Cannabigerol Prevents Quorum Sensing and Biofilm Formation of Vibrio harveyi

Abstract

Cannabigerol (CBG) is a non-psychoactive cannabinoid naturally present in trace amounts in the Cannabis plant. So far, CBG has been shown to exert diverse activities in eukaryotes. However, much less is known about its effects on prokaryotes. In this study, we investigated the potential role of CBG as an anti-biofilm and anti-quorum sensing agent against Vibrio harveyi. Quorum sensing (QS) is a cell-to-cell communication system among bacteria that involves small signaling molecules called autoinducers, enabling bacteria to sense the surrounding environment. The autoinducers cause alterations in gene expression and induce bioluminescence, pigment production, motility and biofilm formation. The effect of CBG was tested on V. harveyi grown under planktonic and biofilm conditions. CBG reduced the QS-regulated bioluminescence and biofilm formation of V. harveyi at concentrations not affecting the planktonic bacterial growth. CBG also reduced the motility of V. harveyi in a dose-dependent manner. We further observed that CBG increased LuxO expression and activity, with a concomitant 80% downregulation of the LuxR gene. Exogenous addition of autoinducers could not overcome the QS-inhibitory effect of CBG, suggesting that CBG interferes with the transmission of the autoinducer signals. In conclusion, our study shows that CBG is a potential anti-biofilm agent via inhibition of the QS cascade.

Keywords: Vibrio harveyi; biofilm; bioluminescence; cannabinoids; motility; quorum sensing.

FIGURE 1

The chemical structure of Cannabigerol (CBG).

FIGURE 2

Anti-quorum sensing activity of CBG on wild-type V. harveyi (BB120). (A) CBG did not affect the planktonic growth of V. harveyi. (B) CBD strongly prevented the bioluminescence of V. harveyi. The graphs represent the average of 3 samples. (C) The relative bioluminescence as determined by the area under the curve (AUC) of the graph presented in (B) N = 3, *p < 0.05.

FIGURE 3

Anti-biofilm activity of CBG on wild-type V. harveyi (BB120). (A) CBG reduced the amount of DNA in the biofilms formed by V. harveyi. V. harveyi were grown in complete AB medium for 24 h in the presence of various concentrations of CBG, and the amount of DNA in the biofilms were quantified by real-time PCR using primers for 16S rRNA. N = 3. *p < 0.05. (B) Scanning electron microscopy of biofilms formed by wild-type V. harveyi in the presence or absence of 20 μg/ml CBG. 0.1% Ethanol (EtOH) served as vehicle control. Two different magnifications are shown (x1000 and x5000).

FIGURE 4

CBG inhibits V. harveyi motility. (A) The motility of V. harveyi in soft agar containing the indicated concentrations of CBG. (B) The relative motility of V. harveyi in the presence of indicated concentrations of CBG as determined by the spreading area shown in (A) N = 3, *p < 0.05.

FIGURE 5

CBG antagonizes the quorum sensing signals delivered by autoinducers. (A) The relative bioluminescence of luxM, luxS double mutant MM77 (AI-1^–, AI-2^–) strain was measured over time in the absence or presence of AI-1, AI-2 and/or 1 μg/ml CBG. The bioluminescence was corrected for differences in bacterial growth by simultaneously measuring the optical density at 595 nm. (B) The relative bioluminescence as determined by the area under the curve (AUC) of the graph presented in (A) N = 3, *p < 0.05. (C) The relative bioluminescence as determined by the area under the curve (AUC) of luxM null mutant BB152 (AI-1^–AI-2⁺) exposed to AI-1 and/or 1 μg/ml CBG. N = 3. *p < 0.05. (D) The relative bioluminescence as determined by the area under the curve (AUC) of luxS null mutant MM30 (AI-1⁺AI-2^–) exposed to the AI-2 precursor DPD (10 μM) and/or 1 μg/ml CBG. N = 3, *p < 0.05.

FIGURE 6

CBG increases the LuxO expression and activity. (A) RNA from wild-type V. harveyi (BB120) that has been incubated with 2 μg/ml CBG for 10 h were analyzed by real-time PCR for luxU and luxO gene expression as well as the expression of the LuxO-regulated genes aphA, hfq, and qrr1-5. The relative expression was compared to untreated bacteria incubated for the same time period using 16S rRNA as internal control. (B) The relative gene expression of luxR in the same samples described in (A). (C) The relative gene expression of autoinducer synthases and autoinducer receptors, N = 3, *p < 0.05.

FIGURE 7

luxS null and luxN null mutants showed diminished response to CBG. (A–F) The bioluminescence of the indicated mutant strains grown in the absence or presence of CBG. The black lines represent control samples of no CBG, the orange lines bacteria treated with 1 μg/ml CBG, and the green lines bacteria treated with 2 μg/ml CBG. Each line is the average of three samples. (G) The relative bioluminescence as determined by the area under the curve (AUC) of the graphs presented in (A–F), N = 3, *p < 0.05 in comparison to CBG-treated wild-type bacteria.

FIGURE 8

Possible action mechanism of CBG. The quorum sensing system is tightly regulated and has many feedback loops. Our data provide evidence that the anti-quorum sensing activity of CBG is caused by antagonizing the signals delivered by AI-1 and AI-2. Based on our observation that LuxN is required for the anti-QS activity of CBG and CBG augments the LuxO activity, we propose that CBG prevents the dephosphorylation of LuxU.