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

Brackman G, Defoirdt T, Miyamoto C, Bossier P, Van Calenbergh S, Nelis H, Coenye T - BMC Microbiol. (2008)

Bottom Line: The active compounds neither interfered with the bioluminescence system as such, nor with the production of AI-2.Mobility shift assays revealed a decreased DNA-binding ability of LuxR.The use of these compounds resulted in several marked phenotypic changes, including reduced virulence and increased susceptibility to stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium. gilles.brackman@ugent.be

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.

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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.
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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.

Mentions: Bioluminescence in Vibrio harveyi BB170 is mainly controlled by AI-2, as this strain is not responsive to AHL stimulation [7]. Hence we limited the possible target of cinnamaldehyde and cinnamaldehyde derivatives to the AI-2 QS system. To determine the molecular target within the AI-2 QS pathway we measured the effect of cinnamaldehyde and cinnamaldehyde derivatives on the bioluminescence in different QS mutants. Vibrio harveyi MM30 has a mutation in the luxS gene, making it incapable of producing AI-2. However, this strain will react to exogenously added AI-2 with activation of the QS transduction system leading to bioluminescence. Inhibition of bioluminescence in this mutant would suggest the absence of an inhibitory effect on LuxS. Further we evaluated the effect of the test compounds on the production of AI-2 in Escherichia coli K12. The Vibrio harveyi JAF553 and JAF483 mutants contain a point mutation in the luxU and luxO genes, respectively, thereby preventing their phosphorelay capacity. Vibrio harveyi BNL258 has a Tn5 insertion in the hfq gene, resulting in a non-functional Hfq protein. Vibrio harveyi strains JAF553, JAF483 and BNL258 are all constitutively luminescent and inhibition of bioluminescence in one of these indicates that the cinnamaldehyde compounds act downstream of the mutated protein. Cinnamaldehyde and 2-NO2-cinnamaldehyde were found to block bioluminescence in Vibrio harveyi MM30 (Fig. 3), suggesting that these compounds do not exert their effect at the level of AI-2 production but rather at the level of the QS transduction system. Affirmatively, the supernatants of Escherichia coli K12 treated with cinnamaldehyde and cinnamaldehyde derivatives revealed no difference in AI-2 activity compared to the control (data not shown). Cinnamaldehyde and 2-NO2-cinnamaldehyde were found to block bioluminescence to the same extent in all other mutants tested (Fig. 3). This suggests that the target of cinnamaldehyde and cinnamaldehyde derivatives is the downstream component of the AI-2 signalling transduction pathway, the transcriptional regulatory protein LuxR.


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

Brackman G, Defoirdt T, Miyamoto C, Bossier P, Van Calenbergh S, Nelis H, Coenye T - BMC Microbiol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551610&req=5

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.
Mentions: Bioluminescence in Vibrio harveyi BB170 is mainly controlled by AI-2, as this strain is not responsive to AHL stimulation [7]. Hence we limited the possible target of cinnamaldehyde and cinnamaldehyde derivatives to the AI-2 QS system. To determine the molecular target within the AI-2 QS pathway we measured the effect of cinnamaldehyde and cinnamaldehyde derivatives on the bioluminescence in different QS mutants. Vibrio harveyi MM30 has a mutation in the luxS gene, making it incapable of producing AI-2. However, this strain will react to exogenously added AI-2 with activation of the QS transduction system leading to bioluminescence. Inhibition of bioluminescence in this mutant would suggest the absence of an inhibitory effect on LuxS. Further we evaluated the effect of the test compounds on the production of AI-2 in Escherichia coli K12. The Vibrio harveyi JAF553 and JAF483 mutants contain a point mutation in the luxU and luxO genes, respectively, thereby preventing their phosphorelay capacity. Vibrio harveyi BNL258 has a Tn5 insertion in the hfq gene, resulting in a non-functional Hfq protein. Vibrio harveyi strains JAF553, JAF483 and BNL258 are all constitutively luminescent and inhibition of bioluminescence in one of these indicates that the cinnamaldehyde compounds act downstream of the mutated protein. Cinnamaldehyde and 2-NO2-cinnamaldehyde were found to block bioluminescence in Vibrio harveyi MM30 (Fig. 3), suggesting that these compounds do not exert their effect at the level of AI-2 production but rather at the level of the QS transduction system. Affirmatively, the supernatants of Escherichia coli K12 treated with cinnamaldehyde and cinnamaldehyde derivatives revealed no difference in AI-2 activity compared to the control (data not shown). Cinnamaldehyde and 2-NO2-cinnamaldehyde were found to block bioluminescence to the same extent in all other mutants tested (Fig. 3). This suggests that the target of cinnamaldehyde and cinnamaldehyde derivatives is the downstream component of the AI-2 signalling transduction pathway, the transcriptional regulatory protein LuxR.

Bottom Line: The active compounds neither interfered with the bioluminescence system as such, nor with the production of AI-2.Mobility shift assays revealed a decreased DNA-binding ability of LuxR.The use of these compounds resulted in several marked phenotypic changes, including reduced virulence and increased susceptibility to stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium. gilles.brackman@ugent.be

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.

Show MeSH
Related in: MedlinePlus