors, including PQS, pyocyanin, and motility,50 but significantly lower than its natural counterpart, vanillin.9 This indicated that Juglone is a potent natural pqs inhibitor with functions similar to those of synthetic derivatives.

Our study showed Juglone as a promising compound that inhibits colistin-induced pqs signaling and biofilm formation in P. aeruginosa. The inhibition of pqs signaling was potentially achieved by inhibition of PqsR by Juglone, resulting in the reduced production of pyocyanin and biofilms. Juglone–colistin combinatorial therapy could effectively treat Medaka fish with P. aeruginosa-infected wounds and reduce inflammation. Notably, the combinatorial therapy was effective against P. aeruginosa clinical isolates, indicating its potential for clinical application. Moreover, Juglone was not cytotoxic to human cells and well-tolerated in the animals, suggesting that Juglone was not toxic at the concentration used in this study to inhibit pqs signaling, which was significantly lower than the toxic concentration for human cells (∼80 μM).44

There are broader implications in our findings using Juglone against P. aeruginosa biofilm infections. The use of Juglone offers many advantages over conventional antimicrobials due to its small molecular size and simple chemical structure, which enables scalable and affordable synthesis. Moreover, our work provides an approach to acquire new insights into microbial physiology, including the response to antibiotics and biofilm formation, where Juglone could target bacterial virulence mechanisms instead of killing bacteria outright. This approach might mitigate concerns about antibiotic resistance by reducing selective pressure on traditional antibiotics.

For future studies, structure–activity relationships (SAR) should be performed to develop Juglone derivatives with enhanced efficacy against P. aeruginosa infections. Furthermore, larger scale studies on Juglone’s safety and efficacy will need to be conducted to ensure understanding of clinical benefits and toxicity before Juglone can be used as a therapeutic drug. Exploring its efficacy against other antibiotic-resistant biofilm-forming P. aeruginosa,51 polymicrobial biofilms or in different infection sites would be valuable. Delivery systems or formulations can be incorporated to enhance Juglone’s bioavailability, stability, and targeted delivery to specific infection sites. Next, understanding Juglone’s mechanism of action and its combination with existing antibiotics paves the way for combination therapies. Further investigations into the effectiveness of Juglone in conjunction with other antibiotics or therapeutic agents could revolutionize treatment strategies for biofilm-associated infections caused by various pathogens.52 In conclusion, Juglone offers an alternative precision antivirulence therapy that can eliminate biofilm-based infections.

Acknowledgments

This research is supported by The Hong Kong Polytechnic University Internal grants (Departmental Startup Grant (BE2B), UALB, ZVVV and ZVMH), Environmental and Conservation Fund (ECF-84/2021), Health and Medical Research Fund (HMRF-201903032), Pneumoconiosis Compensation Fund Board (PCFB-ZJN2), and State Key Laboratory of Chemical Biology and Drug Discovery Fund (1-BBX8).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00354.

  • Juglone was minimally cytotoxic to human lung fibroblasts (Figure S1) and bacterial strains used in the study (Table S1) (PDF)

Author Contributions

Conceptualization: S.L.C. Design and conducting of in vitro experiments: Y.M. and W.S.T. Design and conducting of in vivo experiments: S.Y.L. and B.L.K. Data analysis: Y.M., W.S.T., B.L.K., and S.L.C. Drafting manuscript: Y.M., B.L.K., and S.L.C.

The authors declare no competing financial interest.

Supplementary Material

pt3c00354_si_001.pdf (154.8KB, pdf)

References

  1. Murray C. J. L.; Ikuta K. S.; Sharara F.; Swetschinski L.; Robles Aguilar G.; Gray A.; Han C.; Bisignano C.; Rao P.; Wool E.; et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022, 399 (10325), 629–655. 10.1016/S0140-6736(21)02724-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. a WHO Publishes List of Bacteria for Which New Antibiotics are Urgently Needed, 2019.; b Antibiotic Resistance Threats in the United States, 2019. https://www.cdc.gov/drugresistance/biggest-threats.html. [PubMed]
  3. Bjarnsholt T. The role of bacterial biofilms in chronic infections. APMIS 2013, 121 (s136), 1–58. 10.1111/apm.12099. [DOI] [PubMed] [Google Scholar]
  4. a Li S.; Liu S. Y.; Chan S. Y.; Chua S. L. Biofilm matrix cloaks bacterial quorum sensing chemoattractants from predator detection. ISME J. 2022, 16 (5), 1388–1396. 10.1038/s41396-022-01190-2. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Chan S. Y.; Liu S. Y.; Seng Z.; Chua S. L. Biofilm matrix disrupts nematode motility and predatory behavior. ISME J. 2021, 15 (1), 260–269. 10.1038/s41396-020-00779-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Rice L. B. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J. Infect. Dis. 2008, 197 (8), 1079–1081. 10.1086/533452. [DOI] [PubMed] [Google Scholar]
  6. Abd El-Baky R. M.; Masoud S. M.; Mohamed D. S.; Waly N. G.; Shafik E. A.; Mohareb D. A.; Elkady A.; Elbadr M. M.; Hetta H. F. Prevalence and Some Possible Mechanisms of Colistin Resistance Among Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa. Infect. Drug Resist. 2020, 13, 323–332. 10.2147/IDR.S238811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gogry F. A.; Siddiqui M. T.; Sultan I.; Haq Q. M. R. Current Update on Intrinsic and Acquired Colistin Resistance Mechanisms in Bacteria. Front. Med. 2021, 8, 677720 10.3389/fmed.2021.677720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cummins J.; Reen F. J.; Baysse C.; Mooij M. J.; O’Gara F. Subinhibitory concentrations of the cationic antimicrobial peptide colistin induce the pseudomonas quinolone signal in Pseudomonas aeruginosa. Microbiology 2009, 155 (Pt 9), 2826–2837. 10.1099/mic.0.025643-0. [DOI] [PubMed] [Google Scholar]
  9. Mok N.; Chan S. Y.; Liu S. Y.; Chua S. L. Vanillin inhibits PqsR-mediated virulence in Pseudomonas aeruginosa. Food & Function 2020, 11 (7), 6496–6508. 10.1039/D0FO00046A 10.1039/D0FO00046A. [DOI] [PubMed] [Google Scholar]
  10. Gallagher L. A.; McKnight S. L.; Kuznetsova M. S.; Pesci E. C.; Manoil C. Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa. J. Bacteriol. 2002, 184 (23), 6472–6480. 10.1128/JB.184.23.6472-6480.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lin J.; Cheng J.; Wang Y.; Shen X. The Pseudomonas quinolone signal (PQS): not just for quorum sensing anymore. Front. Cell. Infect. Microbiol. 2018, 8, 230. 10.3389/fcimb.2018.00230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Wilson R.; Sykes D. A.; Watson D.; Rutman A.; Taylor G. W.; Cole P. J. Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium. Infect. Immun. 1988, 56 (9), 2515–2517. 10.1128/iai.56.9.2515-2517.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. de Aldecoa A. L. I.; Zafra O.; González-Pastor J. E. Mechanisms and Regulation of Extracellular DNA Release and Its Biological Roles in Microbial Communities. Front. Microbiol. 2017, 8, 1390 10.3389/fmicb.2017.01390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shah P. L.; Scott S. F.; Knight R. A.; Marriott C.; Ranasinha C.; Hodson M. E. In vivo effects of recombinant human DNase I on sputum in patients with cystic fibrosis. Thorax 1996, 51 (2), 119–125. 10.1136/thx.51.2.119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Abdalla M. Y.; Hoke T.; Seravalli J.; Switzer B. L.; Bavitz M.; Fliege J. D.; Murphy P. J.; Britigan B. E. Pseudomonas Quinolone Signal Induces Oxidative Stress and Inhibits Heme Oxygenase-1 Expression in Lung Epithelial Cells. Infect. Immun. 2017, 85 (9), e00176-17. 10.1128/IAI.00176-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Butler M. S.; Gigante V.; Sati H.; Paulin S.; Al-Sulaiman L.; Rex J. H.; Fernandes P.; Arias C. A.; Paul M.; Thwaites G. E.; et al. Analysis of the Clinical Pipeline of Treatments for Drug-Resistant Bacterial Infections: Despite Progress, More Action Is Needed. Antimicrob. Agents Chemother. 2022, 66 (3), e0199121 10.1128/aac.01991-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lau W. Y. V.; Taylor P. K.; Brinkman F. S. L.; Lee A. H. Y. Pathogen-associated gene discovery workflows for novel antivirulence therapeutic development. eBioMedicine 2023, 88, 104429. 10.1016/j.ebiom.2022.104429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. a Spaulding C. N.; Klein R. D.; Schreiber H. L. t.; Janetka J. W.; Hultgren S. J. Precision antimicrobial therapeutics: the path of least resistance?. npj Biofilms Microbiomes 2018, 4, 4. 10.1038/s41522-018-0048-3. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Paharik A. E.; Schreiber H. L. t.; Spaulding C. N.; Dodson K. W.; Hultgren S. J. Narrowing the spectrum: the new frontier of precision antimicrobials. Genome Med. 2017, 9 (1), 110. 10.1186/s13073-017-0504-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ghosh C.; Sarkar P.; Issa R.; Haldar J. Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance. Trends Microbiol. 2019, 27 (4), 323–338. 10.1016/j.tim.2018.12.010. [DOI] [PubMed] [Google Scholar]
  20. Pushpakom S.; Iorio F.; Eyers P. A.; Escott K. J.; Hopper S.; Wells A.; Doig A.; Guilliams T.; Latimer J.; McNamee C.; et al. Drug repurposing: progress, challenges and recommendations. Nat. Rev. Drug Discovery 2019, 18 (1), 41–58. 10.1038/nrd.2018.168. [DOI] [PubMed] [Google Scholar]
  21. Pereyra C. E.; Dantas R. F.; Ferreira S. B.; Gomes L. P.; Silva-Jr F. P. The diverse mechanisms and anticancer potential of naphthoquinones. Cancer Cell Int. 2019, 19 (1), 207. 10.1186/s12935-019-0925-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rahman M. M.; Islam M. R.; Akash S.; Shohag S.; Ahmed L.; Supti F. A.; Rauf A.; Aljohani A. S. M.; Al Abdulmonem W.; Khalil A. A.; et al. Naphthoquinones and derivatives as potential anticancer agents: An updated review. Chem.–Biol. Interact. 2022, 368, 110198 10.1016/j.cbi.2022.110198. [DOI] [PubMed] [Google Scholar]
  23. Giongo V.; Falanga A.; De Melo C. P. P.; da Silva G. B.; Bellavita R.; De-Simone S. G.; Paixão I. C.; Galdiero S. Antiviral Potential of Naphthoquinones Derivatives Encapsulated within Liposomes. Molecules 2021, 26 (21), 6440. 10.3390/molecules26216440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Song R.; Yu B.; Friedrich D.; Li J.; Shen H.; Krautscheid H.; Huang S. D.; Kim M.-H. Naphthoquinone-derivative as a synthetic compound to overcome the antibiotic resistance of methicillin-resistant S. aureus. Commun. Biol. 2020, 3 (1), 529. 10.1038/s42003-020-01261-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Han Q.; Yan X.; Zhang R.; Wang G.; Zhang Y. Juglone Inactivates Pseudomonas aeruginosa through Cell Membrane Damage, Biofilm Blockage, and Inhibition of Gene Expression. Molecules 2021, 26 (19), 5854 10.3390/molecules26195854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Chua S. L.; Tan S. Y.-Y.; Rybtke M. T.; Chen Y.; Rice S. A.; Kjelleberg S.; Tolker-Nielsen T.; Yang L.; Givskov M. Bis-(3′-5′)-Cyclic Dimeric GMP Regulates Antimicrobial Peptide Resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2013, 57 (5), 2066–2075. 10.1128/AAC.02499-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kesarwani M.; Hazan R.; He J.; Que Y.; Apidianakis Y.; Lesic B.; Xiao G.; Dekimpe V.; Milot S.; Deziel E.; et al. A Quorum Sensing Regulated Small Volatile Molecule Reduces Acute Virulence and Promotes Chronic Infection Phenotypes. PLoS Pathog. 2011, 7 (8), e1002192 10.1371/journal.ppat.1002192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Asfahl K. L.; Schuster M. Additive effects of quorum sensing anti-activators on Pseudomonas aeruginosa virulence traits and transcriptome. Front. Microbiol. 2018, 8, 2654. 10.3389/fmicb.2017.02654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tan S. Y.-Y.; Chua S.-L.; Chen Y.; Rice S. A.; Kjelleberg S.; Nielsen T. E.; Yang L.; Givskov M. Identification of Five Structurally Unrelated Quorum-Sensing Inhibitors of Pseudomonas aeruginosa from a Natural-Derivative Database. Antimicrob. Agents Chemother. 2013, 57 (11), 5629–5641. 10.1128/AAC.00955-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ilangovan A.; Fletcher M.; Rampioni G.; Pustelny C.; Rumbaugh K.; Heeb S.; Cámara M.; Truman A.; Chhabra S. R.; Emsley J.; Williams P. Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR). PLoS Pathog. 2013, 9 (7), e1003508 10.1371/journal.ppat.1003508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Chua S. L.; Liu Y.; Li Y.; Jun Ting H.; Kohli G. S.; Cai Z.; Suwanchaikasem P.; Kau Kit Goh K.; Pin Ng S.; Tolker-Nielsen T.; et al. Reduced Intracellular c-di-GMP Content Increases Expression of Quorum Sensing-Regulated Genes in Pseudomonas aeruginosa. Front. Cell. Infect. Microbiol. 2017, 7, 451 10.3389/fcimb.2017.00451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Fong J.; Yuan M.; Jakobsen T. H.; Mortensen K. T.; Delos Santos M. M. S.; Chua S. L.; Yang L.; Tan C. H.; Nielsen T. E.; Givskov M. Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa. J. Med. Chem. 2017, 60 (1), 215–227. 10.1021/acs.jmedchem.6b01025. [DOI] [PubMed] [Google Scholar]
  33. Ma Y.; Deng Y.; Hua H.; Khoo B. L.; Chua S. L. Distinct bacterial population dynamics and disease dissemination after biofilm dispersal and disassembly. ISME J. 2023, 17, 1290. 10.1038/s41396-023-01446-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rybtke M.; Chua S. L.; Yam J. K. H.; Givskov M.; Yang L.; Tolker-Nielsen T. Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa Using Fluorescence-Based Biosensors. Methods Mol. Biol. 2017, 1657, 87–98. 10.1007/978-1-4939-7240-1_8. [DOI] [PubMed] [Google Scholar]
  35. Liu S. Y.; Leung M. M.-L.; Fang J. K.-H.; Chua S. L. Engineering a microbial ‘trap and release’ mechanism for microplastics removal. Chem. Eng. J. 2021, 404, 127079 10.1016/j.cej.2020.127079. [DOI] [Google Scholar]
  36. Liu Y. S.; Deng Y.; Chen C. K.; Khoo B. L.; Chua S. L. Rapid detection of microorganisms in a fish infection microfluidics platform. J. Hazard. Mater. 2022, 431, 128572 10.1016/j.jhazmat.2022.128572. [DOI] [PubMed] [Google Scholar]
  37. Gallagher L. A.; McKnight S. L.; Kuznetsova M. S.; Pesci E. C.; Manoil C. Functions Required for Extracellular Quinolone Signaling by Pseudomonas aeruginosa. J. Bacteriol. 2002, 184 (23), 6472–6480. 10.1128/JB.184.23.6472-6480.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ilangovan A.; Fletcher M.; Rampioni G.; Pustelny C.; Rumbaugh K.; Heeb S.; Cámara M.; Truman A.; Chhabra S. R.; Emsley J.; Williams P. Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR). PLoS Pathog. 2013, 9 (7), e1003508 10.1371/journal.ppat.1003508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Guo Q.; Kong W.; Jin S.; Chen L.; Xu Y.; Duan K. Pqs R-dependent and Pqs R-independent regulation of motility and biofilm formation by PQS in Pseudomonas aeruginosa PAO1. J. Basic Microbiol. 2014, 54 (7), 633–643. 10.1002/jobm.201300091. [DOI] [PubMed] [Google Scholar]
  40. a Lau G. W.; Hassett D. J.; Ran H.; Kong F. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends Mol. Med. 2004, 10 (12), 599–606. 10.1016/j.molmed.2004.10.002. [DOI] [PubMed] [Google Scholar]; b Hall S.; McDermott C.; Anoopkumar-Dukie S.; McFarland A. J.; Forbes A.; Perkins A. V.; Davey A. K.; Chess-Williams R.; Kiefel M. J.; Arora D.; Grant G. Cellular effects of pyocyanin, a secreted virulence factor of Pseudomonas aeruginosa. Toxins 2016, 8 (8), 236. 10.3390/toxins8080236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Chua S. L.; Yam J. K. H.; Hao P.; Adav S. S.; Salido M. M.; Liu Y.; Givskov M.; Sze S. K.; Tolker-Nielsen T.; Yang L. Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms. Nat. Commun. 2016, 7 (1), 10750 10.1038/ncomms10750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Chua S. L.; Ding Y.; Liu Y.; Cai Z.; Zhou J.; Swarup S.; Drautz-Moses D. I.; Schuster S. C.; Kjelleberg S.; Givskov M.; Yang L. Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels. Open Biol. 2016, 6 (11), 160162 10.1098/rsob.160162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang L.; Jelsbak L.; Marvig R. L.; Damkiær S.; Workman C. T.; Rau M. H.; Hansen S. K.; Folkesson A.; Johansen H. K.; Ciofu O.; et al. Evolutionary dynamics of bacteria in a human host environment. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (18), 7481–7486. 10.1073/pnas.1018249108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Erisen S.; Arasoğlu T.; Mansuroglu B.; Kocacaliskan İ.; Derman S. Cytotoxic and mutagenic potential of juglone: a comparison of free and nano-encapsulated form. Arch. Ind. Hyg. Toxicol. 2020, 71 (1), 69–77. 10.2478/aiht-2020-71-3344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Liu Y. S.; Deng Y.; Chen C. K.; Khoo B. L.; Chua S. L. Rapid detection of microorganisms in a fish infection microfluidics platform. J. Hazard. Mater. 2022, 431, 128572 10.1016/j.jhazmat.2022.128572. [DOI] [PubMed] [Google Scholar]
  46. Gomes M. C.; Mostowy S. The Case for Modeling Human Infection in Zebrafish. Trends Microbiol. 2020, 28 (1), 10–18. 10.1016/j.tim.2019.08.005. [DOI] [PubMed] [Google Scholar]
  47. Kang D.; Turner K. E.; Kirienko N. V. PqsA Promotes Pyoverdine Production via Biofilm Formation. Pathogens 2018, 7 (1), 3 10.3390/pathogens7010003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Chen Y.; Yuan M.; Mohanty A.; Yam J. K. H.; Liu Y.; Chua S. L.; Nielsen T. E.; Tolker-Nielsen T.; Tolker-Nielsen T.; Givskov M.; Cao B. Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa. Environ. Microbiol. Rep. 2015, 7 (3), 498–507. 10.1111/1758-2229.12278. [DOI] [PubMed] [Google Scholar]
  49. Newman D. J.; Cragg G. M. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J. Nat. Prod. 2020, 83 (3), 770–803. 10.1021/acs.jnatprod.9b01285. [DOI] [PubMed] [Google Scholar]
  50. a Soukarieh F.; Liu R.; Romero M.; Roberston S. N.; Richardson W.; Lucanto S.; Oton E. V.; Qudus N. R.; Mashabi A.; Grossman S.; et al. Hit Identification of New Potent PqsR Antagonists as Inhibitors of Quorum Sensing in Planktonic and Biofilm Grown Pseudomonas aeruginosa. Front. Chem. 2020, 8, 204 10.3389/fchem.2020.00204. [DOI] [PMC free article] [PubMed] [Google Scholar]; b Huang X. H.; She M. T.; Zhang Y. H.; Liu Y. F.; Zhong D. X.; Zhang Y. H.; Zheng J. X.; Sun N.; Wong W. L.; Lu Y. J. Novel quinoline-based derivatives as the PqsR inhibitor against Pseudomonas aeruginosa PAO1. J. Appl. Microbiol. 2022, 133 (4), 2167–2181. 10.1111/jam.15601. [DOI] [PubMed] [Google Scholar]
  51. Ma Y.; Aung T. T.; Lakshminarayanan R.; Chua S. L. Biofilm formation and virulence potential of carbapenem-resistant Pseudomonas aeruginosa. Lancet Microbe 2023, 4 (7), e489 10.1016/S2666-5247(23)00097-6. [DOI] [PubMed] [Google Scholar]
  52. Yu M.; Chua S. L. Demolishing the great wall of biofilms in Gram-negative bacteria: To disrupt or disperse?. Med. Res. Rev. 2020, 40 (3), 1103–1116. 10.1002/med.21647. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pt3c00354_si_001.pdf (154.8KB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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