Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin

Abstract

During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.

Keywords: CsgA; amyloids; biofilms; curli; transthyretin.

Fig. 1.

Transthyretin inhibits CsgA amyloid formation in vitro. Purified CsgA was incubated with substoichiometric concentrations of (A) WT-TTR, (B) M-TTR, and (C) BSA and human lysozyme (Lyz). Aggregation kinetics were monitored by ThT fluorescence.

Fig. 2.

Microscopy images documenting fibers and aggregates. Representative TEM images taken at zero time point showed no aggregation in (A) 15 µM CsgA, (B) 1:1 CsgA: WT-TTR, and (C) 1:1 CsgA: M-TTR. Images taken after 15 h of incubation depicted (D) CsgA fibers, (E) thin proto-fibril like structures when CsgA incubated with WT-TTR at 1:1 molar ratio, and (F) unstructured amorphous aggregates when CsgA incubated with M-TTR at 1:1 molar ratio. (Scale bars, 100 nm.)

Fig. 3.

Structural transitions of CsgA in presence of transthyretin. AFM images taken after 15 h showed (A) CsgA (15 µM) fibers and (B) spherical oligomers when CsgA incubated with M-TTR at 1:1 molar ratio. (C) Relative peak intensities of 1D NMR data showing major signal decays in the aliphatic region of CsgA after 76 h of incubation as the protein turns into amyloid fibers. (D) The experiment was repeated in the presence of 1:1 CsgA: M-TTR and 50 µM M-TTR alone.

Fig. 4.

Effect of transthyretin on seeded CsgA aggregation. Polymerization of CsgA in the presence of 2% (wt/vol) preformed and sonicated CsgA fibrils (CsgA seeds) prepared as described in Materials and Methods. (A) CsgA seeds accelerated CsgA polymerization. (B) CsgA seeds accelerated CsgA polymerization in presence of WT-TTR. (C) CsgA seeded polymerization inhibited by M-TTR.

Fig. 5.

Effect of occupied T4 binding site on CsgA amyloid inhibition. CsgA polymerization expressed as T50 in presence of tetrameric TTR (WT-TTR and T119M mutant) incubated without and with small molecules that bind in the T4 site: (A) with tafamidis (taf), (B) with resveratrol (res), (C) with A2. Presence of small molecules in T4 site abolished the inhibitory activity of WT-TTR and T119M mutant. ns, not significant, *P < 0.03, **P < 0.002, ***P < 0.0002.

Fig. 6.

Transthyretin suppressed pellicle-biofilm formation in E. coli UTI89. (A) Concentration-dependent effect of WT-TTR and M-TTR on biofilm formation in E. coli UTI89 grown in YESCA media at 26 °C for 48 h under quiescent conditions. E. coli UTI89 in media (Top), +WT-TTR (Middle), and +M-TTR (Bottom). (B) Biofilm biomass quantification in untreated cells and cells with exogenously added WT-TTR and M-TTR. *P = 0.001 and **P < 0.0001. Representative TEM images of (C) E. coli UTI89 cells, (D) E. coli UTI89 cells with 15 µM WT-TTR, and (E) E. coli UTI89 cells with 15 µM M-TTR. (Scale bars, 300 nm.) The arrow indicates network of curli fibers. (F) Growth curves of E. coli UTI89 in absence and presence of WT-TTR and M-TTR.