Water-soluble cranberry extract inhibits Vibrio cholerae biofilm formation possibly through modulating the second messenger 3', 5' - Cyclic diguanylate level

Abstract

Quorum sensing (QS) and nucleotide-based second messengers are vital signaling systems that regulate bacterial physiology in response to changing environments. Disrupting bacterial signal transduction is a promising direction to combat infectious diseases, and QS and the second messengers are undoubtedly potential targets. In Vibrio cholerae, both QS and the second messenger 3', 5'-cyclic diguanylate (c-di-GMP) play a central role in controlling motility, motile-to-sessile life transition, and virulence. In this study, we found that water-soluble extract from the North American cranberry could significantly inhibit V. cholerae biofilm formation during the development/maturation stage by reducing the biofilm matrix production and secretion. The anti-biofilm effect by water-soluble cranberry extract was possibly through modulating the intracellular c-di-GMP level and was independent of QS and the QS master regulator HapR. Our results suggest an opportunity to explore more functional foods to fight stubborn infections through interference with the bacterial signaling systems.

Fig 1. WCESP inhibits V. cholerae biofilm formation.

The bacterial cultures were incubated for 24 hours without shaking in the absence and presence of 2 mg/ml of WCESP. (A) Biofilm pellicle formed at the air-water interface in the absence of WCESP, but not in the presence of WCESP. (B) Quantification of the pellicle and surface-attached bacterial cells of V. cholerae C6706 and other pathogenic bacteria in the presence and absence of WCESP. Results shown are the average of three independent experiments, and triplicate samples were tested each time; error bars are standard error of the mean. (* indicates p < 0.05). (C) Crystal violet staining results of surface-attached V. cholerae bacterial cells.

Fig 2. qRT-PCR analysis of V. cholerae biofilm-related genes in response to 2 mg/ml WCESP (normalized to 16S rRNA).

Results are the average of three independent experiments, and error bars are standard error of the mean. (* indicates p < 0.05).

Fig 3. WCESP impedes mainly the development stage of V. cholerae biofilm formation.

Inhibition of 4-hr pre-established biofilm formation by 2 mg/ml WCESP. Results shown are the average of three independent experiments, and triplicate samples were tested each time; error bars are standard error of the mean. (* indicates p < 0.05).

Fig 4. WCESP (2 mg/ml) decreases V. cholerae cell-surface hydrophobicity.

The Hydrophobicity Index (HPBI) was calculated using the following formula: HPBI = (OD Initial—OD Final) / OD Initial x 100%. Data shown are the average results from three independent experiments, and triplicate samples were tested each time; error bars are standard error of the mean. (* indicates p < 0.05).

Fig 5. WCESP-mediated biofilm inhibition is independent of HapR and the quorum-sensing pathway.

(A) β-galactosidase assay of hapR transcription using a V. cholerae C6706 strain carrying a chromosomal hapR-lacZ fusion. (B) Biofilm formation in the presence and absence of 2 mg/ml WCESP using the plate count method in V. cholerae C6706 derived mutant strains. Data shown are the average results from three independent experiments, and triplicate samples were tested each time; error bars are standard error of the mean. (* indicates p < 0.05).

Fig 6. Quantification of intracellular c-di-GMP concentration in the presence and absence of 2 mg/ml WCESP using the thiazole orange (TO)-based fluorescent detection method.

The concentration of c-di-GMP was calculated using a calibration curve and normalized to the concentration of a bacterial culture with 1 x 10⁹ cells/ml. Data shown are the average results from three independent experiments, and error bars are standard error of the mean.

Fig 7. Induction of c-di-GMP synthesis partially restored biofilm formation in the presence of WCESP.

The overnight bacterial culture was 1:100 diluted into 5 ml of fresh LB medium and incubated at 37°C with shaking until OD₆₀₀ reached ~ 0.25. The culture was then aliquoted into four tubes with 1 ml each. WCESP and L-arabinose were added to the corresponding tubes with a final concentration of 2 mg/ml and 0.2%, respectively. All the tubes were subsequently incubated at 37°C for 24 hours without shaking. Tube 1–4, VC6706 with the empty vector pBAD33 to serve as controls. Tube 5–8, VC6706 with pAT1662 (pBAD33::VCA0956-His₆).