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

Marcel Stenvang,Morten S Dueholm,Brian S Vad,Thomas Seviour,Guanghong Zeng,Susana Geifman-Shochat,Mads T Søndergaard,Gunna Christiansen,Rikke Louise Meyer,Staffan Kjelleberg,Per Halkjær Nielsen,Daniel E Otzen

doi:10.1074/jbc.m116.739953

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.

Highlights

Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea
Protein composition of the fibrils and sequence analysis suggests that these proteins have specific roles as a fibrillation nucleator (FapB), a ␤-barrel membrane pore used for secretion of FapC and FapB monomers (FapF), and a protease required for processing of the amyloid subunits (FapD) [16, 23]
EGCG and Doxycycline Remodel FapC Amyloid Fibrils—To investigate the possibility that functional amyloids in P. aeruginosa can be remodeled, we screened the effect of EGCG together with three other compound candidates that have shown antiamyloid effects on other proteins: silibinin [50], doxycycline (A␤ and ␤2-microglobulin fibrils) [51, 52], and curcumin (A␤ fibrils) [53]

Summary

Introduction

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. Functional amyloids were first discovered in Escherichia coli and termed curli [14], and several others were later discovered, e.g. TasA in Bacillus subtilis [15] and Fap in Pseudomonas [16, 17] These organisms all produce extracellular fibrils as part of the biofilm. Overexpression of the fap operon in P. aeruginosa PAO1 leads to a highly aggregative and adherent phenotype that forms excessive amounts of biofilm Analysis of this overexpressing strain showed that amyloid formation markedly changes the overall proteome [16, 23]. Fap overexpression reduces the abundance of virulence factors and increases that of biofilm-associated proteins These proteomic changes have several parallels to those observed in chronic infections of cystic fibrosis patients [23]. UK4 [26, 27] has been shown to increase biofilm hydrophobicity and mechanical strength

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