Unravelling the structural and molecular basis responsible for the anti-biofilm activity of zosteric acid

Article abstract of DOI:10.1371/journal.pone.0131519

The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to generate more potent anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to understand the structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Considerations concerning the relationship between structure and anti-biofilm activity revealed that i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the molecule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and, consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instrumental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down approach, combined with mass spectrometry, the herein-defined active structure of ZA was matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase, WrbA, suggesting a possible role of this protein in the biofilm formation process.

Full text of DOI:10.1371/journal.pone.0131519

RESEARCH ARTICLE
Unravelling the Structural and Molecular
Basis Responsible for the Anti-Biofilm Activity
of Zosteric Acid
Cristina Cattò1, Silvia Dell’Orto2, Federica Villa1,3, Stefania Villa2, Arianna Gelain2,
Alberto Vitali4, Valeria Marzano5, Sara Baroni1¤, Fabio Forlani1*, Francesca Cappitelli1
1 Department of Food Environmental and Nutritional Sciences, Università degli Studi di Milano, Milano, Italy,
2 Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milano, Italy, 3 Center for Biofilm
Engineering, Montana State University, Bozeman, MT, United States of America, 4 Institute of Chemistry of
Molecular Recognition-UOS Rome, CNR, Roma, Italy, 5 Institute of Biochemistry and Clinical Biochemistry,
Catholic University of Rome, Roma, Italy
¤ Current address: Start Up Unit, Department of Experimental Oncology and Molecular Medicine,
Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
* fabio.forlani@unimi.it
a11111
Abstract
The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an
alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches.
Despite its potential, the lack of information concerning the structural and molecular mecha-
nism of action involved in its anti-biofilm activity has limited efforts to generate more potent
anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA
scaffold diversity was designed and screened against Escherichia coli to understand the
structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Consid-
erations concerning the relationship between structure and anti-biofilm activity revealed that
i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the mole-
cule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the
anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and,
consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instru-
mental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down
approach, combined with mass spectrometry, the herein-defined active structure of ZA was
matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase,
WrbA, suggesting a possible role of this protein in the biofilm formation process.
OPEN ACCESS
Citation: Cattò C, Dell’Orto S, Villa F, Villa S, Gelain
A, Vitali A, et al. (2015) Unravelling the Structural and
Molecular Basis Responsible for the Anti-Biofilm
Activity of Zosteric Acid. PLoS ONE 10(7): e0131519.
doi:10.1371/journal.pone.0131519
Editor: Alessio Lodola, University of Parma, ITALY
Received: March 24, 2015
Accepted: June 3, 2015
Published: July 1, 2015
Copyright: © 2015 Cattò et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by Fondazione
Cariplo, grant n. 2011-0277 (FC). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Introduction
Direct observation of a wide variety of natural habitats has established that 99% of microorgan-
isms grow in the form of a biofilm, a complex of structured microorganism communities that
aggregate on surfaces and that are embedded in a self-produced extracellular polymeric sub-
stance [1]. It is reported that biofilms are able to colonize any surface that offers minimal
Competing Interests: The authors have declared
that no competing interests exist.
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Structural and Molecular Basis of Zosteric Acid Activity
conditions for life, including all human artefact surfaces such as industrial installations, work
benches, water distribution systems, and also medical devices, with devastating consequences
in terms of social and economic impacts [1]. In the clinical area, especially, the role of biofilm
in the contamination of medical implants is well established; unfortunately, such implants are
associated with a high risk of biofilm infection and are major contributors to the morbidity and
mortality found among hospitalized patients [2, 3].
The most detrimental property of biofilms is that conventional antimicrobial practices have
proven inadequate since, by adopting the sessile mode of life, microorganisms can improve
their resistance to antimicrobial agents up to several orders of magnitude [4–6]. In addition,
the long term and intensive use of antibiotics and biocides has dramatically supported the
development of resistant microbial strains, reducing the possibility of treating biofilm effec-
tively [7]. Increasingly restrictive regulations limiting the use of substances hazardous to
human health and the environment have also resulted in several biocides being banned [8–10].
With this perspective, the development of new improved therapeutic solutions able to
reduce the incidence of biofilm-associated contamination has become imperative. An innova-
tive approach could be the use of biocide-free anti-biofilm agents with novel targets, unique
modes of action and properties that are different from those of currently used antimicrobials
[11]. The use of sublethal doses of bio-inspired molecules able to interfere with specific key
steps involved in biofilm formation could be an interesting strategy to defeat biofilm formation
[12–14]. The use of such strategies might also apply a milder evolutionary pressure in the
development of resistance as most virulence traits are not essential for bacterial survival [15].
Zosteric acid or p-(sulfoxy) cinnamic acid (ZA), a secondary metabolite produced by the
seagrass Zostera marina, might be suitable for implementation as a preventive or integrative
approach against biofilm formation. At sublethal concentrations, ZA is able to reduce both bac-
terial and fungal adhesion, and plays a pivotal role in shaping biofilm architecture. This can be
seen through the reduction of biofilm biomass and thickness, and the thwarting of budded-to-
hyphal-form transition. Additionally, ZA extends the performance of antimicrobial agents,
and shows cytocompatibility towards soft and hard tissue [16–18]. A comparative proteomic
study on E. coli cells exposed to ZA has shown that, in bacterial cells, ZA acts as an environ-
mental cue to global stress, promoting the expression of various protective proteins, the pro-
duction of the signal molecule autoinducer-2, and the synthesis of flagella to escape from
adverse conditions [19].
Despite such promising performance there are currently two main bottlenecks limiting the
exploitation of ZA as a preventive strategy to counteract the formation of biofilm on abiotic
surfaces: i) the structural elements required for anti-biofilm activity remain unclear and ii) the
cellular targets for ZA are still unexplored.
In this follow-up study, a 43-member library of small molecules based on ZA-scaffold diver-
sity was designed and screened against E. coli, used as the model bacterial biofilm system.
Thus, the aim of this study was to identify important structural determinants for the ZA anti-
biofilm activity, and to eventually develop ZA analogues with improved anti-biofilm activity.
In order to functionally exploit the information arising from the molecule library analysis, the
resulting active structure of the ZA-scaffold was matrix-immobilized and its ability to interact
with a cell component was evaluated. On the assumption that the primary events triggering the
processes related to the anti-biofilm activity of ZA rely on ZA’s interaction with a protein, a
protein pull-down approach was developed. The identification of the cellular target for ZA is
thus a crucial step to an understanding of the mode of action, and for the structure-led design
and improvement of any potential therapeutic compound and material. Our work provides not
only the first identification of the cellular target for the active structure of ZA, but also
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 1. Structural formula of zosteric acid (ZA) and cinnamic acid (4).
doi:10.1371/journal.pone.0131519.g001
convincing evidence that its mode of action is likely to result from a synergistic effect arising
from a perturbation of oxidative homeostasis.
Materials and Methods
Zosteric acid related analogues
The compounds used in this study are listed in Figs 1 and 2. Some compounds were purchased
from Sigma-Aldrich, others were prepared following slightly modified literature procedures (S1
Protocol). All the reagents, including solvents, were purchased from Sigma-Aldrich, and were
used without any further purification. Reactions involving air-sensitive reagents were performed
under nitrogen atmosphere and anhydrous solvents were used when necessary. The Biotage-Ini-
tiator microwave synthesizer was used. Reactions were monitored by Thin Layer Chromatogra-
phy (TLC) analysis on aluminium-backed Silica Gel 60 plates (0.2 mm, Merck), and were
visualized under a UV lamp operating at wavelengths of 254 and 365 nm. Visualization was
aided by opportune staining reagents. Intermediates and final compounds were purified by flash
chromatography using Merck Silica Gel 60 (70–230 mesh). The purity of the final compounds
was determined by High Performance Liquid Chromatography (HPLC) analysis and
was > 95%. 1H and 13C Nuclear Magnetic Resonance (NMR) spectra were recorded at room
temperature on a Varian 300 MHz Oxford instrument. CDCl3, CD3OD, D2O, acetone-d6 and
DMSO-d6 were used as deuterated solvents for all spectra runs. Chemical shifts are expressed
in ppm from tetramethylsilane resonance in the indicated solvent (TMS: 0.0 ppm), and coupling
constants (J-values) are given in Hertz (Hz). 1H NMR data are reported in the following
order: ppm, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), and
number of protons. Melting points and NMR data are consistent with literature data.
Synthesis of cinnamic acid derivatives
Zosteric acid was synthesized as already described in a previous work [16]. The cis isomer 38
was obtained starting from cis 4-hydroxycinnamic acid 32 under microwave irradiation in the
presence of sulfur trioxide pyridine complex in acetonitrile (Fig 3). The final product was iso-
lated as sodium salt. Most of the substituted cinnamic acid derivatives were prepared in high
yield (> 90%) by the Knoevenagel-Doebner procedure. In detail, compounds 2, 7, 8, 11 and 17
were obtained through a one-pot reaction between the suitable substituted benzaldehyde and
malonic acid in refluxing pyridine to induce decarboxylation (Fig 4) [20]. The trans geometries
of the ethenyl π-bonds were confirmed by proton-proton coupling constants. Cis cinnamic
acid 37 was synthesized from the commercially available ethyl phenylpropiolate. The subse-
quent hydrogenation of alkyne in the presence of the Lindlar catalyst and pyridine in methanol
led to the corresponding cis-alkene 36. Then the ester group was hydrolyzed under alkaline
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 2. Structures of cinnamic acid analogues used in the present study.
doi:10.1371/journal.pone.0131519.g002
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 3. Synthetic scheme of compounds 23, 24, 33, 34, 35, 38. Reagents and conditions: i) from 1 for 23, from 5 for 24, from 32 for 33: H2SO4, MeOH,
reflux, 1 h; ii) from 32: Py Á SO3, DMF dry, 120°C, 25 min; iii) CH3I, anhydrous K2CO3, dry DMF, reflux, 1.5 h; iv) 1N NaOH, EtOH.
doi:10.1371/journal.pone.0131519.g003
conditions to provide the final compound 37 (Fig 5). Esters of cinnamic acid in cis (33) or in
trans configuration (23, 24) were prepared by Fischer esterification of the carboxylic group
[21]. The protection of the hydroxyl group as methyl ether in the presence of iodomethane in
dry N,N-dimethylformamide provided the compounds 35. The hydrolysis of the ester was per-
formed in alkaline conditions to obtain compound 34 (Fig 3).
Escherichia coli strain and growth condition
The well characterized Escherichia coli K-12 wild-type strain (ATCC 25404) was used as a
model system for the bacterial biofilms. The strain was stored at –80°C in suspensions contain-
ing 20% glycerol and 2% peptone, and was routinely grown in Luria-Bertani broth (LB, Sigma-
Aldrich) at 30°C for 16 h.
Planktonic growth in the presence of ZA-related compounds
Planktonic growth of E. coli in LB medium supplemented with 0 (negative control), 0.183, 1.83,
18.3, 183 and 1830 μM of the synthesized molecules and 3% of DMSO were carried out in
384-well microtiter plates. Growth curves at 30°C were generated using the PowerWave XS2
microplate reader (Biotek). Growth was followed by measuring the absorbance at 600 nm
(A600) every 10 min for over 24 h in wells inoculated with 3 μl (3% vol/vol) of an overnight cul-
ture (final concentration 107 cells/mL). An absorbance-based growth kinetics was constructed
by plotting the A600 of suspensions minus the A600 of the non-inoculated medium against incu-
bation time. The polynomial Gompertz model [22] was used to fit the growth curves to calcu-
late the maximum specific growth rate (A600/min), using GraphPad Prism software (version
5.0, San Diego, CA, USA). Six biological replicates of each treatment were performed. The
obtained data were normalized to the negative control and reported as the mean of these. The
percentage reduction, compared to the control, was also calculated. Molecules able to reduce
the maximum specific growth rate of E. coli planktonic cells with respect to the negative control
were coded as follows: less than 10% as 0, between 10% and 20% as—1, between 20% and 30%
as—2, and more than 30% as—3.
The ability of bacteria to grow with each compound as the sole carbon and energy source
was also tested using a mineral medium (KH2PO4 30 g/L, Na2HPO4 70 g/L, NH4Cl 10 g/L, pH
7) supplemented with 1830 μM of each synthesized molecule and 3% of DMSO. Bacteria were
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 4. Synthetic scheme of compounds 2, 7, 8, 11, 17. Reagents and conditions: i) malonic acid, Py, piperidine, reflux, 80–180 min.
doi:10.1371/journal.pone.0131519.g004
added to a final concentration of 107 cells/mL and grown at 30°C for 72 h. The positive control
was the mineral medium supplemented with glucose at both 1830 μM and 10000 μM concen-
tration. Microbial growth was followed by measuring A600. All the experiments were repeated
three times.
Biofilm assay in the presence of ZA-related compounds
The effects of ZA-related compounds on cell adhesion were assessed quantitatively using fluo-
rochrome-labeled cells in hydrophobic 96-well black sided plates as previously reported by
Villa et al. [16]. Briefly, 200 μL of phosphate buffered saline (PBS, 0.01 M phosphate buffer,
0.0027 M potassium chloride pH 7.4, Sigma-Aldrich) containing 107 cells supplemented with 0
(negative control), 0.183, 1.83, 18.3, 183 and 1830 μM of each synthesized molecule and 3% of
DMSO were placed in microtiter plate wells. Cells were incubated 18 h at 30°C. The microtiter
plate wells were washed twice with 200 μL PBS, and adhered cells were stained using 10 μg/mL
4,6-diamidino-2-phenylindole (Sigma-Aldrich) in PBS for 20 min in the dark at room temper-
ature. Fluorescence intensity was measured using the fluorometer VICTOR X Multilabel Plate
Readers (Perkin Elmer) at excitation wavelength of 335 nm and emission wavelength of 433
nm. A standard curve of fluorescence intensity versus cell number was determined and used to
quantify the anti-biofilm performance of the ZA-related compounds. Six biological replicates
of each condition were performed. The experiment was repeated four times. Obtained data
were normalized to the negative control and reported as the mean of these data. Percentage
reduction in comparison to the control was also calculated. Molecules able to reduce the num-
ber of E. coli adhered cells by less than 20% with respect to the negative control were coded 0,
Fig 5. Synthetic scheme of compounds 36, 37. Reagents and conditions: i) Lindlar catalyst, H2, pyridine, MeOH, r.t., 16 h; ii) 1N NaOH, EtOH/THF (1:1), r.
t., 12 h.
doi:10.1371/journal.pone.0131519.g005
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Structural and Molecular Basis of Zosteric Acid Activity
between 20% and 30% as—1, between 30% and 40% as—2 and more than 40% as—3. Mole-
cules able to increase the number of adhered cells by more than 20% with respect to the nega-
tive control were coded + 1.
Statistical analysis
Analysis of variance (ANOVA) via a software run in MATLAB (Version 7.0, The MathWorks
Inc., Natick, USA) was applied to statistically evaluate any significant differences among the
samples. The ANOVA analysis was carried out after verifying whether the data satisfied the
assumptions of i) independence, ii) normal distribution and iii) homogeneity of variances.
Tukey’s honestly significant different test (HSD) was used for pairwise comparison to deter-
mine the significance of the data. Differences were considered significant for p < 0.05.
E. coli protein extraction
An E. coli culture was prepared as reported in the section ‘Escherichia coli strain and growth
condition’. The E. coli culture was centrifuged at 5000 g for 15 min at 4°C and collected cells
(~ 2.5 g, wet weight) were washed 3 times with PBS and resuspended in 200 mM Tris-HCl,
40 mM NaCl, pH 7.5 (protein extraction buffer) to a final concentration of 250 mg cells/mL.
Soluble protein extract was obtained by sonication (seven 1-min sonication cycles at 22 μm
amplitude followed by 2-min cooling periods, all on ice-bath, in Soniprep 150) and cell debris
was removed by centrifugation at 10000 g for 45 min at 4°C.
Matrix immobilization of p-amino cinnamic acid
Five milliliters of drained NHS-Activated Sepharose 4 Fast Flow (GE Healthcare Life Sciences)
were washed 10 times with 15 mL of cold 1 mM HCl and then suspended in 2.5 mL of 0.2 M
NaHCO3, 0.5 M NaCl, 3% DMSO, pH 8.3 (coupling buffer), containing 0.4 M p-amino cin-
namic acid (p-ACA). The suspension was mixed in a rotary shaker overnight at 4°C. After
removal of the coupling mixture, the remaining unreacted groups were blocked with 0.5 M eth-
anolamine, 0.5 M NaCl, pH 8.3 (5 mL) and 10 subsequent washing steps were performed
according to the manufacturer's instructions. The matrix (p-ACA/matrix) was stored in 20%
ethanol (3 mL) at 4°C. To generate the control matrix (EA/matrix), p-ACA was omitted in the
coupling mixture.
Fluorescence analysis
Fluorescence measurements were carried out in a Perkin-Elmer LS 50B spectrofluorometer
equipped with PTP-1 Fluorescence Peltier System. Excitation and emission spectra (250–600
nm) were recorded at 50 nm/min, with both emission and excitation slit widths set at 3.0 nm,
and at 20°C. Fluorescence spectra of p-ACA free in solution were recorded by dissolving differ-
ent concentrations (0.2–5.0 mM) of p-ACA in 0.4 M NaHCO3, 1 M NaCl (pH 8.3). Fluores-
cence spectra of p-ACA/matrix and EA/matrix suspensions were recorded under continuous
magnetic stirring.
Matrix hydrolysis and analysis of hydrolysates
One mL (drained volume) of the matrix samples was hydrolyzed by treatment with 4 M
NaOH aqueous solution (1 mL). The suspensions were stirred 4 h at room temperature.
Reactions were monitored by thin layer chromatography (TLC) analysis on aluminium-
backed Silica Gel 60 plates (0.2 mm, Merck) and were visualized under UV lamp (λ = 254
nm). Visualization was aided by opportune staining reagents. The matrix was filtered off and
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Structural and Molecular Basis of Zosteric Acid Activity
washed. The aqueous layer was acidified to pH 1 with 2 M HCl and then extracted with ethyl
acetate (3 x 1 mL). The combined organic layers were dried over anhydrous sodium sulfate,
residual solvent was evaporated under reduced pressure and submitted to mass spectrometry
analysis.
Matrix hydrolysates were resuspended in 100% methanol, further diluted with 0.1% formic
acid in water and analysed by liquid chromatography-electrospray ionization-mass spectrome-
try (LC-ESI-MS) on an Ultimate 3000 Micro HPLC apparatus (Dionex, Sunnyvale, CA, USA)
equipped with a FLM-3000-Flow manager module directly coupled to a LTQ Orbitrap XL
hybrid FT mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Reverse-phase
chromatography was performed on a Jupiter C18, 5 μm, 150 x 1.0 mm column (Phenomenex,
Torrance, CA, USA) and a 31 min run (gradient 0 to 40% acetonitrile in water with 0.1% for-
mic acid over 20 min) at a flow rate of 80 μL/min. Mass spectra were collected at 60000 resolu-
tion in the Orbitrap analyser (mass range 50–1000 m/z) in positive ion mode. High resolution
MS data were elaborated manually using the Xcalibur Qual Browser software (version 2.2,
Thermo Fisher Scientific).
Protein pull-down
Functionalized matrix (5 mL, drained volume) was washed once with 5 mL water and then
with 5 mL of protein extraction buffer. To ease the removal of the liquid phase by matrix
decanting, centrifugation at 700 g for 2 min at room temperature was applied after each wash-
ing step. The washed matrix was incubated with freshly-prepared soluble protein extract (*
9.6 mL; * 90 mg protein) for 2.5 h at room temperature in a rotary shaker. The incubation
mixture was then packed into a 15 mL chromatography column (i. d. 10 mm) and washed with
protein extraction buffer until absorbance at 280 nm was null (* 100 mL), at a flow rate of 1.5
mL/min to remove unbound proteins. Proteins specifically bound to the functionalized matrix
were recovered by competitive elution with 60 mM competitor (15 mL; sodium cinnamate or
sodium zosterate) in protein extraction buffer, followed by 50 mL of 200 mM Tris-HCl, 1 M
NaCl, pH 7.5. A compact single beam UV-detector (Uvicord SII, 280 nm filter; GE Healthcare)
was employed for flow-through monitoring and fractions were collected during the course of
the chromatography. To increase the protein concentration, the fraction aliquots were precipi-
tated using cold 13% trichloroacetic acid (TCA), centrifuged at 13000 g for 30 min at 4°C and
subsequently washed with cold acetone.
Other procedures
Protein concentration was determined using the Bradford method, using bovine serum albu-
min as standard [23]. Electrophoretic analysis (SDS-PAGE) was performed under denaturing
and reducing conditions according to Laemmli [24]. TCA-precipitated proteins were dissolved
in Laemmli sample buffer and submitted to thermal denaturation before SDS-PAGE. Coomas-
sie-stained bands were manually excised from gels and subjected to mass spectrometry
analysis.
Mass spectrometry analysis and protein identification
Excised bands and spots were subjected to trypsin digestion as previously described [25] and
the trypsin digested peptides were analyzed by liquid chromatography-electrospray ionization-
tandem mass spectrometry (LC-ESI-MS/MS) using the same HPLC and LTQ Orbitrap mass
spectrometer previously reported in “Matrix hydrolysis and analysis of hydrolysates”. Reverse-
phase chromatography was performed on the same column and at the same flow rate but
under different chromatographic conditions: linear gradient 1.6 to 44% acetonitrile in water
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Structural and Molecular Basis of Zosteric Acid Activity
with 0.1% formic acid over 40 min and total LC-run of 61 min. Mass spectra were collected in
positive ion and data dependent scan mode (MS scan at 60000 of resolution in the Orbitrap,
mass range 300–2000 m/z, and MS/MS scan on the three most intense peaks in the linear ion
trap). Selected peptide charge states were isolated with a width of m/z 2, and were activated for
30 msec using 35% normalized collision energy and an activation q of 0.25. Protein identifica-
tion was achieved using the embedded ion accounting algorithm (Sequest HT) of the software
Proteome Discoverer (version 1.4, Thermo Fisher Scientific) after searching a UniProtKB/
Swiss-Prot Protein Knowledgebase [release 2013_12 of 11-Dec-13; taxonomical restriction:
Escherichia coli (strain K12), 4431 sequence entries]. The search parameters were 10 ppm toler-
ance for precursor ions and 0.6 Da for product ions, 1 missed cleavage, carbamydomethylation
of cysteine as fixed modification, the oxidation of methionine as variable modification and on a
decoy database search calculated false discovery rate under 5%.
Results and Discussion
Chemical perspective of ZA mode of action
The main goal of this study was to identify structural determinants for the anti-biofilm activity
of ZA, and highlight features that could be useful in the design of new derivatives endowed
with better anti-biofilm activity than ZA.
Therefore, a 43-member library of small molecules based on the ZA-scaffold was designed
and screened against Escherichia coli. Compounds were characterized by introduction of sub-
stituents at different positions on the phenyl ring and making several side-chain modifica-
tions, such as removal of unsaturation and substitution of the carboxylic acid with an alcohol,
an aldehyde and ester functionalities. Moreover, both E/Z isomers were prepared in order to
explore the role of the double bond. Of all the concentrations tested (0.183, 1.83, 18.3, 183,
1830 μM), the concentration at which ZA performed its best anti-biofilm activity was
1830 μM [16].
Before evaluating the anti-biofilm activity of ZA derivatives, we investigated both their abil-
ity to act as a carbon and energy source and their potential impact on the planktonic growth of
the model bacterium E. coli. To dissolve the molecules in the aqueous medium it was necessary
to use DMSO, so its possible impact on E. coli planktonic growth and cell adhesion was
assessed. It was found that 3% DMSO successfully dissolved the molecules in the media, and
had no effect on either planktonic growth or cell adhesion.
In the first steps of the study the ability of E. coli to grow with 43 ZA-related compounds at
1830 μM as the sole carbon and energy source was tested and compared with a positive control
using glucose at the same concentration. The E. coli in the positive control did not grow, and
showed an A600 lower than 0.05 after 48 h incubation. Indeed, the minimum glucose concen-
tration in a mineral medium that supported E. coli growth was 10 mM (A600 = 0.201 0.007).
Consequently, E. coli with glucose 10 mM was taken to be the positive control. E. coli did not
grow in the presence of each of the ZA-related compounds as the sole carbon and energy
source, and showed an A600 lower than 0.05 after 48 h incubation with each molecule.
The ability of the 43 ZA-related compounds to affect E. coli planktonic growth was also eval-
uated. The E. coli maximum specific growth rates in the presence of each ZA-related com-
pound at 0.183, 1.83, 18.3, 183 and 1830 μM were calculated and compared to a negative
control grown without the molecule, using the ANOVA statistic test (S1 Table).
On the basis of these results, all the derivatives were evaluated for their ability to affect sur-
face cell adhesion. Thus the number of adhered cells in the presence of each ZA-related com-
pound, at 0.183, 1.83, 18.3, 183 and 1830 μM was assessed quantitatively using fluorochrome-
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Structural and Molecular Basis of Zosteric Acid Activity
labelled cells, and compared to a negative control in the absence of the molecule, using the
ANOVA statistic test (S1 Table).
With the aim of identifying trends, planktonic growth and cell adhesion data were grouped
to obtain a global picture of the biological activity of each ZA-related compound at the different
concentrations (Fig 6). The compounds showed: i) no biological activity (no reduction of the
number of adhered cells and no planktonic growth effect), ii) anti-biofilm activity (reduction of
the number of adhered cells and no planktonic growth effect), iii) increased biofilm formation
(increase in the number of adhered cells and no planktonic growth effect), and iv) biocidal
effect (in contrast to the above trends, planktonic growth was inhibited while the number of
adhered cells either increased or decreased). Five molecules (7, 12, 18, 19, 39) induced no bio-
logical activity at low and middle concentrations, or biocidal effect at the maximum concentra-
tion (Trend 1). Nine molecules (24, 25, 27, 29–31, 40, 42, 43) promoted biofilm formation at
the lowest concentration but showed no biological activity or biocidal effect at middle and high
concentrations (Trend 2). Nineteen molecules (1–6, 8–11, 13–15, 20, 21, 23, 26, 28, 41) had
no biological activity at low concentration, showed an anti-biofilm activity at middle concen-
trations and induced biocidal effect at the maximum concentration (Trend 3). Eight molecules
(ZA, 16, 17, 22, 33–36) promoted biofilm formation at the lowest concentration, showed an
anti-biofilm activity at middle concentrations, and induced a biocidal effect at the maximum
concentration (Trend 4). Finally, molecules 32, 37 and 38 showed a peculiar trend (Trend 5):
molecule 32 showed anti-biofilm activity at the lowest concentration, no biological activity at
middle concentrations and biocidal effect at the highest concentration, molecule 37 promoted
biofilm formation at concentrations 0.183, 1.83 and 183 μM, inhibited biofilm formation at
18.3 μM and induced a biocidal effect at the highest concentration; molecule 38 promoted bio-
film formation at all tested concentrations except for the 183 μM, where there was no biological
activity.
For each ZA-related compound a global anti-biofilm performance value was also calculated
as (sum of cell adhesion codes of all concentrations) - (sum of planktonic growth codes of all
concentrations) (Fig 7). This value takes into account i) the occurrence that the ZA-related
compound induced a different bacterial response depending on the concentration, ii) the posi-
tive contribution of concentrations that reduce the number of adhered cells; iii) the negative
contribution of concentrations that enhances the number of adhered cells; iv) the negative con-
tribution of concentrations that reduces planktonic growth (the inhibition of planktonic
growth negatively affected the anti-biofilm performance). Molecules with an anti-biofilm per-
formance value i) equal to 0 were considered globally without anti-biofilm performance; ii)
below 0 were considered globally able to exert an anti-biofilm activity (= - 1: little anti-biofilm
performance; = - 2: moderate anti-biofilm performance; ꢀ - 3: optimal anti-biofilm perfor-
mance), iii) above 0 were considered globally able to improve biofilm performance (= +1: little
improvement of biofilm performance; = +2: moderate improvement of biofilm performance;
ꢁ+3: optimal improvement of biofilm performance). Four molecules (7, 12, 14, 32) presented
anti-biofilm performance equal to 0. Twenty molecules presented anti-biofilm performance
below 0 (= - 1: 16; = - 2: 5, 6, 13, 21, 23, 28; ꢀ - 3: ZA, 1–4, 8–11, 17, 20, 22, 41). Twenty mole-
cules presented anti-biofilm performance above 0 (= + 1: 19, 26, 31, 33, 36; = + 2: 15, 25, 27,
39, 42, 43; ꢁ + 3: 18, 24, 29, 30, 34, 35, 37, 38, 40).
The molecules grouped on the basis of both their biological activity and anti-biofilm perfor-
mance value allowed the recognition of some chemical structure elements required to exert
anti-biofilm activity.
Interestingly, as reported in the S1A Table, cinnamic acid 4, a natural compound obtained
by cinnamon lacking the sulfate monoester group, showed interesting anti-biofilm activity at a
1000-fold lower inhibitory concentration than ZA (0.183 vs 183 μM).
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 6. Biological activity of ZA-related compounds. Biological activity of each ZA-related compound at the
different tested concentrations. For an understanding of the meaning of each trend refer to the ‘Results and
discussion’ section. Yellow: no biological activity; Green: anti-biofilm activity; Blu: promotion of biofilm
formation; Red: biocidal effect.
doi:10.1371/journal.pone.0131519.g006
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 7. Global anti-biofilm performance of ZA-related compounds and structures with the most
significant anti-biofilm performance. Global anti-biofilm performance value of each ZA-related compound
calculated as (sum of cell adhesion codes of all concentrations)-(sum of planktonic growth codes of all
concentrations). Values equal to 0 were considered without anti-biofilm performance, below 0 were
considered globally able to exert an anti-biofilm activity, and above 0 were considered able to improve biofilm
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Structural and Molecular Basis of Zosteric Acid Activity
performance. Yellow: no anti-biofilm performance; Light orange: little anti-biofilm performance; Medium
orange: moderate anti-biofilm performace; Dark orange: optimal anti-biofilm performance; Light green: little
improvement of anti-biofilm performance; Medium green: moderate improvement of anti-biofilm performance;
Dark green: optimal improvement of anti-biofilm performance.
doi:10.1371/journal.pone.0131519.g007
The substitution pattern of the phenyl ring and the nature of the substituents were found to
have a significant effect on the biofilm inhibitory activity. In the first instance, a broad variety
of substituents with different electronic and steric properties were introduced at position 4 on
the phenyl ring. Compounds (1–3, 5, 6, 8–11) showed interesting activity (Trend 3) compared
to the unsubstituted cinnamic acid 4, meanwhile the presence of cyano (7) and nitro (12)
groups caused a loss in activity (Trend 1).
Comparing the derivatives bearing a hydroxyl group (1, 14), as well as the chlorine atom (9,
13), respectively in positions 4 and 3 on the phenyl ring, highlighted that the substitution at the
para position was preferred (1 and 9 more active than 14 and 13), although the same trend was
still present. On the contrary, the substitution at position 2 was not well tolerated. Indeed, as
for compound 17 the activity was reduced 10-fold compared to 9, while in the other cases the
activity was almost completely abolished. To determine whether the introduction of a second
substituent on the phenyl ring would enhance activity, a series of disubstituted derivatives (15,
16, 19, 20, 22) were synthesized. In general, each disubstituted compound was found to have
different behavior and lower anti-biofilm activity compared to the corresponding monosubsti-
tuted compound. This effect was most evident for the 3,4-dichlorocinnamic acid 15, whose
activity was 10-fold lower than the monosubstituted counterpart 9.
Following this, the side chain modifications were also explored. In general, the unsubsti-
tuted compounds having the carboxylic function protected as esters (26, 28) showed lower
activity in comparison to cinnamic acid 4, and particularly the ethyl ester 28 was less active
than methyl ester 26. Besides, among the para-substituted cinnamic acid esters (23–24), the
presence of a hydroxyl group (23), seemed to be important for the anti-biofilm activity as the
corresponding 4-methoxy derivative 24 was inactive. Moreover, derivatives bearing an alde-
hyde (29, 30) or an alcohol (31) to replace the carboxylic acid functionality were inactive
(Trend 2), confirming that the carboxylic moiety was essential for good anti-biofilm activity.
To investigate the importance of the configuration of the double bond, the majority of cis deriv-
atives (32–38) were synthesized and found to have different behavior (Trends 4, 5) compared
to the trans counterparts. Interestingly, exceptions to this rule were the cis isomer of ZA 38 and
cinnamic acid 37, which had lost their anti-biofilm activity (Trend 5). Additionally, specific
concentrations of 37 and 38 seemed to enhance the biofilm formation.
Similarly, some saturated derivatives (39–42) were chosen to investigate the role of the dou-
ble bond. All the compounds exhibited the same trend (Trend 2) except for compound 41, a
catechol derivative, that did not follow this general behaviour (Trend 3) probably due to a dif-
ferent mechanism of interaction (as compound 20).
Finally, coumarin 43, which could be considered the cyclic derivative of 4, was unable to
inhibit the biofilm growth, confirming the essential role of the side chain.
As for the anti-biofilm performance, some additional considerations could be drawn. Gen-
erally, ZA derivatives followed the same behaviors described for the anti-biofilm activity, with
following exceptions: i) derivatives 5 and 6, which have a methoxy group and a fluorine atom
in the para position on the phenyl ring respectively, showed an anti-biofilm performance
slightly lower than that of the cinnamic acid 4; ii) the anti-biofilm performance of compound 9
turned out to be similar to the corresponding analogue 17, in which the chlorine atom was at
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Structural and Molecular Basis of Zosteric Acid Activity
position 2 on the phenyl ring. However, the general trend, according to which the substitution
at position 2 was not well tolerated, was confirmed; iii) the anti-biofilm performance of the
disubstituted halogen derivatives (15, 16, 19) was in general lower than that of the monosubsti-
tuted counterparts as well as the anti-biofilm activity. The same was not observed for com-
pounds (20, 22), which were characterized by electron donating substituents, whose
monosubstituted analogues showed a decreased anti-biofilm performance; iv) the esters 26 and
28 showed the opposite behaviour, since the methyl ester 26 exhibited an anti-biofilm perfor-
mance lower than that of the ethyl ester 28; v) the majority of cis derivatives (32–38) were
found to have a global anti-biofilm performance promoting biofilm formation.
Our data clearly indicate that: i) the para-sulfoxy ester group is not necessary for exploiting
the anti-biofilm activity of the molecule, but rather the cinnamic acid scaffold is responsible for
the anti-biofilm performance (condition 1); ii) the anti-biofilm activity of ZA derivatives
depends upon the presence of a carboxylate anion and consequently by its hydrogen-donating
ability. Basically, the acidity (and thus the ability to donate hydrogen) of ZA derivatives
depends on the nature of electron-donating (EDG) and electron-withdrawing (EWG) groups
on the phenyl ring. EDG increases the charge density of the carboxylate anion and therefore
decreases acidity. Conversely, EWG withdraws negative charge from the carboxylate anion and
thus increases acidity (condition 2); iii) the conjugated aromatic system is instrumental for the
anti-biofilm activities of ZA and its analogues since the presence of the double bond stabilized
the carboxylate anion (condition 3).
As for condition 1, [26] hypothesized that the antifouling effect of ZA lies in the high affinity
for water shown by its sulfate group which increases the cell surface hydrophility. In contrast,
Villa et al. [16] demonstrated that the activity of methyl zosterate, in which the carboxylate
moiety was replaced by a methyl ester, had no anti-biofilm ability, suggesting that the activity
of ZA sodium salt is not due to the presence of the sulfate ester as confirmed by the present
study. It is likely that the sulfate ester makes the active cinnamic moiety more soluble and less
toxic. Phytochemical investigation revealed that in several seagrass species sulfotransferase
enzymes catalyze the sulfation of a wide range of compounds, including phenolic acid com-
pounds [27, 28]. Sulfoconjugation converts compounds into more water soluble metabolites
than a non-sulfated molecule. Thus it is more mobile in the water-based sap of the vascular
transport system during their translocation from local leaf to systemic leaves. As a conse-
quence, ZA facilitated excretion from the plant and a more rapidly local and systemic plant
response against the pathogen attack [29]. Moreover, sulfonated compounds are generally less
toxic to plant cells than non-sulfonated forms. In fact cinnamic acids are rarely found free in
plants but generally in the less toxic form of esters [30]. Cinnamic acid is reported to be a
potent auxin-inhibitor, affecting both the development of root hairs and phloem tissues and
thus limiting the conduction of water, minerals and nutrients, which lead to growth reduction
and changes in morphology of plants [31]. Indeed, the addition of a sulfoxy group to cinnamic
active moiety is a strategy that plants exert to protect themself from the toxic effect of the active
molecule [29].
In line with the abovementioned conditions 2 and 3, an overall view of the obtained results
highlighted that compounds typically bearing a carboxylate anion in a conjugated system,
showed the best anti-biofilm activity and were effective at sublethal concentrations. Further-
more, derivatives with para substituents (1–3, 5–12) on the phenyl ring displayed different
degrees of E. coli inhibition growth. Since the strength of a carboxylic acid depends mainly on
the extent of its ionization, analogues 7 and 12, with strong EWG on the phenyl ring (nitro and
cyano groups, respectively), reduced the electron density of the oxygen atoms, leading to loss
of activity. On the contrary, compounds bearing moderate and strong EDG (compounds 2 and
5, respectively methyl and methoxy derivatives) enhance the negative charge and exhibited
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Structural and Molecular Basis of Zosteric Acid Activity
good values of inhibition. Moreover, our results demonstrated that compounds in which the
conformation of the double bond was changed (4 vs 37, trans/cis isomers), as well as saturated
derivatives (5 vs 39, unsaturated/saturated derivatives) decreased the ability to inhibit biofilm
formation. Thus, the more the electron density of the carboxylic group of the ZA derivatives
increases, the more effective the molecules are as anti-biofilm compounds at sublethal
concentrations.
Indeed, our work highlighted the structural determinants for the ZA anti-biofilm activity,
and provided the prospects to generate more potent derivatives based on the same ZA active
scaffold and with a better anti-biofilm activity compared to ZA.
The ZA active scaffold is functional in protein interaction
Assuming that primary events triggering the processes related to the anti-biofilm activity relied
on an interaction with a protein, the whole E. coli soluble proteome was screened in order to
evaluate the functional ability of the anti-biofilm ZA active scaffold in targeting a protein. To
this purpose, a pull-down system combined with mass spectrometry-based approach was
developed. The pull-down approach consisted essentially of: i) a batch incubation with the pro-
tein extract to favour protein binding to the ZA active scaffold grafted on a solid support; ii)
column-packing of the incubated solid support in a gravity-flow chromatographic system and
extensive washing of the unbound material; iii) elution of bound protein.
The results obtained from the library screening were instrumental for the application of the
affinity pull-down approach. In fact, structure-activity relationship data are necessary to
immobilize the anti-biofilm compound on an affinity matrix in a site-selective manner, without
compromising the biological activity of the molecule under investigation.
Taking into account the results from the library analysis, we considered: i) the cinnamic
acid scaffold responsible for the anti-biofilm performance of ZA (ZA active scaffold), and ii)
the para position on the phenyl ring as a good anchoring point for matrix immobilisation.
Thus, the p-aminocinnamic acid (p-ACA; 10) was chosen as the proper molecule to covalently
bound via an amide bond the ZA active scaffold to a sepharose matrix bearing 6-aminohexa-
noic spacer arms activated by N-hydroxysuccinimide esterification (p-ACA/matrix).
Starting from the evidence that p-ACA shows a good fluorescent signal (Fig 8A), fluores-
cence analysis was carried out on suspensions of the sepharose matrix in order to monitor the
effect of the coupling reaction. The p-ACA/matrix suspension showed fluorescence, whereas
no fluorescence was detectable in suspensions of the control matrix (EA/matrix) prepared by
omitting p-ACA in the coupling reaction (Fig 8B). Emission and excitation maxima were 426
nm and 350 nm for the p-ACA/matrix suspension, and 443 nm and 380 nm for the p-ACA
solution. Considering that fluorescence of free p-ACA was conserved after the immobilisation
and that the shift of emission wavelength can be due likely to the different conditions of the
samples (suspension vs solution), fluorescence data strongly suggest that the coupling reaction
did not alter the fluorogenic structure of the p-aminocinnamate group.
In order to verify the presence of the grafted ZA active scaffold, p-ACA/matrix was hydro-
lysed and the mixture was submitted to thin layer chromatography (TLC) analysis. A TLC spot
co-migrating with p-ACA was exhibited (data not shown). The hydrolysate was also analyzed
by mass spectrometric techniques. Mass spectrometry analysis registered a monoisotopic single
charged mass ([M+H]+) of 164.069 m/z in the hydrolysates produced by p-ACA/matrix (Fig
9). The theoretical [M+H]+ for p-ACA is reported to be 164.071 m/z. There is significant evi-
dence that p-ACA was released during hydrolysis (experimental mass error of 0.002 Da) and
consequently p-ACA/matrix was successfully functionalized with the ZA active scaffold bound
in the para position via the hydrolysable amide.
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 8. Fluorescence analysis of matrix functionalization. A) Emission spectrum (λexc 380 nm) of 5 mM p-ACA in 0.4 M NaHCO3, 1 M NaCl (pH 8,3). B)
Emission spectra (λexc 350 nm) of suspensions of p-ACA/matrix (50 μl, drained volume; solid line) and EA/matrix (50 μl, drained volume; dashed line) in 2 mL
of 0.4 M NaHCO3, 1 M NaCl (pH 8,3). A. U., arbitrary units.
doi:10.1371/journal.pone.0131519.g008
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 9. Mass spectrometry analysis of hydrolyzed p-ACA/matrix. The eXtracted Ion Current (XIC) profile
at 164.069 m/z, recorded during the LC-MS run, is shown. LC retention time on main peak is evidenced. The
164.069 m/z ion corresponds (ΔM = 0.002 Da) to the theoretical m/z value of the [M+H]+ of p-ACA. NL:
normalization level.
doi:10.1371/journal.pone.0131519.g009
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Structural and Molecular Basis of Zosteric Acid Activity
Fig 10. SDS-PAGE analysis of protein pull-down. Coomassie Blue stained SDS-PAGEs of representative protein-pull-down experiments carried out using
sodium cinnamate (CA) or sodium zosterate (ZA) as elution competitors and matrixes functionalized (p-ACA/matrix) or not (EA/matrix) with the ZA active
scaffold are reported. CE, crude extract (35 μg); U, unbound fraction (35 μg); W1, W2, washing step fractions; E1-E3, elution step fractions. The W and E
samples were TCA-precipitated from 870 μL fraction aliquots. The Mrs and positions of marker proteins are reported on left side.
doi:10.1371/journal.pone.0131519.g010
The pull-down approach was carried out with soluble protein extract from E. coli, and protein
material bound to the p-ACA/matrix was eluted by competition with sodium cinnamate or
sodium zosterate. Collected fractions were analysed by SDS-PAGE (Fig 10). One main band at
Mr *25000 was always observable in the SDS-PAGE profiles of fractions eluted using either
sodium cinnamate or sodium zosterate as competitors in independent pull-down experiments.
No further bands were observable in the fractions collected when NaCl concentration was
increased up to 1 M after the end of the elution by competition. Moreover no bands were eluted
when the control matrix (EA/matrix) replaced p-ACA/matrix in the pull-down approach.
According to an estimation based on the band intensity, *10 μg were pull-down from 90 mg
soluble protein extracted from 2.5 g cell wet pellet (500 mL E. coli culture). All together the results
from the SDS-PAGE analysis showed that i) the immobilized ZA active scaffold is functional in
protein binding, ii) mainly a single protein (Mr *25000) from the E. coli soluble protein extract
is bound, and iii) the bound protein is released by competition with either ZA or cinnamic acid.
The proteins pulled-down from the p-ACA/matrix by competition with either cinnamic
acid or ZA and resolved by SDS-PAGE were excised from the gel and submitted to mass spec-
trometric analysis for protein identification. The identified proteins and their characteristics
are reported in Table 1 and S2 Table. In all cases mass spectrometric analysis gave back a result
with an high score and coverage percentage, suggesting an unequivocal identification of the
protein. Mass spectrometric analysis revealed that NADH:quinone dehydrogenase (WrbA;
accession number: P0A8G6) was pulled-down by competition with either sodium cinnamate
or sodium zosterate, thus showing that the ZA active scaffold is functional in the interaction
with a specific protein from the E. coli soluble extract.
WrbA is an FMN-dependent flavoprotein bearing NADH:quinone oxidoreductase (NQO)
activity [32]. Crystallographic studies reported that WrbA is characterized by a tetrameric qua-
ternary structure with a hydrophobic active site pocket that provides an ideal stacking
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Structural and Molecular Basis of Zosteric Acid Activity
Table 1. Mass spectrometry protein identification.
Compa
Accessionb
Genec
Proteind
Scoree
Coveragef
# Peptideg
# PSMh
MW (KDa)i
Aask
CA
ZA
P0A8G6
P0A8G6
wrbA
wrbA
NAD(P)H quinone dehydrogenase
NAD(P)H quinone dehydrogenase
189.78
235.56
70.20
89.90
11
15
85
93
20.8
20.8
198
198
a) competitor used in the elution step;
b) alphanumeric unique protein sequence identifier provided by UniProtKB/Swiss-Prot protein knowledge base;
c) gene name;
d) protein name;
e) protein identification’s SEQUEST HT Score;
f) percentage of protein sequence covered by identified peptides;
g) total number of identified peptide sequences (peptide spectrum matches);
h) number of identified peptides matching to the protein;
i) theoretically calculated molecular weight;
k) number of amino acids.
doi:10.1371/journal.pone.0131519.t001
environment for aromatic moieties [33]. FAD-dependent homodimeric NQOs (e.g. human
and rat NQO1) are inhibited by certain coumarins and flavones (e.g. dicoumarol, warfarin,
esculetin, phenindione, chrysin, curcumin) [34–36]. The crystal structure of the human Nqo1
in complex with dicoumarol was reported, and it was shown that one of the coumarin rings of
dicoumarol was stacked parallel to the isoalloxazine ring of FAD in the active site [37]. The
crystal structure of E. coli WrbA with oxidized FMN bound reveals a close relationship to the
mammalian Nqo1 [38]. Indeed it appears that the NQO active site is well adapted to accommo-
date phenolic compounds such as ZA and cinnamic acid, suggesting that interaction of the ZA
active scaffold with WrbA may involve the active site function.
The transcription factor CsgD (curlin subunit gene D) from E. coli is considered the master
regulator of the biofilm formation. The wrbA gene was shown to be one of the regulation tar-
gets of CsgD and the distribution of the CgsD-binding regions in the gene suggests that the
expression of wrbA is upregulated by CgsD supporting a WrbA role in biofilm formation [39].
Moreover, the wrbA gene expression was recognized to be, among few other stress-related
genes, under the regulon of the rpoS-encoded alternative sigma factor σS [40, 41]. The rpoS
gene appears to be directly involved in the biosynthesis of signal molecules such as the trypto-
phan metabolite indole which has recently been proved to participate in various aspects of bac-
terial life including virulence induction, cell cycle regulation, stress resistance, genetic stability,
control of metabolic feedback and it is also proposed to be a quorum sensing signal with an
important role in the biofilm formation process [42, 43]. Indole is generated by the cytoplasmic
enzyme, tryptophanase, which hydrolyses tryptophan to produce indole, pyruvate and ammo-
nia, a reaction that occurs exclusively in bacteria. In E. coli, this process is catalysed by TnaA
[44]. In particular, RpoS appears to promote tryptophan degradation by inducing the trypto-
phanase gene tnaA, which results in the production of indole, and by stimulating expression of
the WrbA protein, which might strengthen negative regulation of the trp biosynthetic operon
by an unknown mechanism [41, 45]. The E. coli WrbA (W, i.e. tryptophan, Repressor Binding
protein) was reported to promote complex formations between the tryptophan repressor TrpR
and its DNA operator sequences [40]. Subsequent experiments addressed the influence of
WrbA on the TrpR-DNA complex, and as these did not show any effect, the involvement of
WrbA in transcription regulation was revoked [46]. Although a physical connection to the
tryptophan repressor was actually retracted, a physiological connection between WrbA and
TrpR cannot be ruled out [46]. It is reported that either too little or too much indole can
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Structural and Molecular Basis of Zosteric Acid Activity
abolish the ability to increase biofilm formation, in a dose dependent manner and with strain
specific activity [42]. In a previous study [19], it was shown that E. coli, grown in the presence
of ZA, exhibited enhanced tryptophanase activity and an increased amount of indole. It can be
hypothesized that the ZA active scaffold interacts with WrbA with subsequent consequences
on the indole production.
WrbA has been reported to be involved in maintaining quinones in a fully reduced state
[32]. Quinones are compounds generally tethered to the plasma membrane or freely traversing
the lipid bilayer. Although these compounds are essential for normal electron transport by
cycling between the oxidized (hydroquinone) state and the two-electron reduced (hydroqui-
nol) state, it has been demonstrated that such quinonoids also participate in deleterious redox
cycling through direct interactions with single-electron acceptors such as O2, leading to the
accumulation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and the
hydroxyl radical responsible for a devastating consequence in the cells [47]. In order to guard
against the production of ROS from one-electron redox cycling, cells have evolved NQOs to
maintain quinones in a reduced state as a measure of protection against oxidative stress. A pre-
vious study [19] demonstrated that ZA makes E. coli cells more prone to accumulate ROS lead-
ing to a stress condition, to which the bacterium responds by activating defensive mechanisms
against oxidative damage. We can hypothesize that the ZA active scaffold may negatively mod-
ulate the oxidoreductase activity of WrbA leading to a ROS accumulation inside cells affecting
biofilm formation. Recent data proved the ability of ROS to modulate quorum sensing and
showed their important role in biofilm formation [48, 49]. The upset of the ROS level caused
by the ZA active scaffold might interfere with the quorum sensing signal destabilizing the bio-
film formation. Interestingly, it was suggested that ROS play a role in modulating the indole
signaling pathway by the induction of the TnaA expression [50]. Kuczyńska-Wisnik et al. [51]
demonstrated that an E. coli mutant strain that experiences endogenous oxidative stress shows
an enhanced expression of tryptophanase and an increased indole production that delays the
biofilm formation. In addition, antibiotics that promote ROS formation inhibit development of
E. coli biofilm in an indole-dependent way, which means that bacteria also use indole signals to
modulate pathways dealing with the oxidative stress, and resulting in biofilm repression [52].
A BLAST search against all non-redundant databases of Bacteria using WrbA as the query
protein sequence returned 5792 sequences with identity between 40% and 100% to WrbA
(expect-value threshold ꢀ 10-5), most of which belonged to different E. coli strains, Shigella
ssp., Enterobacter ssp., Salmonella ssp., Klebsiella ssp., Cronobacter ssp., Yersinia ssp., Serratia
ssp., Burkholderia ssp. and Pseudomonas ssp.. A similar search inside Fungi databases returned
490 sequences with identity between 36% and 91% to WrbA (expect-value threshold ꢀ 10-5)
from many species, including Candida spp., Clavispora spp., Aspergillus spp. and Fusarium
spp.. The BLAST search revealed that WrbA sequences are widespread suggesting an impor-
tant role of this protein in the microorganism’s life, possibly associated to the response against
environmental stresses. The presence of WrbA-like proteins in other microorganisms, includ-
ing those involved in human infections or agricultural diseases, may suppose that ZA active
scaffold derivatives could interact with these proteins leading to a possible effect on the biofilm
development of these microorganisms. In this direction, it was demonstrated that ZA is able to
significantly reduce Pseudomonas putida biofilm [18] and Candida albicans biofilm [17].
Thus this part of the work highlighted the functional role of the ZA active scaffold in the
interaction with WrbA and, on the basis of the above considerations, an altered production of
both indole and ROS can be envisaged as the consequence of the interaction, affecting the bio-
film formation.
We envision that the findings gained from this work will be used to covalently couple the
anti-biofilm molecules to a supporting material in such a way as to deliver the active moiety to
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Structural and Molecular Basis of Zosteric Acid Activity
the cellular target. In this approach, no molecule would be leached from the surface, thus side-
stepping the problem of the compound kinetics release, providing long-term protection against
bacterial colonization, and reducing the risk of developing resistant microbial strains, as the
concentration of the anti-biofilm agent would be constantly below the lethal concentration
while maintaining a bioactive molecule.
Conclusion
In this study, the bottlenecks limiting the exploitation of ZA as a preventive strategy to counteract
biofilm formation, were successfully explored and overcome. The screening of the 43-member
library of small molecules based on ZA scaffold against E. coli biofilm highlighted the key role of
cinnamic scaffold as specific structural feature responsible for the ZA anti-biofilm performance.
Moreover structure-anti-biofilm activity relationship considerations revealed that the anti-biofilm
activity of ZA derivatives depended upon the presence of a carboxylate anion and, consequently,
on its hydrogen-donating ability. In addition, the conjugated aromatic system was instrumental
to the anti-biofilm activities of ZA and its analogues since the presence of the double bond stabi-
lized the carboxylate anion. Therefore this study has evidenced that the cinnamic acid scaffold
can be considered a potential template for a class of promising anti-biofilm compounds.
The evaluation of the functional ability of the ZA active scaffold to interact with a protein
led to the discovery of WrbA as a molecular target involved in ZA anti-biofilm activity, suggest-
ing a possible role of this protein in the biofilm formation process. However, it could be possi-
ble that there is a mechanism triggered by ZA interaction with this protein, sofurther
clarification studies are necessary. The confirmation of a relation between biofilm formation
and WrbA would open the way for the development of new and more effective therapies
against biofilm formation.
Supporting Information
S1 Protocol. General procedure for the synthesis of compounds 2, 7, 8, 11, 17, 23, 24, 33,
34, 36–38.
(PDF)
S1 Table. Planktonic growth and cell adhesion in the presence of ZA and compounds 1–43.
Percentage reduction respect to the negative control is calculated as (ZA-related compound
data—control data) x 100 / control data. a) Percentage reduction of the maximum specific
growth rate: code (0) x >- 10%; code (- 1)- 10% ꢀ x < -20%; code (- 2)- 20% ꢀ x < -30%; code
(- 3) x ꢀ - 30%; b) Percentage reduction of the number of adhered cells: code (+ 1) x > + 20%;
code (0) + 20% ꢀ x < -20%; code (- 1)- 20% ꢀ x < -30%; code (- 2)- 30% ꢀ x < -40%; code (-
3) x ꢀ - 40%. According to post hoc analysis (Tukey’s HSD, p < 0.05), data sharing the same
letter indicate no significant difference.
(PDF)
S2 Table. Mass spectrometry peptide sequencing.
(PDF)
Acknowledgments
We wish to thank Dr. F. Iavarone (Institute of Biochemistry and Clinical Biochemistry, Catho-
lic University of Rome, Italy) for assistance in mass spectrometry experiments, and Federica
Larocca for assistance in the pull-down experiments.
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Structural and Molecular Basis of Zosteric Acid Activity
Author Contributions
Conceived and designed the experiments: FF FV AG FC. Performed the experiments: CC SDO
AV VM SB. Analyzed the data: CC SDO FV SV AG AV VM FF FC. Contributed reagents/
materials/analysis tools: FC SV FF. Wrote the paper: CC SDO SV FV FF. Designed the experi-
ments: CC SV SB.
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