Potassium 2-methoxy-4-vinylphenolate: A novel hit exhibiting quorum-sensing inhibition in: Pseudomonas aeruginosa via LasIR/RhlIR circuitry

Article abstract of DOI:10.1039/c9ra06612h

The emergence of multidrug-resistant (MDR) bacterial strains in the last decade is astonishingly alarming. Many of the widely used antibiotics have failed to exhibit clinical efficacy against such strains. Eventually we will exhaust all the resources in our antibiotic armamentarium. As a need of the hour, novel strategies are desperately required not only to curb, but also to reverse, the development of resistance in these pathogens, thereby maintaining their sensitivity towards current antibiotics. Intervention of bacterial virulence, rather than killing them, by inhibiting specific pathways/targets has emerged as a novel approach to tackle the drug resistance problem. The bacterial virulence is regulated via quorum-sensing, a cell-cell communication process precisely controlled by autoinducer molecules such as acyl homoserine lactone (AHL). The present study aimed at identifying promising quorum-sensing inhibitors in Pseudomonas aeruginosa, an opportunistic human pathogen especially associated with nosocomial infections, yielding four potential hits. Out of these, potassium 2-methoxy-4-vinylphenolate was the most potent quorum-sensing inhibitor targeting P. aeruginosa LasIR/RhlIR circuitry. It also inhibited biofilm formation, various virulence factors like LasA protease, LasB elastase and pyocyanin, and motility of bacteria like swarming and twitching.

Full text of DOI:10.1039/c9ra06612h

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Potassium 2-methoxy-4-vinylphenolate: a novel hit
exhibiting quorum-sensing inhibition in
Pseudomonas aeruginosa via LasIR/RhlIR circuitry†
b
Cite this: RSC Adv., 2019, 9, 40228
Mayank D. Shah,a Prashant S. Kharkar,
Niteshkumar U. Sahu,b Zoya Peerzadaa
c
*
and Krutika B. Desai
The emergence of multidrug-resistant (MDR) bacterial strains in the last decade is astonishingly alarming.
Many of the widely used antibiotics have failed to exhibit clinical efficacy against such strains. Eventually
we will exhaust all the resources in our antibiotic armamentarium. As a need of the hour, novel strategies
are desperately required not only to curb, but also to reverse, the development of resistance in these
pathogens, thereby maintaining their sensitivity towards current antibiotics. Intervention of bacterial
virulence, rather than killing them, by inhibiting specific pathways/targets has emerged as a novel
approach to tackle the drug resistance problem. The bacterial virulence is regulated via quorum-sensing,
a
cell–cell communication process precisely controlled by autoinducer molecules such as acyl
homoserine lactone (AHL). The present study aimed at identifying promising quorum-sensing inhibitors
in Pseudomonas aeruginosa, an opportunistic human pathogen especially associated with nosocomial
infections, yielding four potential hits. Out of these, potassium 2-methoxy-4-vinylphenolate was the
most potent quorum-sensing inhibitor targeting P. aeruginosa LasIR/RhlIR circuitry. It also inhibited
biofilm formation, various virulence factors like LasA protease, LasB elastase and pyocyanin, and motility
of bacteria like swarming and twitching.
Received 22nd August 2019
Accepted 19th November 2019
DOI: 10.1039/c9ra06612h
rsc.li/rsc-advances
common nosocomial pathogen) in general and ventilator-asso-
ciated pneumonia (VAP) (the second most common pathogen), in
particular.4,5 The emergence of multidrug-resistant (MDR) strains
of Pseudomonas aeruginosa have created havoc in the not-so-
distant past.6,7 Several conventional and emerging strategies have
been and are being utilized to combat the disastrous situation.8–11
One such strategy is to inhibit a phenomenon named quorum
sensing in these deadly pathogens.12
Quorum sensing is a bacterial cell-to-cell communication
mechanism mediated by a set of chemicals known as auto-
inducers.13 At the core of quorum sensing process is the
production, release, accumulation and population-wide detec-
tion of the autoinducers. The Gram-negative bacteria use small-
molecules while Gram-positive bacteria use oligopeptides as
autoinducers. These molecules accumulate in the environment
as the bacterial population density increases, and this infor-
mation is monitored by the bacteria to track variations in their
cell numbers and ultimately to control the gene expression.
Such coordinated activities brought about via quorum sensing
include bioluminescence, secondary metabolites such as anti-
biotics production, biolm formation, and virulence factor
secretion, to name a few.14,15
1. Introduction
The past decade has witnessed a surge in antibiotic resistance.
Several, otherwise antibiotic-sensitive microbes, have become
resistant, leading to utter therapeutic failure. The World Health
Organization (WHO) went to the extent of calling the 21st century
as the ‘Post-antibiotic era’.1 The WHO fact sheet on antimicrobial
resistance (released on February 15, 2018) is concerning.2 The
overall statistics indicate the gravity of the situation. Moreover,
the emergence of multidrug- and pan-drug-resistant pathogens,
particularly the Gram-negative ones, has made the matter worse.3
Among these pathogens, mostly responsible for the nosocomial
infections, Klebsiella pneumoniae, Acinetobacter baumannii, and
Pseudomonas aeruginosa are the front-runners. In particular,
Pseudomonas aeruginosa is most dangerous. It is an aerobic,
Gram-negative, opportunistic human pathogen majorly respon-
sible for healthcare-associated infections (the sixth most
aSunandan Divatia School of Science, SVKM's NMIMS (Deemed to be University),
Mumbai, 400056, India
bInstitute of Chemical Technology, Category I Deemed to be University (MHRD/UGC),
Mumbai-400019, India
In Pseudomonas aeruginosa, there are two circuits as integral
parts of quorum sensing mechanism which are LasI/R and RhlI/
R (Fig. 1), resembling LuxI/R system in Vibrio scheri.16 The LasI/
R and RhlI/R produce small-molecule autoinducers, N-3-oxo-
cMithibai College of Arts & Science & Amrutben Jivanlal College of Commerce &
Economics, Mumbai, 400056, India. E-mail: krutika.desai@mithibai.ac.in
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra06612h
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Fig. 1 Quorum sensing in Pseudomonas aeruginosa; AHL: acyl homoserine lactone; 1: N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL);
2: N-butanoyl-L-homoserine lactone (C4-HSL).
dodecanoyl homoserine lactone (3OC12-HSL) and C4-homo- production in P. aeruginosa signicantly.37 Baicalein (avonoid
serine lactone (C4-HSL), respectively.17,18 These molecules bind from the roots of Scutellaria baicalensis) was demonstrated to
to their cognate receptors forming the autoinducer–receptor inhibit several virulence factors and biolm formation.38
complex, which ultimately regulates various downstream Another interesting study revealed that [6]-gingerol, a pungent
molecules/processes including virulence factors like LasA active from fresh ginger which is structurally analogous to
protease, LasB elastase, pyocyanin (PCN), swarming and 3OC12-HSL, reduced biolm formation, virulence factors such
twitching activity and biolm formation. Given the importance as pyocyanin, exoprotease and rhamnolipid and mice
of quorum sensing in the bacterial virulence and biolm mortality.39 The in silico studies conrmed that [6]-gingerol
formation, its inhibition has emerged as an important strategy bound specically to LasR receptor. The QS genes involved in
to tackle the menace of MDR and pan-drug-resistant (PDR) production of virulence factors were specially repressed as seen
bacterial pathogens.19,20 The concept is very simple. By inter- from the transcriptome analysis.
fering with their virulence factors and not killing them via
Working on the similar lines, Gala et al. (author's lab) iden-
inhibition of quorum sensing, we are in a way slowing down the tied active components from Tinospora cordifolia (family:
emergence of MDR and PDR strain. Thanks to the weaker Menispermaceae, common name: Guduchi) following isolation
selective pressure exerted on bacteria by these mechanisms!
and identication using column chromatography, gas chroma-
Due to the promise offered by quorum sensing inhibition, it tography-mass spectrometry (GC-MS) and in silico molecular
is not surprising that a variety of small-molecules (synthetic as studies as potential anti-quorum sensing inhibitors.40 Four
well as natural products) with signicant antiquorum sensing molecules, namely 2,3,4-triacetoxybutyl acetate (3), 2-(5-ethenyl-5-
and antibiolm activities in Gram-negative and Gram-positive methyloxolan-2-yl)propan-2-ol (4), methyl hexadecanoate (5) and
bacteria have been reported.21–24 Literature is full of reports 2-methoxy-4-vinylphenol (6) (Fig. 2) were postulated to inhibit QS
particularly on quorum sensing inhibition in Pseudomonas via either LasI or LasR.41 We were the rst to report these mole-
aeruginosa.25–36
cules as potential QS inhibitors. Interestingly, the partial struc-
Carvacrol (essential oil) was reported to inhibit biolm tural similarity of 5 and 6 with previously reported [6]-gingerol
formation in S. aureus and P. aeruginosa and pyocyanin was intriguing, which further strengthened our belief that these
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Fig. 2 Previously reported lead structures from Tinospora cordifolia: 2,3,4-triacetoxy-butyl acetate (3), 2-(5-ethenyl-5-methyloxolan-2-yl)
propan-2-ol (4), methyl hexadecanoate (5) and 2-methoxy-4-vinylphenol (6).
leads would provide us newer chemotypes for QS inhibitors. In Innity 1290 series (Agilent Technologies, USA) instrument
the present study, we report further proling of these leads (3–6, coupled with quadrupole time of ight (iFunnel Q-TOF LC-MS
Fig. 2) by quorum quenching studies against P. aeruginosa. 6550) and equipped with electrospray ionization (ESI) mode.
Although the major issue with compound 6 was its signicantly
Pseudomonas aeruginosa PAO1 strain was used as a standard
lesser aqueous solubility. We were determined to improve its strain for evaluation of all antiquorum, antibiolm and anti-
aqueous solubility by suitable structural and/or chemical modi- virulence activity studies. Staphylococcus aureus 737 was used
cation. Herein, we describe the complete proling of modied for LasA protease study. Chromobacterium violaceum ATCC
compound 6 as a potential QS inhibitor in a battery of assays 12472 and Escherichia coli MG4/pKDT17 were used as a reporter
validating the original observations and the hypothesis.
strain in variety of assays. Luria Bertani (LB) broth and agar
powder were purchased from Himedia Laboratories, Mumbai,
India. o-Nitrophenyl-4-D-galactopyranoside (ONPG) was
purchased from Sisco Research Laboratories (SRL), Mumbai,
India. Rest of the chemicals were purchased from Qualigens
Fine Chemicals, Mumbai, India. E. coli MG4/pKDT17 was grown
in LB medium containing 100 mg ampicillin. All other bacterial
2. Material and methods
2.1. General
All the chemicals such as 2-(5-ethenyl-5-methyloxolan-2-yl)
propan-2-ol (4, Fig. 2), erythritol, 2-methoxy-4-vinyl phenol and
elastin congo red (ECR), propidium iodide, solvents and
reagents were purchased from the approved vendors such as
Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Ger-
many), Spectrochem (Mumbai, India), Sigma-Aldrich (Stein-
heim, Germany, St. Louis, MO, USA) or Merck (Darmstadt,
ꢀ
strains were incubated overnight at 37 C, wherever required.
2.2. Synthesis of test compounds
2.2.1. 2,3,4-Triacetyloxybutyl acetate (3). A mixture of
Germany) and used without further purication unless other- erythritol (1 g, 8.19 mmol), anhydrous NaOAc (6.7 g, 81.9 mmol,
wise indicated. SYTO-9 dye was purchased from Thermo Fisher, 10 equiv.) and acetic anhydride (7.75 mL, 81.9 mmol, 10 equiv.)
Waltham, MA. Reactions were carried out under dry N2 atmo- and was heated on water-bath at 90 ꢀC until a clear solution was
sphere and thin-layer chromatography (TLC), wherever obtained (approx. 2 h). The reaction was monitored by LC-MS.
required, was performed using an aluminum plate coated with Once completed, the reaction mixture was poured on crushed
silica gel 60 F254 (Merck Millipore, Billerica, MA, USA). 2- ice and stirred for further 15 min. The precipitated solid was
Methoxy-4-vinyl phenol was purchased from Alfa Aesar by ltered under vacuum, washed with cold water and dried in
Thermo Fisher Scientic (Tewksbury, MA). Melting points were oven. The crude product was recrystallized from 95% EtOH to
recorded using conventional Thiele tube and are uncorrected. get pure compound. Yield: 0.72 g; melting point: 86–88 ꢀC; MS
The NMR spectra were recorded in either in CDCl3 or DMSO-d6 (ESI): 308.10 [M + H2O]+; FT-IR (KBr, cmꢁ1): 2964 (C–H
with tetramethylsilane (TMS) as internal standard. FT-IR stretching), 1743 (C]O stretching), 1242, 1220 (C–O–C
spectra were recorded on PerkinElmer RX1 instrument (Wal- stretching).
1
tham, MA). H-NMR spectra were obtained on Bruker Advance
2.2.2. Methyl palmitate (5). Palmitic acid (5 g) was dis-
400 (400 MHz) spectrometer. Mass spectra (MS) were recorded solved in MeOH HPLC and few drops of conc. H2SO4 added. The
either on a Shimadzu 8040 LC-MS/MS system (Japan), or Agilent reaction mixture was reuxed on water-bath for 5 h. The
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reaction was monitored by GC. Aer completion, the solvent dryness under N2 atmosphere. The dried extract was mixed in
was evaporated and the syrupy liquid was taken up in dry DCM. LB broth and 2.0 mL of overnight grown E. coli MG4 culture and
The organic layer was washed with sat. NaHCO3 (2 ꢂ 20 mL), incubated for 5 h at 37 ꢀC for induction of b-galactosidase. The
followed by water (2 ꢂ 20 mL). The organic layer was dried enzyme activity was determined as described by Miller using
(anhyd. Na2SO4) and the solvent removed in vacuo to obtain ONPG as substrate.40,42
ꢀ
pure product. Yield: 4.67 g; melting point: 32–34 C; MS (ESI):
271.27; FT-IR (KBr, cmꢁ1): 2924 (C–H stretching), 1744 (C]O
2.4. Effect of the compound on P. aeruginosa biolm
stretching), 1171, 1198 (C–O–C stretching).
formation as analyzed by crystal violet assay
2.2.3. Potassium 2-methoxy-4-vinylphenolate (7) (Scheme
1). A mixture of 2-methoxy-4-vinyl phenol (6) (1 g, 3.33 mmol),
anhydrous K2CO3 (0.24 g, 1.67 mmol) and anhydrous toluene
(20 mL) was reuxed for 12 h with continuous stirring attached
using Dean–Stark apparatus. The reaction was monitored by
TLC. Aer complete consumption of the starting phenol, the
reaction mixture was cooled and the ꢀsolid ltered, washed with
anhydrous toluene and dried at 60 C for 24 h, when slightly
yellow-colored amorphous and free-owing powder was ob-
Biolm formation was checked using 96-well at-bottom
microtiter plate, as per the modied method reported by
Mathur et al. (2006).43 Overnight culture of P. aeruginosa was
diluted with fresh LB broth supplemented with 0.5% glucose,
up to 0.2 OD600. Then control/test compound was added. The
positive control, azithromycin dihydrate, was dissolved in
PBS : EtOH (1 : 1), pH 6.0, and 50% DMSO/PBS : EtOH (1 : 1)
was used as negative control. Microtiter wells were lled with
200 mL aliquots of the diluted cultures as described above,
uninoculated broth and uninoculated broth containing the
1
tained. Yield: 0.8 g; HRMS (ESI): 187.0406 [M ꢁ H]ꢁ; H-NMR
(DMSO-d6) d ppm 6.9 (m, 2H), 6.6 (m, 1H), 5.61 (d, 1H), 5.55 (s,
1H), 5.2 (d, 1H), 3.9 (s, 3H); FT-IR (KBr, cmꢁ1): 2929 (C–H
stretching), 1630 (C]C stretching), 1124 (C–O–C stretch).
ꢀ
compound. The plates were incubated for 24 h, at 37 C. The
wells were aspirated, washed with deionized water to remove
the planktonic bacteria, and dried. The biolm, formed in
microtiter plate wells, was xed with 200 mL MeOH for 15 min.
Then the solvent was removed and the plates were dried. Next,
the biolm was stained using 200 mL of 1% crystal violet dye for
15 min. Excess stain was removed and the plate washed with tap
water and dried. The bound dye was resolubilized in 95% EtOH
(180 mL) for 5 min and the absorbance at 490 nm recorded using
BioTek Microplate reader Epoch™ 2.40
2.4.1. Confocal microscopy. Overnight culture of P. aeru-
ginosa (wild type) was diluted with fresh LB broth up to 0.2
OD600. Culture with the compound was inoculated on a clean,
sterile glass slide under sterile condition and incubated for 24
h. Next day, the cells were washed twice with PBS (pH 7.0) to
remove unattached cells. SYTO-9 (lex/lem 480/500 nm) and
propidium iodide (lex/lem 490/635 nm) dyes were used for
confocal study in 2 : 1 concentration. The mixture of dyes (5 mL)
was added onto the slide and coverslip was xed with trans-
parent nail paint. Samples were observed under Spinning disc
confocal laser microscope (CLSM, Zeiss LSM 800, Carl Zeiss,
Jena) at 40X.
2.3. Anti-quorum sensing activity
The test compounds, dissolved in 50% DMSO at specied
concentration, i.e., 4 mg mLꢁ1, were screened for short acyl-HSL
and long acyl-HSL inhibition according to the protocols given
below. The corresponding concentrations of the test
compounds were 13.7 mM (3), 14.7 mM (4), 23.4 mM (5) and
21.4 mM (7).
2.3.1. Inhibition of short-chain AHL. Agar well-diffusion
assay was performed to estimate the inhibition of short-chain
AHL by the test compounds. The overnight grown culture of
Chromobacterium violaceum ATCC12472 was mixed in 15 mL LB
agar (1.5%). Test compounds were added in the agar well.
DMSO (50%) was used as negative control. Azithromycin dihy-
drate (AZ) (4 mg mLꢁ1 in PBS : EtOH 1 : 1 as solvent) was used as
positive control and PBS : EtOH (1 : 1) as its negative control.
Production of pigment inhibition was checked aer 24 h incu-
bation.40 To support this assay quantitative analysis of violacein
pigment inhibition was done. Growth curve analysis was done
to conrm that compound 7 possess anti-quorum sensing
activity not anti-bacterial. Bacterial growth curve was observed
by measuring cell density at 600 nm at various time point until
it reached to stationary phase.
2.5. Anti-virulence assays
2.5.1. Swarming activity. Test compound was mixed with
LB agar with positive control (azithromycin 4 mg mLꢁ1) and
2.3.2. Inhibition of long-chain AHL. The ability of negative control [50% DMSO/PBS : EtOH (1 : 1)]. The mixtures
compounds to inhibit long acyl-HSL in P. aeruginosa was tested were poured onto plates and allowed to solidify. P. aeruginosa
using reporter strain E. coli MG4/pKDT1719 (ref. 41) where this (10 mL) was point inoculated in the middle of the plate, which
strain is used to detect 3-oxo- or 3-hydroxy substituted or were then incubated at 37 ꢀC for 48 h. The degree of swarming
unsubstituted C8–C14 long acyl-HSLs wherein the long acyl- was determined by measuring the diameter of the swarm and
HSL induces the expression of the b-galactosidase gene. The compared with control.44
decrease in expression of b-galactosidase is indicative the
2.5.2. LasA protease. Overnight culture of P. aeruginosa
inhibition of long-chain acyl-HSL. The bacterial culture was (wild type) was diluted with fresh LB broth up to 0.2 OD600. Test
grown along with test compounds or only the solvent (negative compound (0.1 mL, dose dependent concentration in 50%
control) or azithromycin dihydrate (4 mg mLꢁ1) (positive DMSO)/azithromycin (4 mg mLꢁ1 in PBS : EtOH 1 : 1)/50%
control) for 24 h. Culture supernatant was then extracted with DMSO/PBS : EtOH (1 : 1) was added to the diluted culture (4.9
ꢀ
EtOAc twice and the pooled organic layers were evaporated to mL) and further incubated at 37 C for 24 h. The culture was
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centrifuged at 10 000 rpm under cold (4 ꢀC) condition for 15 fold changes in gene expression. The fold change of target genes
min. The culture supernatant (0.5 mL) was mixed with 1.5 mL of for each group with respect to the control group was calculated
15 min boiled suspension of Staphylococcus aureus in 0.02 M using the DDCt method.
Tris–HCl buffer (pH 8.5). The OD600 was measured at 0 and 3 h
with UV-Vis spectrophotometer (PerkinElmer Lambda 25) post
3. Results and discussion
incubation at 37 C.45,46
ꢀ
2.5.3. LasB elastase. Overnight culture of P. aeruginosa
In our previous report, we demonstrated the anti-QS and anti-
biolm activities of the cold EtOAc extract of Tinospora cordifolia
stem along with probable chemical constituents of the extract.40
We selected four of these constituents (3–6, Fig. 2) based the
molecular docking studies. The compounds were either
synthesized (3 or 5) or procured from the vendors (4 and 6).
While working with compound 6, we observed solubility issues
(precipitation) at the concentrations tested. To circumvent
these issues, the potassium salt of 6 (7, Scheme 1) was subse-
quently prepared, thoroughly characterized and then used in
further studies.
(wild type) was diluted with fresh LB broth up to 0.2 OD600
.
Then, 0.1 mL of test compound (dose dependent concentrations
in 50% DMSO)/azithromycin (4 mg mLꢁ1 in PBS : EtOH 1 : 1)/
50% DMSO/PBS : EtOH (1 : 1) was added to the diluted culture
(4.9 mL) and incubated at 37 ꢀC for 24 h. The culture was
ꢀ
centrifuged at 10 000 rpm (4 C). Buffer (0.1 mL, 0.1 M Tris, 1
mM CaCl2, and pH 7.5) containing ECR substrate (10 mg) was
mixed with culture supernatant (0.5 mL), incubated at 37 ꢀC for
24 h. The tubes were centrifuged at 10 000 rpm (4 ꢀC) for 10
min, and OD495 of the supernatant was measured.47
2.5.4. Pyocyanin assay. Overnight culture of P. aeruginosa
(wild type) was diluted with fresh King's medium B up to 0.2
OD600. Test compound (0.1 mL, dose dependent concentration
in 50% DMSO)/azithromycin (4 mg mLꢁ1 in PBS : EtOH 1 : 1)/
50% DMSO/PBS : EtOH (1 : 1) were added to the diluted culture
(4.9 mL) and incubated for 48 h at 37 ꢀC. The culture was
centrifuged at 10 000 rpm (4 ꢀC) for 15 min. To the culture
supernatant (5 mL), CHCl3 (3 mL) was added, vortexed to extract
blue pyocyanin in CHCl3, followed by absorbance measurement
at 690 nm. Pyocyanin concentration was calculated as mg mLꢁ1
using the formula, OD690/16.40,48
3.1. Evaluation of anti-quorum sensing activity
3.1.1. Inhibition of short-chain AHL. Chromobacterium
violaceum, a large, Gram-negative bacterium, produces short-
chain AHL (C6-HSL) which regulates the production of natural
antibiotic, a dark purple pigment – violacein (violet nondiffus-
ible pigment). Reduction or absence of violacein is, thus,
indicative of inhibition of short-chain AHL in C. violaceum. All
four test compounds (3–5, 7) were evaluated for their ability to
affect violacein production. As seen from Fig. 3(i), of all the
compounds tested, only 7 could inhibit violacein production
signicantly (Fig. 3(i)(d) well 1, diameter of zone of pigment
inhibition: 23 mm) which was better than AZ (Fig. 3(i)(d) well 3,
diameter of zone of pigment inhibition: 20 mm) at the
concentration tested, i.e., 4 mg mLꢁ1. Dose–response studies
involving compound 7 at concentrations 4, 6 and 8 mg mLꢁ1
exhibited zone of pigment inhibition diameter as 22 ꢃ 1, 25 ꢃ 1
and 28 ꢃ 1 mm, respectively. Inhibition of pigment production
2.5.5. Twitching motility assay. Compound 7 (4 mg mLꢁ1
in 50% DMSO)/AZ (4 mg mLꢁ1 in PBS : EtOH 1 : 1) (positive
control)/negative control (50% DMSO/PBS : EtOH (1 : 1)) was
mixed with LB agar. The mixtures were poured into plates and
allowed to solidify. The Pseudomonas aeruginosa culture (PAO1)
was stabbed to bottom in the middle of the plate. Plates were
ꢀ
then incubated at 37 C for 24 h. The extent of twitching was
determined by measuring the diameter of growth zone (in mm),
followed by comparison with the control.49
was not observed at lower concentrations, i.e., 2 mg mLꢁ1
.
Compound 7, from among the four test compounds, was a clear
winner which inhibited short chain AHL production as seen
from the presence of a colorless zone of inhibition against the
purple lawn of C. violaceum. Though there was visible growth
around the well (Fig. 3(i)(d)), there was no pigmentation at all,
demonstrating inhibition of short-chain AHL molecule in rhl QS
circuitry with no antibacterial activity. Quantitative analysis of
violacein pigment inhibition showed signicant inhibition
which support the anti-quorum sensing activity of compound 7.
Growth curve analysis showed no antimicrobial activity at dose
dependent manner of compound 7 (Fig. S1†).
2.6. Gene regulation
Overnight culture of P. aeruginosa PAO1 (OD600: 0.2) was inoc-
ulated with LB medium supplemented with different concen-
tration of compound 7 (2 mg mLꢁ1 to 8 mg mLꢁ1) and
ꢀ
incubated at 37 C. Total RNA was extracted using TriZol and
concentration and purity were determined by UV absorption
(260/280 nm) using Take 3 plate in Epoc2 (BioTek). cDNA was
synthesized from puried mRNA sample using PrimeScript
reagent kit (TaKaRa). Real Time PCR was carried out using SYBR
green master mix (Thermo scientic, USA) with StepOne
Applied Biosystems (California, USA). The reaction procedure as
follows: 95 ꢀC for 30 s, 40 cycles of 95 ꢀC for 5 s, and 60 ꢀC for 5 s,
and a nal melting curve analysis from 65 ꢀC to 95 ꢀC, with
increments of 0.5 ꢀC every 5 s. Real-time PCR amplications
were conducted in triplicate. Primer sequences (Sigma-Aldrich,
USA) for P. aeruginosa QS genes were used as described in Table
S1.† 50 The ribosomal gene rpsL was chosen as a housekeeping
gene to normalize the qRT-PCR data and to calculate the relative Scheme 1 Synthesis of potassium 2-methoxy-4-vinylphenolate (7).
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Fig. 3 (i) Inhibition of short-chain AHL in C. violaceum. (a) compound 3 – well 1: 4 mg mLꢁ1, well 2: 6 mg mLꢁ1, well 3: 8 mg mLꢁ1, well 4: 50%
DMSO (negative control); (b) compound 4 – well 1: 4 mg mLꢁ1, well 2: 6 mg mLꢁ1, well 3: 8 mg mLꢁ1, well 4: 50% DMSO; (c) compound 5 – well
1: 4 mg mLꢁ1, well 2: 6 mg mLꢁ1, well 3: 8 mg mLꢁ1, well 4: 50% DMSO; (d) well 1 (compound 7): 4 mg mLꢁ1, well 2: 50% DMSO, well 3 (azi-
thromycin, positive control): 4 mg mLꢁ1, well 4: PBS : EtOH (1 : 1) (negative control for AZ); (ii) quantitative analysis violacein pigment (a) tube
method in dose dependent manner (4 mg mLꢁ1, 6 mg mLꢁ1, 8 mg mLꢁ1); (b) graphical representation of violacein pigment inhibition. (iii)
Inhibition of long-chain AHL assay in P. aeruginosa (a) by the test compounds 3 (4 mg mLꢁ1), 4 (4 mg mLꢁ1), 5 (4 mg mLꢁ1), 7 (4 mg mLꢁ1) and AZ
(4 mg mLꢁ1) (n ¼ 6); (b) dose–response studies of compound 7 (n ¼ 3). Statistically significant difference between the test compounds compared
to azithromycin (AZ) was analysed using Dunnett's multiple comparison test (*** ¼ p < 0.001).
detects 3-oxo- or 3-hydroxy C8–C14 long-chain AHL, which in
turn, induces the expression of b-galactosidase gene. The
decrease in b-galactosidase expression is, thus, indicative of
3.1.2. Inhibition of long-chain AHL. The ability of
a compound to inhibit long-chain AHL in P. aeruginosa was
evaluated using a reporter strain, E. coli MG4/pKDT17, which
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long-chain AHL inhibition. Previously, Liu et al. have demon- the molecular docking analysis, it made perfect sense that 7
strated similar activity, i.e., inhibition of 3OC12-HSL production would inhibit biolm formation in Pseudomonas aeruginosa by
in Pseudomonas aeruginosa, by small-molecule heterocyclic fur- interfering one of the QS circuitries (las, rhl or pqs), preferably
anones.51 Of the several compounds tested, three compounds las. In summary, these preliminary results with 7 encouraged us
showed promising activity on LasIR.
to further evaluate it in the anti-virulence assays.
As seen from Fig. 3(iii)(a), it was clearly evident that
To gain further insights into structural requirements for
compound 7, compared to 3–5 and the positive control AZ (65 ꢃ biolm inhibition, we studied literature reports detailing the
0.43%), showed remarkable inhibition (70 ꢃ 0.62%) of long- structure–activity relationship (SAR) around 2-methoxyphenols
chain AHL at the concentration tested, i.e., 4 mg mLꢁ1. The bearing 4-substituent [H (Guaiacol), ethyl (4-ethylguaiacol), allyl
observed activity, i.e., inhibition of long-chain AHL in P. aeru- (eugenol), 2-methylvinyl (isoeugenol) and propyl (2-methoxy-4-
ginosa, was indicative of the compound effect on the las QS propylphenol)] which can easily be compared with compound 7
circuitry. Further, the dose–response studies of compound 7 at (Fig. S2†). The report52 discussed the inhibition of biolm
4, 6 and 8 mg mLꢁ1 were carried out. The results precisely formation in one of the Gram-negative microorganisms, E. coli
indicated the dose-dependent increase in % inhibition as 69 ꢃ O157:H7 by essential oils (containing eugenol-like compounds).
0.52% (4 mg mLꢁ1), 72 ꢃ 0.68% (6 mg mLꢁ1) and 75 ꢃ 0.69% (8 It was found that the presence of double bond as part of 4-
mg mLꢁ1). Overall, the results were encouraging.
substituent, clearly imparted signicant biolm inhibitory
activity to the studied compounds. The order of activity with
respect to biolm inhibition was – eugenol > isoeugenol > 2-
methoxy-4-propylphenol > 4-ethylguaiacol > guaiacol. The
3.2. Evaluation of anti-biolm forming activity
During biolm formation, bacteria are covered in a matrix of hydrophobic substitution at position 4 on the 2-methoxyphenol
extracellular polymeric substances (EPS) that hold microbial led to increased activity. Further potentiation of activity was
cells together onto a surface. Biolm formation cycle of P. aer- observed when the substituent with unsaturation was used. The
uginosa PAO1 can be divided into ve major phenotypic steps. study53 provided important clues with respect to structural
The progression commences by the reversible adhesion of requirement for biolm inhibitory activity in Gram-negative
planktonic bacteria onto the surface suitable for growth (Stage microbes. The presence of C]C bond at position 4 on the
I), followed by irreversible attachment of bacteria, which aromatic ring, keeping other substituents constant, made the
thereaer form microcolonies in the EPS matrix (Stage II). major impact on biolm formation. Given the strict structural
Gradually, these microcolonies expand and their conuence requirements at position 4, it can be postulated that the 4-
leads to a more structured phenotype with noncolonized space substituent played an important role in modulating the biolm
(Stage III). Further, the noncolonized spaces are lled with inhibition possibly by interacting with appropriate target(s) in
bacteria, which nally cover the entire surface (Stage IV). the QS circuitry in the Gram-negative microbes.
Finally, the bacteria disperse from the sessile structure and re-
enter in the planktonic state to spread and colonize other
surfaces (Stage V).52
3.3. Anti-virulence assays
Compound 7 demonstrated statistically signicant (p <
3.3.1. Swarming activity. Pseudomonas aeruginosa is
0.001) dose-dependent increase in the anti-biolm forming capable of concerted and rapid movement across a semisolid
activity (as assessed by crystal violet staining) in 24 h treated or more precisely, viscous surfaces, i.e., swarming motility,
culture of Pseudomonas aeruginosa PAO1 (Fig. 4(i)) compared to signicantly affected by a lot of genes. It is typied by the
the positive control, AZ. The corresponding values were AZ (66 translocation of the bacterial population across growth media.
ꢃ 0.3%), 7 – 4 mg mLꢁ1 (76 ꢃ 0.24%), 6 mg mLꢁ1 (79 ꢃ 0.28%) In this assay, the test compound, in specied concentrations,
and 8 mg mLꢁ1 (82 ꢃ 0.2%). The results were in line with earlier were added to the media and the bacterial culture was point-
anti-QS activity outcome (Fig. 3). The spinning-disc confocal inoculated. Swarming motility (measured in mm) was
microscopy studies corroborated the results from anti-QS and observed on the semisolid agar plate, as seen in Fig. 5. The
anti-biolm forming activity studies (Fig. 4(ii)). As precisely Pseudomonas aeruginosa PAO1 cells, in the absence of the test
seen in Fig. 4(ii)(A–C), the number of dead cells (red stained) compound, formed tendrils migrating outwards or away from
increased while the viable cell (green stained) number the inoculation point, with continued branching during this
decreased in dose-dependent manner compared to the process. In contrast, in presence of the test compound,
untreated control (Fig. 4(ii)(D)), which was attributed to the bacterial cells grew to form a localized colony in the center of
increased cell-killing (in the absence of antibacterial activity of the plate, with no signs of swarming. The ability of several
7) due to biolm inhibition. Azithromycin, at the concentration synthetic compounds to disrupt the QS-regulated swarming
tested, also showed inhibition of biolm formation, albeit to motility in P. aeruginosa was demonstrated previously.54 Thus,
lesser extent as assessed qualitatively from Fig. 4(ii)(E). The the manifestation of anti-QS property in the form of inhibition
higher number of red stained (dead) cells signies a greater of swarming motility in P. aeruginosa further unequivocally
number of static cells present onto the surface, which in turn, supported the proposed quorum quenching property of 7 in
denote the reduced biolm formation.
dose-dependent manner.
In consonance with our previous report,40 where we postu-
3.3.2. Inhibition of LasA protease and LasB elastase. LasA
lated the hits (compound 6) would bind to LasR as supported by protease is a staphylolytic endoprotease enzyme regulated by
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Fig. 4 (i) Dose-dependent anti-biofilm forming activity of 7 in 24 h-treated culture of Pseudomonas aeruginosa PAO1 (n ¼ 9). The asterisk above
bars indicate statistically significant difference compared to AZ (4 mg mLꢁ1) based on Dunnett's multiple comparison test (*** ¼ p < 0.001). (ii)
Spinning-disc confocal laser microscopy micrographs of 24 h old P. aeruginosa PAO1 biofilms treated with various concentrations of 7. Bacterial
viability was determined using SYTO-9 and propidium iodide dyes. Exposure to (A) 4 mg mLꢁ1, (B) 6 mg mLꢁ1, (C) 8 mg mLꢁ1, (D) no exposure, (E)
AZ (4 mg mLꢁ1). Cells stained red are dead while cells stained green are viable cells. The scale bar represents 20 mm.
LasIR circuit in P. aeruginosa. During the assay, S. aureus Compound 7 (4 mg mLꢁ1) and AZ (4 mg mLꢁ1) inhibited LasA
suspension was used as a substrate, wherein the enzymatic protease enzyme signicantly, i.e., 75 ꢃ 0.8% and 82.5 ꢃ 0.6%,
activity was measured spectrophotometrically at 600 nm. respectively (Fig. 6(i)). Further, the dose–response study
Fig. 5 Inhibition of swarming activity of P. aeruginosa by compound 7 in dose-dependent manner – (A) 4 mg mLꢁ1 (16.5 ꢃ 1 mm), (B) 6 mg mLꢁ1
(15 ꢃ 1.3 mm), (C) 8 mg mLꢁ1 (14 ꢃ 1 mm) along with (D) negative control (50% DMSO) (20.5 ꢃ 1.2 mm) and (E) untreated P. aeruginosa (21.5 ꢃ 1
mm).
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Fig. 6 Dose-dependent activity of compound 7 on virulence factors – (i) LasA protease; (ii) LasB elastase and (iii) pyocyanin in P. aeruginosa
PAO1 (n ¼ 3). The asterisk above bars indicate statistically significant difference compared to AZ using Dunnett's multiple comparison test (*** ¼
p < 0.001, ** ¼ p < 0.05).
involving 7 exhibited marginal increase in LasA protease %
inhibition from 4 mg mLꢁ1 to 8 mg mLꢁ1 (Fig. 6(i)). On the
similar lines, LasB elastase enzyme degrades elastin and
collagen proteins in the lung tissues. For the evaluation of
extent of LasB elastase inhibition by the test compounds,
elastin Congo red (ECR) was employed as a substrate, where
LasB degraded the elastin and Congo red was liberated. The red
color produced by the liberated congo red was measured spec-
trophotometrically at 495 nm. Compound 7 (4 mg mLꢁ1) and AZ
(4 mg mLꢁ1) signicantly inhibited LasB elastase enzyme (65.5 ꢃ
0.6% and 69.5 ꢃ 0.87%, respectively) (Fig. 6(ii)). Slight increase
in the LasB elastase % inhibition was observed with increasing
doses (4 to 8 mg mLꢁ1) (Fig. 6(ii)). Protease and elastase
(regulated by the las circuitry) play vital roles in colonizing host
tissues.
3.3.3. Pyocyanin assay. Pyocyanin, a blue-green pigment
produced by P. aeruginosa, is regulated by rhl QS circuitry and is
implicated in both acute and chronic infections causing
multiple devastating effects majorly by reactive oxygen species
(ROS) generation. King's B medium is used specically for
pyocyanin pigment production. Previously, O'Loughlin et al.
demonstrated that m-bromothiolactone (mBTL) signicantly
inhibited pyocyanin pigmentation in P. aeruginosa in vitro and
in vivo.56 Test compound 7 (4 mg mLꢁ1, 68.5 ꢃ 0.56%) and AZ (4
mg mLꢁ1, 71 ꢃ 0.61%) signicantly inhibited pyocyanin
pigment production (Fig. 6(iii)). Further, marginal increase in
%
inhibition of pyocyanin production was noted with
increasing dose (4, 6 and 8 mg mLꢁ1) of 7, indicating Pseudo-
monas aeruginosa virulence inhibition.
3.3.4. Twitching motility. Twitching motility in P. aerugi-
nosa is a agellum-independent mode of surface translocation
under humid conditions on semisolid/solid surfaces, which
requires functional type IV pili (T4P). Twitching motility is
necessary for P. aeruginosa biolm development.57 It is the rst
step towards biolm formation wherein the twitching motility
Hentzer et al. synthesized halogenated furanones (produced
by Australian red macro-alga Delisea pulchra) and checked anti-
quorum sensing and anti-virulence activity in P. aeruginosa. As
a result of that study, furanones showed inhibition in quorum
sensing and virulence factors like LasA protease and elastase.55
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Fig. 7 Twitching motility exhibited by compound 7 following treatment with (A) 4 mg mLꢁ1 (24.5 ꢃ 1.5 mm); (B) 6 mg mLꢁ1 (22 ꢃ 1 mm); (C) 8 mg
mLꢁ1 (17 ꢃ 1.5 mm); (D) AZ (positive control, 23.5 ꢃ 1 mm) and (E) P. aeruginosa PAO1 (30.5 ꢃ 1.5 mm).
aids bacterial attachment to the solid surfaces forming the hits, compound 7 was a clear winner. It exhibited signicant
biolm. Thus, this phenomenon is so clearly associated with anti-QS, anti-biolm forming and anti-virulence activities in
high cell densities and cell–cell communication, which are the relevant assays in dose-dependent manner. Compound 7 also
hallmarks of QS systems in bacteria.58 In the macroscopic down regulated all four genes responsible for quorum sensing
twitching assay, Pseudomonas aeruginosa culture is stabbed in P. aeruginosa in dose dependent manner. The activities were
through a layer of agar to the base of the plate, usually plastic, comparable or even better than the positive control AZ at the
and the large halo of interstitial colony expansion is observed in doses tested. Given the tiny structure of 7, there is so much
the plate. As seen in Fig. 7, compound 7, with increasing scope for further structural modications and functional
concentration (4, 6 and 8 mg mLꢁ1), reduced the twitching studies centered on it. These results were in line with other
motility in Pseudomonas aeruginosa, which was comparable to reports with respect to the structural requirements for biolm
the positive control AZ (Fig. 7(B) and (D)) or even better than AZ inhibitory activity by 4-substituted 2-methoxyphenols. The
(Fig. 7(C) and (D)). It was clearly evident from Fig. 7 that 7 discovery of hit 7 is just the beginning of the story. The results
signicantly reduced the twitching motility in Pseudomonas reported in the present study are likely to motivate researchers
aeruginosa in dose-dependent manner.
in the eld to further explore it.
3.4. Gene regulation
Funding
Studies on down regulation of LasIR and RhlIR circuitry have
been done using real time PCR. Phenotypic assays showed
inhibition of biolm and virulence factors in P. aeruginosa. To
conrm the same at genetic level, real time PCR was performed
and observed signicant down regulation (in terms of fold
change) of all four genes viz lasI, rhlI, lasR and rhlR as concen-
tration of compound 7 increase (2 mg mLꢁ1, 4 mg mLꢁ1, 6 mg
mLꢁ1 and 8 mg mLꢁ1) in the presence of housekeeping gene
(rpsL) (Fig. S3†). Result conrms the inhibition of both circuitry
LasIR and RhlIR in P. aeruginosa PAO1. In fact, down regulation
of these genes conrms the inhibition of biolm and other
virulence factors. The lasI–lasR and rhlI–rhlR quorum sensing
systems regulate expression of various virulence genes and play
an important role in biolm formation. Luo et al. reported that
baicalin (5,6,7-trihydroxyavone, avonoid monomer puried
from Scutellaria baicalensis) signicantly repressed all four gene
in Pseudomonas aeruginosa strain.59 Similar study was conduct-
ed by Hossain et al. where methyl gallate (phenolic compound)
signicantly down regulated all four genes.60
This research did not receive any specic grant from funding
agencies in the public, commercial, or not-for-prot sectors.
Conflicts of interest
We do not have any conicts of interest to declare.
Acknowledgements
The authors deeply acknowledge Sophisticated Analytical
Instrument Facility (SAIF), Indian Institute of Technology
Bombay (IIT-B), Mumbai, India for confocal analysis. MS thanks
SVKM's NMIMS (Deemed to be University), Mumbai, India for
providing the necessary facilities. We also thanks Mr Mitesh
Joshi, SVKM's NMIMS, Sunandan Divatia School of Science for
his help with the artwork.
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