Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids

Abstract

The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The Biofilm Associated Protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.

Figure 1

Inhibitory effect of polyphenols in the biofilm formation assay. (a) Biofilm formed by S. aureus V329 (amyloid-based biofilm) and S. aureus 15981 (polysaccharide-based biofilm) in the presence of sub-MICs concentrations of polyphenols (1/500; 1/100; 1/50). The bacterial cells were stained with crystal violet. 2% of DMSO was added as control (Ø). Bacteria cultured in TSB-glu media were used as positive control (−). (b) Quantification of the crystal violet after addition of 200 μl of ethanol-acetone and determination of absorbance at 595 nm. S. aureus V329 (white bars). S. aureus 15981 (grey bars). The error bars represent the standard deviation of the results of three repetitions. Statistical significance was determined with one-way ANOVA followed by multiple comparison test (*P < 0.05; **P < 0.01; ***P < 0.001). QC Quercetin, MC myricetin, BC baicalein, SC scutellarein, ER eriodictyol, GN genistein, AG gallic acid, RV resveratrol, CC curcumin.

Figure 2

Antibiofilm activity of polyphenols against 3 non-related S. aureus strains that express Bap (C104, V858 and Newman-Bap) and other staphylococcal species (S. hycus, S. simiae and S. saprophytycus) that form Bap-dependent biofilms. MBIC were used as 10 μg/ml for quercetin (QC), 10 μg/ml for myricetin (MC) and 5 μg/ml for scutellarein (SC). 2% of DMSO was added as control (Ø). Bacteria cultured in TSB-glu media were used as positive control (−). Crystal violet was quantified by measuring absorbance at 595 nm. Statistical significance was determined with one-way ANOVA followed by multiple comparison test (*P < 0.05;**P < 0.01; ***P < 0.001).

Figure 3

Biofilm growth of S. aureus V329 (a) and 15981 (b) in the presence of polyphenols. Graphs show total biofilm mass through time, as determined by impedance measurements in the absence (black line) and presence of quercetin (QC) 10 μg/ml, myricetin (MC) 10 μg/ml, scutellarein (SC) 5 μg/ml and baicalein (BC) 10 μg/ml. Quantification of biofilm growth was recorded every 10 min at 37 °C. Each line represents the mean of three biological replicates. For statistical differences in biofilm CI values, regression analysis was assessed by a linear model between 15 and 25 h of biofilm growth, using the lm library in the R statistical package version 1.0.7.1. (*P < 0.05). SDs are not shown for clarity.

Figure 4

Western immunoblot showing the effect of polyphenols on the expression and aggregation of Bap. (a) GFP protein levels of S. aureus V329 with pCN52-P_bap:GFP plasmid in presence of MBIC of : 10 μg/ml for quercetin (QC), 10 μg/ml for myricetin (MC) and 5 μg/ml for scutellarein (SC). (b) Bap protein levels of S. aureus V329 in presence of MBICs of polyphenols. (c) Native immunoblotting of cell surface extracts S. aureus V329 cultured in presence of MBICs of polyphenols. Bap-related insoluble aggregates are indicated by a dashed line box. Untreated bacterial cells were used as positive control (Ø).

Figure 5

Assembly of Bap_B using the curli-dependent amyloid generator (C-DAG) in the presence of polyphenols. (a) Representative scheme of C-DAG. Bap_B domain is cloned under the control of the inducible PBAD promoter and the signal sequence of CsgA. CsgG is expressed under the control of an IPTG-inducible promoter and forms a pore in the OM for externalization of Bap_B. Bap_B forms amyloid-like fibers (left panel). Polyphenols (green circles) can interrupt the assembly of Bap_B into amyloid fibers (right panel). Outer membrane (OM), cytoplasmic membrane (CM), periplasmic space (PS). (b) Quantification of CR bound to E. coli cells expressing Bap_B from S. aureus and S. saprophytycus in the presence of MBIC of polyphenols. Ø, without polyphenols. The relative effect of the polyphenols was calculated as OD_500nm Bap_B − OD_500nm Bap_A. Bars represent standard deviations of the results of five independent experiments (n = 5). Statistically significant differences were determined using Mann–Whitney test *P < 0.05. (c) Transmission electron micrographs of negatively stained fiber-like structures formed by E. coli cells that express S. aureus Bap_B. Extracellular fibrous structures are not observed in the presence of polyphenols.

Figure 6

Polyphenols inhibit aggregation of a recombinant rBap_B. (a) Kinetic aggregation of recombinant rBap_B protein at 0.1 mg/ml in presence of 200 µM of the polyphenols in phosphate-citrate buffer pH 4.4. Th-T amyloid-dye was added and emission spectra were recorded in the range of 460–600 nm. (b) The basal fluorescence of polyphenols themselves were recorded. (c) Kinetic aggregation assays of rBap_B in the in presence of polyphenols, followed by solution turbidity. Turbidity was measured as absorbance at 360 nm every 10 min. Black dots: positive aggregation control (only rBap_B).

Figure 7

Efficacy of polyphenols in S. aureus biofilm infection model. Two catheters infected with S. aureus strain V329 were implanted at the subscapular space of groups of mice (n = 6), followed by subcutaneous administration of quercetin (QC) 100 mg/kg, myricetin (MC) 20 mg/kg, scutellarein (SC) 17.5 mg/kg post-infection and repeated every day for 10 days. Animal were killed and bacteria were recovered from implanted catheters and counted. The plots display values obtained from individual catheters and the mean is represented by horizontal bars. Statistical significance was determined with one-way ANOVA followed by Dunn’s multiple comparison test comparing to mice that received DMSO as control, Ø. (**P < 0.01; ***P < 0.001).