Exploring the Quorum Quenching Properties of Premna serratifolia on Pseudomonas aeruginosa

Guide:

Dr. Reshma S.V.

Chairperson & Professor,

Department of Biotechnology

PES University

Team members:

Harshini S - PES1UG22BT021

Subhangi Swain - PES1UG22BT048

Tejal Mahesh - PES1UG22BT050

INTRODUCTION

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Quorum sensing in Pseudomonas aeruginosa

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• Quorum sensing (QS) - communication mechanism among bacteria, for coordinating behaviors in a population through autoinducers (Timotius et al.,2018).
• Autoinducers (AHLs) aid these interactions, influencing various physiological processes in both bacteria and plants (Majdura et al.,2023).
• Chemical signaling molecules like N-acyl homoserine lactones,AI-2 are produced by bacteria for communication and are detected by specific receptors in the bacteria.
• Bacteria that utilize QS can produce plant growth-promoting substances, such as indole-3-acetic acid (IAA) (Jung et al.,2020)

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• Traditional Use: The plant has been used in traditional medicine for treating bacterial infections.
• Phytochemical Profile: Contains terpenoids, flavonoids, and other potential antibacterial compounds (Simamora et al., 2020).
• Focus: Study focuses on Pseudomonas aeruginosa infections, including Otitis, Keratitis, Hot tub rash, and Burn wound infection.
• Inhibitory activities: Antioxidant activity, Antimicrobial activity, Anti-inflammatory activity, Anticancer activity, Anti-arthritic activity, Neuroprotective activity, Skin protection and melanogenesis inhibition, Immunomodulatory activity (Timotius., 2021).

LITERATURE REVIEW

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Author & Year

Published

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• A novel diterpene compound with a previously unreported skeleton (5-methyl-10-demethyl-abietane type) was isolated from the root bark.
• Structural elucidation was confirmed by advanced 2D NMR (COSY, HMBC, NOESY) and ESI-MS analysis, identifying 20 carbon atoms with high aromaticity.
• Unlike leaf or root wood extracts, only the root bark showed strong bioactivity—highlighting its phytochemical richness.

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Methodology and paper findings

• Quorum-sensing inhibitors (QSIs) can suppress P. aeruginosa virulence and biofilm formation without killing the bacteria, offering a promising strategy to combat antibiotic resistance.
• Natural and synthetic QSIs targeting the Las, Rhl, and Pqs systems like flavonoids, terpenoids, and AHL analogues disrupt quorum sensing hierarchies and reduce virulence gene expression.
• Several FDA-approved drugs, including erythromycin estolate and nitrofurazone, were repurposed as effective QS inhibitors, showing potential for anti-virulence therapy without promoting resistance.

The Molecular Architecture of Pseudomonas aeruginosa Quorum-Sensing Inhibitors

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Qiaoqiang et al., 2022

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Phytochemicals of Premna serratifolia inhibits quorum sensing in Pseudomonas aeruginosa

HYPOTHESIS

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Image Source: https://apps.lucidcentral.org/rainforest/text/entities/premna_serratifolia.htm

Image Source: https://apps.lucidcentral.org/rainforest/text/entities/premna_serratifolia.htm

OBJECTIVES

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• In Silico Screening of Potential Drug Ability of compounds obtained from Premna serratifolia.

2. Screening of antimicrobial properties of extracted biochemicals.

3. Testing for quorum quenching properties of the extracts.

PATHWAY

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Image Source:

https://www.kegg.jp/pathway/pae02025

In Silico

Molecular Docking

Simulations

Target Identification

METHODOLOGY

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DOCKING & COMPUTATIONAL TOOLS USED

• BIOVIA Discovery Studio – for protein cleaning, ligand preparation, minimization, hydrogen addition, and interaction visualization.
• PyRx – for virtual screening and running AutoDock Vina docking in a high-throughput manner.
• PyMOL – for protein structure inspection, removal of water molecules, chain selection, and binding-site visualization.
• AutoDock Vina – for consensus scoring, binding affinity prediction, and blind docking.
• AutoDock 4 (AD4) – for grid-based docking with the Lamarckian Genetic Algorithm and refined pose optimization.
• SwissDock (EADock DSS engine) – for cross-validation of Vina/AD4 results and identification of alternate binding pockets.
• OpenBabel – for file format conversion, ligand protonation, geometry optimization, and generation of PDBQT files.
• SwissADME – for drug-likeness screening, Lipinski rule evaluation, and ADME/Tox filtering of phytochemicals.
• LigPlot+ – for 2D interaction mapping of hydrogen bonds, hydrophobic contacts, and ligand–residue interactions.

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Tertiary Structures of Target Proteins

Ursolic acid & RetS

Premnazole & LasR

Baicalien & PqsR

Erythromycin & RetS

Friedilin & LasR

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Best Docking Results against Standard Drug (Erythromycin)

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Best Docking Results against Compounds found in Premna serratifolia

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SWISSADME

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• BAICALEIN

2. PREMNAZOLE

3. FRIEDELIN

4. URSOLIC ACID

LIGPLOT

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LasR with Ursolic acid

RetS with Baicalein

PqsR with Premnazole

LIGPLOT

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PqsR with Ursolic acid

RetS with ursolic acid

LasR with friedelin

PCA Plot of Standard drug, Target Protein and Standard drug + Target Protein

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Violin plot and Box Plot

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Heatmap of Standard drugs against Target proteins

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Ridge plot

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Volcano plot

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Volcano plot

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Volcano plot

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HEATMAP OF PREMNA COMPOUNDS AGAINST TARGET PROTEINS

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HEATMAP OF PREMNA COMPOUNDS AGAINST TARGET PROTEINS

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HEATMAP OF PREMNA COMPOUNDS AGAINST TARGET PROTEINS

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HEATMAP OF PREMNA COMPOUNDS AGAINST TARGET PROTEINS

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Simulation Result

Baicalein & LasR

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Simulation Result

Baicalein & LasR, Erythromycin & LasR

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Simulation Result

Baicalein & PqsR

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Simulation Result

Baicalein & PqsR, Erythromycin & PqsR

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Simulation Result

Baicalein & RetS

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Simulation Result

Baicalein & RetS, Erythromyicn & RetS

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Simulation Result

Premnazole & LasR

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Simulation Result

Premnazole & LasR, Erythromycin & LasR

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Simulation Result

Premnazole & PqsR

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Simulation Result

Premnazole & PqsR, Erythromycin & PqsR

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Simulation Result

Premnazole & RetS

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Simulation Result

Premnazole & RetS, Erythromycin & RetS

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Simulation Result

Ursolic acid & LasR

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Simulation Result

Ursolic acid & LasR

Erythromycin & LasR

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Simulation Result

Ursolic acid & RetS

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Simulation Result

Ursolic acid & RetS

Erythromycin & RetS

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Simulation Result

Ursolic acid & PqsR

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Simulation Result

Ursolic acid & PqsR

Erythromycin & PqsR

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Simulation Result

Friedilin & PqsR

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Simulation Result

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Friedilin & PqsR

Erythromycin & PqsR

Simulation Result

Friedilin & LasR

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Simulation Result

Friedilin & LasR

Erythromycin & LasR

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Simulation Result

Friedilin & RetS

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Simulation Result

Friedilin & RetS

Erythromycin & Rets

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Simulation Result

Erythromycin & LasR

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Simulation Result

Erythromycin & PqsR

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Simulation Result

Erythromycin & RetS

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MMPBSA

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• MMPBSA :Molecular Mechanics/Poisson-Boltzmann Surface Area.

• A computational method to calculate the binding free energy between a small molecule and a protein.

• Molecular Mechanics (MM): Calculates the internal energy and interaction energy of the molecules in a vacuum. This includes forces like van der Waals and electrostatic interactions.
• Poisson-Boltzmann Surface Area (PBSA): Accounts for the effect of the solvent (e.g., water) on the binding energy.
• Binding Free Energy: The final value that combines MM energy, solvation energy, and entropy. A more negative value indicates a stronger binding

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AMBER

• AMBER (Assisted Model Building with Energy Refinement) is a widely used family of force fields and a software suite for performing molecular dynamics simulations.

MMPBSA

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Baicalein with LasR

MMPBSA

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Baicalein with PqsR

MMPBSA

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Baicalein with RetS

MMPBSA

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Premnazole with LasR

MMPBSA

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Premnazole with PqsR

MMPBSA

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Premnazole with RetS

MMPBSA

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Ursolic acid with LasR

MMPBSA

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Ursolic acid with PqsR

MMPBSA

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Ursolic acid with RetS

MMPBSA

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Friedelin with LasR

MMPBSA

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Friedelin with PqsR

MMPBSA

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Friedelin with RetS

MMPBSA

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Erythromycin with LasR

MMPBSA

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Erythromycin with PqsR

MMPBSA

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Erythromycin with RetS

SASA analysis

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• SASA: Solvent Accessible Surface Area
• Measures how much of a protein or ligand surface is exposed to the solvent (usually water).
• Calculated by rolling a water-sized probe (1.4 Å) over the molecular surface.
• Higher SASA = more surface exposed (often flexible or hydrophilic regions).
• Lower SASA = more buried surface (stable or hydrophobic core).
• In binding studies, SASA usually decreases because parts of the ligand become buried inside the protein.
• Used in: protein stability, binding analysis, MMGBSA/MMPBSA calculations.

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AMBER

• SASA is calculated using algorithms like LCPO (Linear Combination of Pairwise Overlaps).

Commonly used in:

• MM/PBSA and MM/GBSA
• Stability checks
• Conformational analysis

SASA Results

SASA Results

SASA Results

pkCSM analysis

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• pkCSM: Predicting Pharmacokinetics and Toxicity Using Graph-Based Signatures
• A computational tool used to predict ADMET properties of small molecules.
• Uses graph-based molecular fingerprints to model how a compound behaves in the body.

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Predicts key ADMET parameters:

• Absorption (e.g., intestinal absorption, Caco-2 permeability)
• Distribution (e.g., BBB permeability, VDss)
• Metabolism (CYP450 inhibition)
• Excretion (renal clearance)
• Toxicity (hepatotoxicity, mutagenicity, LD50)

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• Helps evaluate drug-likeness early before in-vitro testing.
• Often used in docking and MD workflows to filter potential lead compounds.

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pkCSM results

pkCSM results

pkCSM results

In Vitro

Bacterial Inoculation

Antibiotic Resistance

Compound Extraction

Plant Inoculation

Quorum Sensing

Quorum Quenching

Antibacterial Assays

METHODOLOGY

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PLANT INOCULATION

Media preparation in MH agar

Plant inoculation

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EXTRACTION (COLD METHOD)

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Sample Preparation:

• Dried plant leaves (1.0 g)
• Chilled ethanol (100 mL, 4 °C)

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Primary Extraction: Macerated leaves in chilled ethanol for 24 h at 4 °C.

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Filtered to separate:

• Green ethanolic filtrate → Crude ethanolic extract (CCE, used for all compound isolations).
• Spent plant residue → retained for potential secondary extraction (hexane or aqueous) to recover non-polar or highly polar constituents.

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Concentration:

Reduced ethanol volume (rotary evaporation ≤40 °C or gentle hood evaporation) to yield a small-volume concentrate or dry residue

EXTRACTION (COLD METHOD)

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Downstream Use:

• CCE aliquots subjected to specific isolation procedures:
• Premnazole → Acid–base extraction (alkaloids)
• Friedelin → Hexane extraction (defatting)
• Ursolic Acid → EtOAc extraction + acid hydrolysis
• Baicalein → Aqueous ethanol extraction + partitioning

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Key Point for All Targets:

• One common ethanol maceration serves as the starting point.
• Spent leaves remain valuable for non-polar or aqueous extractions if further recovery is needed.

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ANTIBIOTIC RESISTANCE ASSAY

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Objective:

• To verify and cultivate an erythromycin-resistant strain of P. aeruginosa for testing quorum-quenching activity of plant extracts.

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Procedure:

• Wild-type P. aeruginosa [strain PAO1] uniformly spread on nutrient agar plates.
• Four erythromycin disks (15 µg/disc) placed equidistantly on each plate.
• Plates incubated at 37 °C for 24 h to allow antibiotic diffusion.
• Colonies growing within or near inhibition zones were isolated and transferred to agar containing 50 µg/mL erythromycin.
• Resistant colonies subcultured for five generations on erythromycin-supplemented media to stabilize the resistance.
• MIC of erythromycin determined periodically by broth dilution to confirm increased tolerance.
• Final stable isolate designated as Pseudomonas aeruginosa ER (erythromycin-resistant) for further assays.

ANTIBIOTIC RESISTANCE ASSAY

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P. aeruginosa grown on nutrient agar media with disks of Erythromycin, with very few strains of growth on antibiotic inhibition zones.

AGAR WELL DIFFUSION ASSAY

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Objective:

• To determine the antibacterial activity of plant extracts against erythromycin-resistant Pseudomonas aeruginosa.

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Procedure:

• Mueller–Hinton agar plates inoculated with resistant P. aeruginosa.
• Wells (6 mm) made aseptically using a sterile cork borer.
• 100 µL of plant extract (25–100 mg/mL) added to each well.
• Erythromycin used as positive control, DMSO as negative control.
• Plates incubated at 37 °C for 24 h.
• Diameter of inhibition zones measured in millimeters.

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Interpretation:

• Larger inhibition zone = greater antibacterial potential of the plant extract.

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DISK DIFFUSION ASSAY

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Objective:

• To evaluate antibacterial potency and diffusion efficiency of plant extracts.

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Procedure:

• Sterile filter paper disks (6 mm) impregnated with 100 µL plant extract.
• Disks air-dried and placed on MHA plates inoculated with resistant strain.
• Erythromycin and DMSO disks used as positive and negative controls.
• Plates incubated at 37 °C for 24 h.
• Zone diameters measured and compared to standard antibiotic.

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Interpretation:

• Zone size reflects the extract’s ability to inhibit bacterial growth via diffusion.

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BROTH DILUTION ASSAY

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Objective:

• To determine the minimum inhibitory concentration (MIC) of plant extracts using test tube dilutions.

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Procedure:

• Prepare two-fold serial dilutions of the plant extract (0.5–100 mg/mL) in sterile nutrient broth in 15 mL test tubes.
• Add 9 mL of sterile broth to each tube and 1 mL of the bacterial suspension (~10⁵ CFU/mL).
• Include one positive control (bacteria without extract) and one negative control (broth only).
• Mix gently and incubate at 37 °C for 24 hours under static conditions.
• Examine tubes for visible turbidity or pellet formation to assess bacterial growth.
• The lowest concentration showing no visible growth is recorded as the MIC.

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Interpretation:

• MIC represents the minimum concentration of the plant extract required to inhibit visible bacterial growth.

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SWARMING MOTILITY ASSAY

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Objective:

• To examine the effect of plant extracts on bacterial motility (quorum sensing behavior).

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Procedure:

• Swarming medium (nutrient agar with 0.5% agar) prepared.
• Sub-inhibitory concentration (½ MIC) of extract incorporated. Alternate plate with no extract is also prepared.
• Center of plates inoculated with 5 µL of bacterial culture.
• Plates incubated at 30 °C for 24 h.
• Diameter of swarming zone measured.

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Interpretation:

• Reduced swarming indicates interference with quorum-sensing–regulated motility.

SWARMING MOTILITY ASSAY

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P. aeruginosa inoculated on LB media with no extract.

P. aeruginosa inoculated on LB media with plant extract.

PYOCYANIN QUANTIFICATION ASSAY

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Objective:

• To assess the effect of plant extracts on pyocyanin production, a quorum-sensing regulated pigment.

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Procedure:

• Resistant strain grown in King’s A broth with and without plant extract (½ MIC).
• Cultures centrifuged, supernatant extracted with chloroform (3:1).
• Chloroform layer re-extracted with 0.2 N HCl.
• Absorbance measured at 520 nm using calorimeter.

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Interpretation:

• Reduced pyocyanin levels = successful quorum sensing inhibition by plant extract.

PYOCYANIN QUANTIFICATION ASSAY

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BIOFILM INHIBITION ASSAY (CRYSTAL VIOLET METHOD)

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Objective:

• To evaluate the ability of plant extracts to inhibit biofilm formation in 15 mL test tubes.

Procedure:

• 5 mL of sterile nutrient broth was added into each sterile 15 mL test tube.
• Each tube was inoculated with 0.1 mL of bacterial suspension (~10⁶ CFU/mL).
• Plant extract was added at ½ MIC concentration, and a control tube was maintained without extract.
• The tubes were incubated vertically at 37 °C for 24 h without shaking to allow biofilm formation on the tube walls.
• After incubation, planktonic cells were gently discarded, and the tubes were rinsed three times with sterile PBS.
• The adhered biofilm was stained with 0.1% crystal violet for 15 min, and excess stain was removed by rinsing.
• 5 mL of 95% ethanol was added to solubilize the bound dye.
• The absorbance of the ethanol extract was measured at 570 nm using a spectrophotometer.

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BIOFILM INHIBITION ASSAY (CRYSTAL VIOLET METHOD)

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SCANNING ELECTRON MICROSCOPY (SEM) ANALYSIS

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Objective:

To visualize and compare biofilm morphology and surface structure of Pseudomonas aeruginosa treated and untreated with plant extract.

Procedure:

• Biofilms grown on small sterile glass or coverslip fragments placed in 15 mL test tubes containing nutrient broth.
• Tubes inoculated with resistant P. aeruginosa and treated with plant extract (½ MIC); control tube left untreated.
• After 24 h incubation at 37 °C, glass pieces gently removed and rinsed with phosphate-buffered saline (PBS) to remove planktonic cells.
• Samples fixed with 2.5% glutaraldehyde for 2 h at 4 °C, then washed three times with PBS.
• Dehydrated sequentially with graded ethanol series (30%, 50%, 70%, 90%, 100%) for 10 min each.
• Dried samples mounted on aluminum stubs, sputter-coated with gold, and examined under a Scanning Electron Microscope (SEM).

SCANNING ELECTRON MICROSCOPY (SEM) ANALYSIS

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Interpretation:

• Control samples: Dense, multilayered biofilm structure.
• Treated samples: Reduced cell attachment, disrupted matrix, and altered surface morphology indicating effective quorum quenching.

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DOCKING & SWISSADME

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​

https://docs.google.com/spreadsheets/d/1K6ScV_xEHUP9Uel5B04paihza7Vdiq4afFFy6UecF2o/edit?usp=sharing

Document of other 30 proteins analyzed using Swissadme:

Excel sheet of docking results:

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https://docs.google.com/spreadsheets/d/1K6ScV_xEHUP9Uel5B04paihza7Vdiq4afFFy6UecF2o/edit?usp=sharing

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CONCLUSION

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• The in silico findings support the hypothesis that compounds found in Premna serratifolia can interact effectively with quorum sensing regulatory proteins in Pseudomonas aeruginosa.
• Compounds such as ursolic acid, baicalein, and friedelin demonstrated strong binding affinities to key QS proteins like LasR, PqsR, and RetS, often outperforming standard antibiotics in docking simulations.
• ADME profiling further validated their potential as drug-like molecules with acceptable pharmacokinetics.

Previously asked questions

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1) Why 3 docking trials?

Molecule is present in local minima, so to remove it from there we perform 3 trials.

2) What is the selected target proteins?

LasR, PqsR, RetS

3) How is biofilm formation relevant to QS?

QS is a cell-to-cell communication system in bacteria where cells coordinate their behavior based on population density, and this coordination often leads to biofilm formation. Biofilms are communities of bacteria encased in a self-produced matrix, and the formation of these structures is a key factor in bacterial pathogenicity and antibiotic resistance.

4) Is Pseudomonas aeruginosa a Gram-negative or Gram-positive bacterium, and how is it related to quorum quenching?

Pseudomonas aeruginosa is a Gram-negative bacterium that uses quorum sensing (QS), a cell-to-cell communication system, to coordinate collective behaviors like forming biofilms and producing virulence factors. Quorum quenching (QQ) disrupts this communication by targeting and inactivating the signaling molecules (autoinducers) or their receptors, thereby reducing the bacteria's pathogenicity without killing them.

Previously asked questions

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5) Why was dry lab done first and not wet lab?

Dry-lab screening was done first because GC–MS and HPLC profiling of Premna serratifolia had not yet been performed, so we relied on literature-reported compounds such as Baicalein, Premnazole, Ursolic acid, and Friedelin. Using these known phytochemicals, computational docking allowed rapid prediction of their inhibitory potential against the major Pseudomonas aeruginosa quorum-sensing proteins LasR, PqsR, and RhlR. This in-silico step helped shortlist only the most promising molecules before investing time, cost, and resources in wet-lab validation.

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6) Why wasn’t the tertiary structure of the molecules shown in the presentation ?

We relied on the final result of the molecule that is the simulation and MMPBSA analysis, since both the analysis was fully completed putting the intial result that is the ligand binding with the target protein wasn’t best suited for the approach.

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7) Why was Pseudomonas aeruginosa and Premna Serratifolia chosen specifically for the research project, what was the reason for the novelty?

P. aeruginosa was selected because it is a major drug-resistant pathogen, and Premna serratifolia was chosen because its phytochemicals have never been tested against its quorum-sensing targets. The novelty comes from being the first to explore Premna compounds against LasR, PqsR, and RhlR.

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1.) Ratnaweera, Pamoda B., et al. "Antimicrobial constituents of Hypocrea virens, an endophyte of the mangrove-associate plant Premna serratifolia L." (2016).

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2.) YEE, Khin Su, and Uthai Sotanaphun. Analysis of volatile constituents from post-harvesting handling and optimization of extractive solvent on the quality of the leaves of Premna serratifolia L. Diss. Silpakorn University, 2022.

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3.) Francis, Steffy, V. Anand Gideon, and S. John Britto. "Antibacterial and GC-MS analysis of stem and leaf of Premna paucinervis (CB Clarke) gamble (Lamiaceae)—An endemic and rediscovered species." Int. J. Bot. Stud 6 (2021): 282-292.

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4.) SL, Varsha, et al. "In Vitro Pharmacological Activity of Endophyte Aspergillus austwickii isolated from the Leaves of Premna serratifolia." Archives of Razi Institute 79.5 (2024).

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5.) Singh, Chandrashekhar, et al. "Green synthesis of silver nanoparticles by root extract of Premna integrifolia L. and evaluation of its cytotoxic and antibacterial activity." Materials Chemistry and Physics 297 (2023): 127413.

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REFERENCES

6.) Sarma, Gopa, Manswi More, and Roshan Shirke. "Qualitative and Quantitative Phytochemical Screening, TLC Fingerprinting, HPTLC, and Proximate Analysis of Premna serratifolia, a Medicinally Important Plant." International Journal of Research in Engineering, Science and Management 4.8 (2021): 190-196.

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7.) Simamora, Adelina, et al. "Antioxidant activity, enzyme inhibition potentials, and phytochemical profiling of Premna serratifolia L. leaf extracts." International Journal of Food Science 2020.1 (2020): 3436940.

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8.) Timotius, K. H. "Health benefits of edible leave of Premna serratifolia L." Journal of Food Science and Nutrition, 7(5), 122 (2021).

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9.) Sanya, D.R.A., Onésime, D., Vizzarro, G. et al. Recent advances in therapeutic targets identification and development of treatment strategies towards Pseudomonas aeruginosa infections. BMC Microbiol 23, 86 (2023). https://doi.org/10.1186/s12866-023-02832-x

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10.) Tunney, M. M., Ramage, G., Field, T. R., Moriarty, T. F., & Storey, D. G. (2004). Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa. Antimicrobial agents and chemotherapy, 48(5), 1879–1881. https://doi.org/10.1128/AAC.48.5.1879-1881.2004

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REFERENCES

THANK YOU

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