Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in Vibrio spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR
© Brackman et al; licensee BioMed Central Ltd. 2008
Received: 23 April 2008
Accepted: 16 September 2008
Published: 16 September 2008
To date, only few compounds targeting the AI-2 based quorum sensing (QS) system are known. In the present study, we screened cinnamaldehyde and substituted cinnamaldehydes for their ability to interfere with AI-2 based QS. The mechanism of QS inhibition was elucidated by measuring the effect on bioluminescence in several Vibrio harveyi mutants. We also studied in vitro the ability of these compounds to interfere with biofilm formation, stress response and virulence of Vibrio spp. The compounds were also evaluated in an in vivo assay measuring the reduction of Vibrio harveyi virulence towards Artemia shrimp.
Our results indicate that cinnamaldehyde and several substituted derivatives interfere with AI-2 based QS without inhibiting bacterial growth. The active compounds neither interfered with the bioluminescence system as such, nor with the production of AI-2. Study of the effect in various mutants suggested that the target protein is LuxR. Mobility shift assays revealed a decreased DNA-binding ability of LuxR. The compounds were further shown to (i) inhibit biofilm formation in several Vibrio spp., (ii) result in a reduced ability to survive starvation and antibiotic treatment, (iii) reduce pigment and protease production in Vibrio anguillarum and (iv) protect gnotobiotic Artemia shrimp against virulent Vibrio harveyi BB120.
Cinnamaldehyde and cinnamaldehyde derivatives interfere with AI-2 based QS in various Vibrio spp. by decreasing the DNA-binding ability of LuxR. The use of these compounds resulted in several marked phenotypic changes, including reduced virulence and increased susceptibility to stress. Since inhibitors of AI-2 based quorum sensing are rare, and considering the role of AI-2 in several processes these compounds may be useful leads towards antipathogenic drugs.
Vibriosis, caused by Vibrio spp., is a major disease of marine fish and shellfish and is an important cause of economic loss in aquaculture [1, 2]. Until recently prophylactic antibiotics were extensively used in aquaculture [3, 4]. However, overuse of antibiotics resulted in increased rates of resistance so that novel approaches are required to manage vibriosis . A possible novel target is the bacterial communication system. Bacteria monitor their population densities through the production and sensing of small signal molecules called autoinducers, a process entitled as quorum sensing (QS). To date three types of QS systems have been identified in Vibrio spp. . Acylated homoserine lactones (AHL) are used as signal molecules in the LuxM/N QS system , while in the CqsA/S system, (S)-3-hydroxytridecan-4-one ("Cholera autoinducer 1", CAI-1) is used . A third QS system appears to be shared by many Gram-positive and Gram-negative bacteria and is based on a mixture of interconvertible molecules collectively referred to as autoinducer-2 (AI-2) . A key enzyme in the production of AI-2 is LuxS. LuxS catalyzes the cleavage of S-ribosylhomocysteine to homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD) . DPD will subsequently undergo spontaneous derivatizations, forming a mixture of molecules, including (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran (R-THMF) and (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate (S-THMF-borate) . Although not all QS systems are present in all Vibrio spp., most of them contain the AI-2 based QS system . In Vibrio spp. AI-2 binds to LuxP, a periplasmic AI-2 receptor that is associated with the LuxQ sensor kinase-phosphatase. At low population density only basal amounts of diffusible signal molecules are produced, and in this situation LuxQ will act as a kinase resulting in a phosphorylation of the response regulator LuxO through a cascade involving LuxU. Phosphorylation activates LuxO resulting in the production of small regulatory RNAs . These small RNAs, together with the chaperone protein Hfq, will destabilize mRNA encoding the response regulator LuxR. However, when population density is sufficiently high, AI-2 will bind to LuxP and as a result LuxQ will act as a phosphatase, leading to a dephosphorylation of LuxO . Since dephosphorylated LuxO is inactive, no small regulatory RNAs will be formed and the LuxR mRNA remains stable, resulting in the production of LuxR and ultimately an altered gene expression pattern. AI-2 based QS was found to play an important role in regulating the production of several virulence factors, biofilm formation and stress responses in several Vibrio spp. [15–17] and it was found to be associated with virulence as shown in several in vivo assays [18, 19]. In contrast, in Vibrio cholerae, CAI-1 was found to be the principle signal molecule in virulence regulation . Because of this involvement in virulence, inhibitors of AI-2 based QS have been proposed as novel antipathogenic agents. While there is a growing interest in and evidence for the use of these antipathogenic substances to interfere with interspecies QS in the control of virulence and biofilm formation, only a few inhibitors of AI-2 based QS are known, including halogenated furanones and cinnamaldehyde [20–23]. Halogenated furanones have previously been shown to disrupt AHL and AI-2 based quorum sensing in Vibrio spp. by decreasing the DNA-binding activity of the response regulator LuxR [24–26]. Halogenated furanones can attenuate the virulence of several Vibrio spp. in gnotobiotic brine shrimp Artemia franciscana and their use results in a reversal of the negative effects of Vibrio harveyi BB120 towards rotifers [27, 28]. Unfortunately, the toxicity of halogenated furanones towards both brine shrimp and rotifers limits their use. In contrast, cinnamaldehyde is a non-toxic synthetic flavouring substance that is widely used in food, beverages, chewing gum, and the perfume and food chemistry, and is generally recognised as safe . Cinnamaldehyde concentrations in food range from 4 ppm to more than 300 ppm . Although cinnamaldehyde is known to be a QS-inhibitor , its exact mechanism of action remains to be elucidated. The goal of the present study was to determine the mechanism of action of cinnamaldehyde and to evaluate its effect on virulence of Vibrio spp. in vitro and in vivo.
Results and discussion
Effect of cinnamaldehyde and cinnamaldehyde derivatives on microbial growth
Effect of cinnamaldehyde and 2-NO2-cinnamaldehyde on bioluminescence
To rule out direct interference with the bioluminescence system of Vibrio harveyi, a constitutively bioluminescent strain was constructed. A plasmid containing luxCDABE genes under lacZ promotion was conjugated into Escherichia coli DH5α (a strain defective in AI-2 production). The bioluminescence was not inhibited by cinnamaldehyde and cinnamaldehyde derivatives (data not shown) and these results indicate that the enzymes of Vibrio harveyi involved in bioluminescence are not inhibited by cinnamaldehyde or cinnamaldehyde derivatives.
Effect of cinnamaldehyde and cinnamaldehyde derivatives on AI-2 based QS
Effect of cinnamaldehyde and cinnamaldehyde derivatives on the bioluminescence of Vibrio harveyiQS mutants
Effect of cinnamaldehyde on LuxR protein levels and on LuxR DNA-binding activity
Effect of cinnamaldehyde and cinnamaldehyde derivatives on Vibrio anguillarumprotease and pigment production
Effect of cinnamaldehyde and cinnamaldehyde derivatives on biofilm formation
Protection of Artemia from Vibrio harveyi
Effect of cinnamaldehyde on the starvation response
Effect of cinnamaldehyde on antibiotic susceptibility
Cinnamaldehyde and several derivatives were shown to interfere with AI-2 based QS by decreasing the ability of LuxR to bind to its target promoter sequence. These compounds, used in sub-inhibitory concentrations, did not only affect in vitro the production of multiple virulence factors and biofilm formation, but also reduced in vivo the mortality of Artemia shrimp exposed to Vibrio harveyi BB120. In addition, cinnamaldehyde reduced the ability to cope with stress factors like starvation and exposure to antibiotics. Our results indicate that cinnamaldehyde and cinnamaldehyde derivatives are potentially useful antipathogenic lead compounds for treatment of vibriosis.
Cinnamaldehyde and cinnamaldehyde derivatives
Cinnamaldehyde (Sigma-Aldrich, Bornem, Belgium) and cinnamaldehyde derivatives [4-MeO-cinnamaldehyde (VWR International, West Chester, USA), 2-MeO-cinnamaldehyde (Wako Pure Chemical Industries, Osaka, Japan), 4-NO2-cinnamaldehyde, 2-NO2-cinnamaldehyde and 4-Me2N-cinnamaldehyde (Acros Organics, Geel, Belgium)] (Fig. 1), were diluted in DMSO (0.5% v/v). The stock solutions were stored at -20°C. Control solutions (CS) contained the same amount of DMSO, without cinnamaldehyde or cinnamaldehyde derivatives.
Bacterial strains, plasmid and growth conditions
Strains and plasmid used in this study.
Reference or source
Vibrio harveyi strains
Wild type from which strains BB152, BB170, MM30, JAF553, JAF483 and BNL258 are derived
luxU H58A linked to KanR
luxO D47A linked to KanR
Isolated from young sea trout (Salmo trutta)
Isolated from tankwater from eelfarm
Escherichia coli strains
AI-2 - strain
pBluelux polylinker and luxCDABE
Effect of cinnamaldehyde and cinnamaldehyde derivatives on bioluminescence
To determine whether any of the compounds had an effect on bioluminescence not related to inhibition of QS, Escherichia coli DH5α was transformed with the pBluelux plasmid, containing luxCDABE under control of a lacZ promoter and the effect on bioluminescence was measured. The pBluelux plasmid was transformed in Escherichia coli DH5α as follows. Overnight cultures were suspended in a 50 mM CaCl2 solution at 0°C. The pBluelux plasmid was added and the solution was incubated for 15 min. After this, the solution was transferred to 42°C for 90 sec and the cell suspension was plated on Trypton soy agar (TSA) (Oxoid, Basingstoke, Hampshire, UK) containing 100 μg/ml ampicillin (Sigma-Aldrich) for selection of transformants. For the bioluminescence assay an overnight culture was diluted to OD590 nm of approximately 0.1 and 100 μl of cell suspension was added to each well of a black 96-well microtiter plate (Perkin Elmer). The effect on bioluminescence for the active compounds was compared to controls not receiving the active molecules.
Bioassay for LuxS inhibition
In order to determine whether any of the compounds tested had an effect on production of AI-2, AI-2 activity was measured in supernatants of Escherichia coli K12 cultures grown for 16 h with or without compounds. Overnight cultures of Escherichia coli K12 were centrifuged (5000 rpm, 5 min, room temperature) and filter sterilised (0.22 μm, Whatman GmbH, Dassel, Germany). The supernatants were used immediately or stored at -20°C. AI-2 levels were determined in a Vibrio harveyi BB170 assay as described previously . In brief, an overnight culture of the reporter strain was diluted 1:5000 into fresh sterile MB medium and 90 μl of this cell suspension was added to the wells of a black 96-well microtiter plate (Perkin Elmer). Ten μl of the appropriate sterile supernatants was then added to the wells, the microtiter plates were incubated at 30°C and bioluminescence was measured hourly using a Wallac Victor2 multilabel counter (Perkin Elmer). Bioluminescence was expressed as the fraction of bioluminescence measured in the positive control reaction. Confirmation of these results was obtained using Vibrio harveyi MM30, a Δ LuxS mutant, instead of Vibrio harveyi BB170. The effect on bioluminescence for the active compounds was compared to controls not receiving the active molecules.
Other Vibrio harveyibioassays
Using Vibrio harveyi strains BB120, JAF553, JAF483 and BNL258, we determined whether the molecular target of our compounds was located in the AI-2 signalling transduction pathway. The bioluminescence assay as described above was used with minor modifications. In brief, the positive control reaction received 10 μl of Escherichia coli K12 supernatant, without addition of the test molecule. Negative control reactions received 10 μl sterile MB-medium. Other wells received 10 μl of Escherichia coli K12 supernatants (containing AI-2) and appropriate amounts of the test molecule.
Effect of cinnamaldehyde on LuxR protein levels and DNA-binding activity
Mobility shift assays and SDS-PAGE assays were performed as described previously  with minor modifications. Vibrio harveyi BB120 cells were grown in the presence and absence of cinnamaldehyde and all cell lysates were taken at different optical densities (OD600 nm = 1.2, 1.6, 1.8 and 2.1). Previously purified LuxR  was used for mobility shift and SDS-PAGE assay. For SDS-PAGE the following protein standard (Bio-rad) was used: 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa.
Quantification of protease activity
Vibrio anguillarum LMG 4411 was grown overnight in MB. Protease activity was quantified following inoculation of cultures into medium containing 2.0% Bacto agar (Oxoid), 2.0% NaCl (Novolab, Geraardsbergen, Belgium) and 3.0% Skim Milk powder (Oxoid). Appropriate amounts of test compounds and CS were added to the mixtures, 0.5 ml of these mixtures was added to the wells of a 24-well microtiter plate (TPP, Trasadingen, Switzerland) and the plate was incubated at 30°C. Clearing was measured spectrophotometrically with a Wallac Victor2 multilabel counter after 24 h.
Quantification of pigment production
Vibrio anguillarum LMG 4411 was grown overnight at 30°C in MB. The overnight culture was then diluted to OD590 nm = 0.05 in Tryptone Soy Broth (TSB) (Oxoid) containing 5 mM L-tyrosine (Sigma-Aldrich) with or without test compound and incubated at 30°C with shaking. At various time points, 3 ml samples were taken from the cultures and supernatants were collected by centrifugation (5000 rpm, 4 min, room temperature), followed by filter sterilisation (0.22 μm). Pigment production was followed by measuring the absorbance at 405 nm.
Biofilm formation assay
Vibrio anguillarum LMG 4411 (doubling time Td: 3.2 h) and Vibrio vulnificus LMG 16867 (Td: 5.3 h) were grown overnight in MB, centrifuged, resuspendend in double concentrated Marine Broth (2xMB) and diluted to an OD590 nm = 0.1 in 2xMB. Fifty μl of the diluted bacterial suspension was transferred to the wells of a round-bottomed 96-well microtiter plate (TPP). Negative controls received 50 μl of CS. Positive controls received 50 μl of the test compound in appropriate concentrations. Bacteria were allowed to adhere and grow without agitation for 4 h at 30°C. After 4 h, plates were emptied and washed with sterile physiological saline (PS). After this washing step, negative control wells were filled with 50 μL 2xMB and 50 μl CS. Other wells were filled with 50 μl 2xMB and 50 μl compound solution and the plate was incubated for 24 h at 30°C. Biofilm biomass was quantified by crystal violet (CV) staining, as described previously . In brief, plates were rinsed with sterile PS, biofilms were fixed by adding 100 μl 99% methanol for 15 min, after which the methanol was removed and plates were air-dried. Biofilms were then stained with 100 μl CV (Pro-lab Diagnostics, Richmond Hill, ON, Canada). After 20 min, CV was removed and wells were filled with 150 μl 33% acetic acid (Sigma-Aldrich). The absorbance was measured at 590 nm using a Wallac Victor2 multilabel counter and results were expressed as the percentages compared to the signal of the control not receiving treatment. For quantification of the number of metabolically active (i.e. living) cells in the biofilm, a resazurin assay was used . In brief, wells were rinsed after 24 h biofilm formation and 100 μl PS was added, followed by addition of 20 μl CellTiter-Blue (CTB) (Promega, Leiden, The Netherlands) solution. After 60 min, fluorescence (ex560 nm/em590 nm) was measured using a Wallac Victor2 multilabel counter. For the quantification of EPS, a Calcofluor white staining (Sigma-Aldrich) was used. In brief, wells were rinsed after 24 h biofilm formation and 100 μl phosphate buffered saline (PBS) containing 0.5 μl 5 mM CFW was added to the wells. After 60 min, fluorescence (ex405 nm/em500 nm) was measured using a Wallac Victor2 multilabel counter.
All experiments were performed with high quality hatching cysts of Artemia franciscana (EG® Type, batch 6940, INVE Aquaculture, Baasrode, Belgium). 200 mg of cysts were hydrated in 18 ml of tap water during 1 h. Sterile cysts and nauplii were obtained via decapsulation as described previously . Challenge tests were performed as described previously  with minor modifications. Briefly, after hatching, groups of 20 nauplii were transferred to new sterile 50 ml tubes that contained 20 ml of 0.22 μm filtered and autoclaved artificial seawater. Vibrio harveyi BB120 was washed in filtered and autoclaved artificial seawater after incubation and added to the Artemia culture water at a concentration of approximately 105 CFU/ml. A suspension of autoclaved LVS3 bacteria in filtered and autoclaved artificial seawater was added as feed in a concentration of approximately 107 CFU/ml culture water. After the addition of 100 μM or 150 μM of cinnamaldehyde or 2-NO2-cinnamaldehyde (or an appropriate volume of solvent), the falcon tubes were put back on the rotor and kept at 28°C. Artemia cultures to which only autoclaved LVS3 bacteria were added were used as controls. The survival of Artemia was scored 48 h after the addition of the strains. All manipulations were done under a laminar flow hood in order to maintain sterility of the cysts and nauplii. Each treatment was done in triplicate.
Vibrio anguillarum LMG 4411 and Vibrio vulnificus LMG 16867 strains were grown overnight in MB, the cells were collected by centrifugation (5000 rpm, 4 min), washed in PS and resuspended in artificial seawater (ASW)  containing 0.1% MB (with and without test compound). These suspensions were incubated at 30°C without shaking. At various time points, 1 ml samples were taken and the number of culturable cells was determined by plating serial dilutions on TSA (Oxoid) plates containing 2% NaCl. Results were expressed as the percentage survival compared to the untreated control.
Effect of cinnamaldehyde on antibiotic resistance
Fifty μL of double concentrated TSB (2xTSB) containing 4% NaCl with or without chlorampenicol (Sigma-Aldrich) or doxycycline (Sigma-Aldrich) (added in the range of 0.001 μg/ml – 25 μg/ml) were dispensed into flat-bottomed 96-well microtiter plates (TPP). An equal amount of cinnamaldehyde was added (final concentration of 100 μM). For the controls, equal amounts of CS were added to the wells. Vibrio vulnificus LMG 16867 or Vibrio anguillarum LMG 4411 was added in a final concentration of 105 CFU/ml. The plates were incubated overnight at 30°C and growth was evaluated after 24 h by absorbance measurements at 590 nm using a Wallac Victor2multilabel counter.
Independent samples t-tests were performed using the SPSS software, version 15.0 (SPSS, Chicago, IL, USA).
The authors like to thank Dr. S. Atkinson for kindly providing the pBlueLux plasmid. This work was supported by the 'Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen' (GB) and FWO-Vlaanderen (TC).
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