Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in Vibrio spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Cinnamaldehyde and cinnamaldehyde derivatives used in this study.

Figure 2

Effect of cinnamaldehyde and cinnamaldehyde derivatives on AI-2 based QS. Bioluminescence in Vibrio harveyi BB170 as a function of the concentration of cinnamaldehyde and cinnamaldehyde derivatives. Bioluminescence measurements were performed 6 h after the addition of the compounds. Bioluminescence of the control (without addition of compound) was set at 100% and the responses for other samples were normalised accordingly. The error bars represent the standard deviation.

Figure 3

Effect of cinnamaldehyde and 2-NO₂-cinnamaldehyde on the bioluminescence of wild type Vibrio harveyi BB120 and the different Vibrio harveyi QS mutants. The percentage of bioluminescence of the Vibrio harveyi wild type BB120 and the mutants MM30, JAF553, JAF483 and BNL258 with 100 μM cinnamaldehyde (white bars) or 100 μM 2-NO₂-cinnamaldehyde (black bars) are presented. Measurements were performed 6 h after the addition of the compounds. Bioluminescence of the control (without addition of compound) was set at 100% and the response for the other samples were normalised accordingly. The error bars represent the standard deviation.

Figure 4

LuxR DNA-binding as determined by mobility shifts and LuxR protein levels as determined by SDS-PAGE. A. Autoradiograph after 5% polacrylamide gel electrophoresis of 32P-labelled LuxR promoter DNA containing the LuxR binding sites, mixed with purified LuxR in the presence (0.19, 0.75 and 1.9 mM) and absence of cinnamaldehyde. B. SDS-PAGE of purified LuxR protein in the presence (0.19, 0.75 and 1.9 mM) or absence of cinnamaldehyde.

Figure 5

Effect of cinnamaldehyde and cinnamaldehyde derivatives on the protease activity of Vibrio anguillarum LMG 4411. Cinnamaldehyde and cinnamaldehyde derivatives were tested at 100 μM, except 4-NO₂-cinnamaldehyde (25 μM). The effect of cinnamaldehyde or cinnamaldehyde derivatives on protease activity was compared to an untreated control. The error bars represent the standard deviation. *: Signal significantly different from the control (p < 0.05).

Figure 6

Effect of cinnamaldehyde and 2-NO₂-cinnamaldehyde on the pigment production of Vibrio anguillarum LMG 4411. Cinnamaldehyde and 2-NO₂-cinnamaldehyde were tested at 100 μM. Vibrio anguillarum LMG 4411 was allowed to produce pigment in the absence (solid symbol) or presence of cinnamaldehyde (open symbol, square) or 2-NO₂-cinnamaldehyde (open symbol, triangle). Three ml samples were taken at multiple time points. The effect of cinnamaldehyde or 2-NO₂-cinnamaldehyde on pigment production was estimated by measuring the absorbance at 405 nm. The error bars represent the standard deviation.

Figure 7

Effect of cinnamaldehyde and cinnamaldehyde derivatives on Vibrio spp. biofilms. Biomass was quantified through CV staining. Cell-viability was quantified through CTB staining. CV signals are presented as a percentage compared to 100% control not receiving treatment (black bars = Vibrio vulnificus; white bars = Vibrio anguillarum). CTB signals are presented as a percentage compared to a 100% control not receiving treatment (vertical striped bars = Vibrio vulnificus; horizontal striped bars = Vibrio anguillarum). *: Signal significantly different compared to 100% control (p < 0.05).

Figure 8

Effect of cinnamaldehyde and 2-NO₂-cinnamaldehyde on the survival of Artemia. White bars represent the survival of Artemia without challenge with Vibrio harveyi BB120. Black bars represent the percentage survival of Artemia after challenge with Vibrio harveyi BB120 in untreated conditions. Striped bars represent the percentage of survival of Artemia after challenge with Vibrio harveyi BB120 when treated with cinnamaldehyde or 2-NO₂-cinnamaldehyde (horizontal: 100 μM stripes; vertical stripes: 150 μM, respectively). *: Survival significantly different from the treatment with pathogen alone (p < 0.01).

Figure 9

Effect of cinnamaldehyde on Vibrio spp. starvation response. The cells were allowed to starve in the presence (horizontal striped bars = Vibrio vulnificus; vertical striped bars = Vibrio anguillarum) and absence (black bars = Vibrio vulnificus; white bars = Vibrio anguillarum) of cinnamaldehyde. The number of CFU/ml was determined after 24 h and 48 h on TSA plates containing 2% NaCl. Data are presented as a percentage of the initial count. Error bars represent standard deviations.

Figure 10

Effect of cinnamaldehyde on antibiotic susceptibility of Vibrio vulnificus LMG 16867. Effects of chloramphenicol (squares) and doxycycline (triangles) on the growth of Vibrio vulnificus LMG16867 in the presence (open symbols) and absence (solid symbols) of cinnamaldehyde (100 μM) are presented. The absorbance at 590 nm was measured after 24 h of growth. Error bars represent standard deviations.