Inhibitory effects of Myricetin derivatives on curli-dependent biofilm formation in Escherichia coli

Abstract

Biofilms are well-organised communities of microbes embedded in a self-produced extracellular matrix (e.g., curli amyloid fibers) and are associated with chronic infections. Therefore, development of anti-biofilm drugs is important to combat with these infections. Previously, we found that flavonol Myricetin inhibits curli-dependent biofilm formation by Escherichia coli (IC₅₀ = 46.2 μM). In this study, we tested activities of seven Myricetin-derivatives to inhibit biofilm formation by E. coli K-12 in liquid culture. Among them, only Epigallocatechin gallate (EGCG), a major catechin in green tea, inhibited biofilm formation of K-12 (IC₅₀ = 5.9 μM) more efficiently than Myricetin. Transmission electron microscopy and immunoblotting analyses demonstrated that EGCG prevented curli production by suppressing the expression of curli-related proteins. Quantitative RT-PCR analysis revealed that the transcripts of csgA, csgB, and csgD were significantly reduced in the presence of EGCG. Interestingly, the cellular level of RpoS, a stationary-phase specific alternative sigma factor, was reduced in the presence of EGCG, whereas the rpoS transcript was not affected. Antibiotic-chase experiments and genetic analyses revealed that EGCG accelerated RpoS degradation by ATP-dependent protease ClpXP in combination with its adaptor RssB. Collectively, these results provide significant insights into the development of drugs to treat chronic biofilm-associated infections.

Figure 1

Chemical structures of Myricetin-derivatives used in this study.

Figure 2

Effects of Myricetin-derivatives on E. coli biofilm formation. (a) Biofilms of E. coli BW25113 were formed in YESCA medium supplemented with Myricetin-derivatives at the indicated concentrations at 30 °C for 7 days. As a control, 1% DMSO without any compounds was supplemented in the medium. A photograph of the 0.2% CV-stained biofilms is shown. (b) Biofilm biomass was quantified by measuring absorbance at 595 nm. (c) Growth of E. coli BW25113 was monitored in the presence of EGCG at the indicated concentrations. As a control, 1% DMSO was added to the culture. (b,c) The means and standard deviations from at least triplicate determinations are represented. (d) Basic chemical structure of the compounds used in this study and summary of structure-function relationship among them are shown. IC₅₀ was calculated as previously described. DB, double bond; Rham, rhamnose; Gal, gallate.

Figure 3

Effect of EGCG on biofilm formation by a clinically isolated strain of E. coli. (a) Biofilms of E. coli O157:H7 Sakai were formed in the presence of the indicated concentrations of EGCG and quantified as described in Fig. 2. (b) Effect of EGCG on growth of the strain was investigated at 30 °C in YESCA medium supplemented with the indicated concentrations of EGCG. As a control, 1% DMSO without any compounds was supplemented in the medium. Absorbance at 600 nm every 30 min was measured. The means and standard deviations from at least triplicate determinations are represented.

Figure 4

Effects of EGCG and Myricetin on curli production in liquid cultures. E. coli BW25113 were grown in YESCA medium in the presence of Myricetin or EGCG (10 μM each) at 30 °C for 48 h, stained with uranyl acetate, and observed by TEM. As a control, the medium was supplemented with 1% DMSO. Scales, 500 nm.

Figure 5

Effects of EGCG on cellular levels of curli related proteins. E. coli BW25113 cells were grown at 30 °C for 48 h in YESCA medium supplemented with the indicated concentrations of EGCG. As a control, the medium was supplemented with 1% DMSO. Curli-related proteins (CsgA, CsgD, CsgG, and RpoS) in the cells were analysed by immunoblotting. RpoD was detected as a loading control. Cell lysates of the indicated mutant strains grown in the presence of 1% DMSO were also used as controls. Full-size scans of immunoblots are shown in Supplementary Fig. S2.

Figure 6

Effects of EGCG on transcription of curli-related genes. Quantitative RT-PCR analysis was performed to detect the transcripts of curli-related genes in E. coli BW25113 grown in YESCA liquid medium in the presence of EGCG at the indicated concentrations. As a control, the medium was supplemented with 1% DMSO. The transcript levels were normalized using ftsZ transcripts as internal standards. Relative mRNA levels are calculated as fold ratios relative to control cells. The means and standard deviations from at least triplicate determinations are represented.

Figure 7

Effects of EGCG on the stability of RpoS in vivo. (a) E. coli BW25113 wild type and its isogenic mutant cells (∆rpoS and ∆clpX) were grown to stationary phase (24 h) in YESCA medium supplemented with 50 μM EGCG. As a control, 1% DMSO was added to the medium. At various time points after the addition of Spectinomycin, cellular proteins were analysed by SDS-PAGE and immunoblotting with anti-RpoS, anti-RpoD (loading control), anti-ClpX, and anti-ClpP antibodies. In the panels of ClpX, the upper band indicated by an arrow corresponds to ClpX and the lower one is a non-specific protein. (b) Band intensities of RpoS in immunoblots in a were measured with the LAS-4000 Image Analyser. (c) Half-lives of RpoS in the presence of DMSO and EGCG were calculated from data in (b). The means and standard errors from at least triplicate determinations are represented. (d) Cellular RpoS levels in BW25113 wild type and mutant strains were analysed by immunoblotting as in a. These strains were harvested at 24 h without supplementation of Spectinomycin. Band intensities of RpoS in immunoblots were measured with the LAS-4000 image analyser. (e,f) Spectinomycin-chase experiments were also performed with ∆clpX. *P < 0.05; **P < 0.01; ***P < 0.001 (compared with DMSO control). Full-size scans of immunoblots are shown in Supplementary Figs S7–9.

Figure 8

A schematic model for mode of action of EGCG. Under normal conditions, RpoS activates the csgDEFG operon and expressed CsgD promotes expression of the csgBAC operon. CsgE/F/G assists translocation of CsgA/B to outside the cell and assembly of curli amyloid fibers on the surface of the cell, leading to robust biofilm formation. EGCG may bind to unknown target molecules, which can repress the expression of iraP. The reduction of IraP derespresses RssB-ClpXP protease that extensively degrades RpoS, which downregulates the expression of the csg operons. The outcomes of the addition of EGCG are indicated as red arrows. Actions of EGCG reported by Serra et al. are illustrated by blue bot lines. Combination of these effects remarkably can decrease curli production and biofilm formation.