Inhibition of Escherichia coli CFT073 fliC expression and motility by cranberry materials

Abstract

In humans, uropathogenic Escherichia coli (UPEC) is the most common etiological agent of uncomplicated urinary tract infections (UTIs). Cranberry extracts have been linked to the prevention of UTIs for over a century; however, a mechanistic understanding of the way in which cranberry derivatives prevent bacterial infection is still lacking. In this study, we used a fliC-lux reporter as well as quantitative reverse transcription-PCR to demonstrate that when UPEC strain CFT073 was grown or exposed to dehydrated, crushed cranberries or to purified cranberry-derived proanthocyanidins (cPACs), expression of the flagellin gene (fliC) was inhibited. In agreement with these results, transmission electron microscopy imaging of bacteria grown in the presence of cranberry materials revealed fewer flagella than those in bacteria grown under control conditions. Furthermore, we showed that swimming and swarming motilities were hindered when bacteria were grown in the presence of the cranberry compounds. Because flagellum-mediated motility has been suggested to enable UPEC to disseminate to the upper urinary tract, we propose that inhibition of flagellum-mediated motility might be a key mechanism by which cPACs prevent UTIs. This is the first report to show that cranberry compounds inhibit UPEC motility via downregulation of the fliC gene. Further studies are required to establish whether these inhibitors play a role in vivo.

Fig. 1.

Growth curve for E. coli CFT073 grown in the presence of cranberry compounds. The graph shows the OD₆₀₀ − initial OD₆₀₀ versus time for cultures of E. coli CFT073 grown in M9 medium (▪) or in M9 medium supplemented with cPACs at 0.1 mg/ml (♦) or with CP at 5 (▴), 10 (×), 15 (solid line), or 20 (★) mg/ml. Results are representative of at least three independent experiments. Abbreviations: CPx, cranberry powder at x mg/ml (e.g., CP5 indicates cranberry powder at 5 mg/ml); cPAC, cranberry proanthocyanidins at 0.1 mg/ml.

Fig. 2.

Downregulation of fliC expression by cranberry materials. (a) Cultures of CFT073 P_fliC-lux were grown in the presence (♦) or absence (▪) of 0.1 mg/ml cPACs or with CP at 5 (▴), 10 (×), 15 (solid line), or 20 (★) mg/ml. Expression of the fliC gene was calculated as follows: normalized luminescence = [luminescence/(OD₆₀₀ − initial OD₆₀₀)]. Three independent experiments were performed, and the results of a typical experiment are presented. (b) Expression of the fliC gene assessed on the mRNA level by quantitative reverse transcription-PCR. Relative mRNA expression of fliC was first normalized to that of a housekeeping gene, gapA, and then related to the normalized expression level of the same gene in the control treatment. Results represent mean values ± standard deviations (SD) for three independent experiments. (c) fliC expression versus time for CFT073 P_fliC-lux cultures harvested at mid-log phase and spiked with sterile water (▪), 0.1 mg/ml cPACs (♦), or CP at 5 (▴), 10 (×), 15 (solid line), or 20 (★) mg/ml. Similar results were obtained in at least three other experiments.

Fig. 3.

Electron microscopic images of CFT073 cells. Bacteria were cultured in LB broth alone (a and c) or supplemented with 10 mg/ml CP (b and d). Magnification, ×43,700. Bars, 500 nm.

Fig. 4.

Effects of cranberry materials on motility. (a) Characterization of the effects of CP or cPACs on swimming motility in soft agar plates. *, P < 0.001 by one-way analysis of variance for comparison with the control. (b) Representative images of swimming motility plates for the control (CON), CP at 10 mg/ml (CP10), and cPACs at 0.1 mg/ml (cPAC). An image of the result obtained with a fliC mutant is included. (c) Swarming motility of CFT073 cells. Error bars represent the SD for triplicate samples. (d) Representative images of swarming motility plates for the control, CP at 5 and 10 mg/ml, and cPACs at 0.1 mg/ml.