Epigallocatechin Gallate Remodels Overexpressed Functional Amyloids in Pseudomonas aeruginosa and Increases Biofilm Susceptibility to Antibiotic Treatment

Abstract

Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea. It has antimicrobial properties and disrupts the ordered structure of amyloid fibrils involved in human disease. The antimicrobial effect of EGCG against the opportunistic pathogen Pseudomonas aeruginosa has been shown to involve disruption of quorum sensing (QS). Functional amyloid fibrils in P. aeruginosa (Fap) are able to bind and retain quorum-sensing molecules, suggesting that EGCG interferes with QS through structural remodeling of amyloid fibrils. Here we show that EGCG inhibits the ability of Fap to form fibrils; instead, EGCG stabilizes protein oligomers. Existing fibrils are remodeled by EGCG into non-amyloid aggregates. This fibril remodeling increases the binding of pyocyanin, demonstrating a mechanism by which EGCG can affect the QS function of functional amyloid. EGCG reduced the amyloid-specific fluorescent thioflavin T signal in P. aeruginosa biofilms at concentrations known to exert an antimicrobial effect. Nanoindentation studies showed that EGCG reduced the stiffness of biofilm containing Fap fibrils but not in biofilm with little Fap. In a combination treatment with EGCG and tobramycin, EGCG had a moderate effect on the minimum bactericidal eradication concentration against wild-type P. aeruginosa biofilms, whereas EGCG had a more pronounced effect when Fap was overexpressed. Our results provide a direct molecular explanation for the ability of EGCG to disrupt P. aeruginosa QS and modify its biofilm and strengthens the case for EGCG as a candidate in multidrug treatment of persistent biofilm infections.

Effects of pharmacological modulators of α-synuclein and tau aggregation and internalization

“…This is consistent with previous reports in which treatment with EGCG was performed in already formed aggregates 16,17,56 . This effect was not only reported for aSyn, but also for other amyloidogenic proteins, such as AB and γ-synuclein (gSyn) 16, 56,57 . Such effects are in agreement with the proposed mechanism of action for EGCG, whereby it mediates the reorientation of bonds between ordered protein molecules, leading to amyloid remodeling and the appearance of unordered, amorphous protein aggregates 17,58 .…”

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

“…EGCG also inhibits PAO1 FapC amyloid formation by stabilizing pre-fibrillar oligomers, and it structurally remodels existing fibrils in vitro through the formation of non-amyloid aggregates [13]. To investigate the effect of EGCG on fibril formation of FapC WT and R3, desalted protein stocks were diluted in Tris buffer with various concentrations of EGCG.…”

“…Biofilm-associated bacteria, in particular, utilize amyloids as a building material to reinforce the EM and resist dispersion by chemical or mechanical agents [8, 11]. These fibrils may also promote antibiotic resistance and act as a reservoir for small molecules involved in quorum sensing [12, 13]. Amyloid fibrils polymerize in the absence of an energy source, so they serve as a metabolically advantageous molecular scaffold despite the limited resources of the EM environment [14].…”

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c

“…When P1 and/or WapA are completely absent due to gene deletion, amyloid inhibitors have a diminished impact on biofilm formation compared to untreated cells because their targets of activity are gone, and the glucans can mediate cell-cell and cell-substratum interactions unhindered by monomeric or oliogomeric P1 and WapA. It has been reported that EGCG disrupts the ability of P. aeruginosa Fap to form amyloid fibrils by stabilizing protein oligomers [59]. In addition, these authors found that treatment of P. aeruginosa biofilms with EGCG decreased the minimal bactericidal concentration of tobramycin, an effect that was amplified when fap genes were overexpressed.…”