Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation

Abstract

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

Figure 1

Curlicide inhibition of in vivo amyloid biogenesis in E. coli. (a) BibC10 (1), FN075 (2), BibC6 (3) and VA028 (4) are ring-fused 2-pyridones that differ in their phenyl ring modifications and, for 3, in the position of naphthyl substitution. (b) CsgA, CsgB, CsgF and CsgG protein profiles obtained by western analysis of MC4100 grown on curlicide-amended agar for 48 h. Each curlicide was tested at 0.1, 1.0 and 2.5 mM, and the positive control sample corresponds to cells grown in DMSO-amended agar. MC4100Δcsg was the negative control. Curli production was abolished at 1.0 mM compound 2, 2.5 mM compound 3. Only a faint CsgA band was observed at 2.5 mM compound 4. (c) Curlicides were effective at the designated micromolar concentrations in blocking curli biogenesis in UTI89 growing in shaking YESCA broth (100 rpm) for 48 h at 28 °C. (d) Representative high-resolution EM images of UTI89 prepared as in c. Titratable reductions in bacterial curliation were observed for cells grown in the presence of curlicides. The scale bar in the first electron micrograph represents 0.2 μm and applies to all images.

Figure 2

Curlicide inhibition of in vitro CsgA polymerization. (a) The fluorescence of freshly purified CsgA mixed with 25 μM ThT, and respective curlicides, was measured at 495 nm after excitation at 438 nm. Data points corresponding to 60-min intervals are displayed. (b) Reductions in ThT fluorescence corresponded to reduced CsgA polymerization. Soluble CsgA levels were confirmed by SDS-PAGE and western analysis using anti-CsgA antibodies. Polymerized CsgA is SDS insoluble, and migration is impeded during electrophoresis.

Figure 3

Curlicide inhibition of curli-dependent UTI89 pellicle formation. (a) Macroscopic observation of curli-dependent pellicles. Pellicle biofilms were visible in wild-type UTI89 culture 48 h post-inoculation in YESCA broth. Deletion of csgA (UTI89ΔcsgA) abolished pellicle formation, and the pellicle was restored by providing csgA in trans (UTI89ΔcsgA/pLR5). (b) Three-dimensional confocal microscopy reconstructions revealed thick pellicles with complex architectures. Pellicles were stained with nucleic acid dye STYO9 (green). Each grid unit is 14.3 μm. (c) Three-dimensional reconstructions of confocal microscopy images showed that GFP reporter gene expression (green) driven by the csgB; csgA promoter is active in UTI89 pellicle. Bacteria were counterstained with nucleic acid dye SYTO83 (red). Grid units were 7.4 μm for the top panel and 14.3 μm for the bottom panel. (d) Curli were detected in the pellicle by Congo red uptake in bright field (DIC) images merged with the red fluorescence channel. Congo red uptake was apparent by macroscopic examination of pellicle (inset). (e) Immunostaining of pellicle segments with anti-CsgA antisera illustrated homogeneous CsgA presence among aggregated cells, whereas planktonic bacteria were unlabeled (top right panel). Calcofluor staining indicated that β-glucans such as cellulose colocalize in the pellicle (bottom panels). (f) Curlicides blocked pellicle formation in a 24-well plate assay in which UTI89 was grown in Congo red–containing YESCA medium at 30 °C (rows 2 and 3). Congo red had no effect on the results but permitted facile display of pellicle roughness. Equivalent carrier volumes of ethanol had no effect on pellicle formation (top row), and pellicle was unaffected by pilicide BibC10, 1 (bottom row).

Figure 4

Curlicide inhibition of UTI89 amyloid-dependent and type 1 pili-dependent biofilm formation on PVC. (a) Curli were dispensable for UTI89 biofilm formation in LB but were required for YESCA-grown biofilms. (b) UTI89 biofilm formation in YESCA in 96-well PVC plates was inhibited in the presence of curlicides, as quantified by crystal violet staining. (c) FN075 inhibited UTI89 biofilm formation on PVC in LB in a dose-dependent manner. Percent biofilm inhibition represents the decrease in biofilm formation in the presence of curlicide relative to biofilm formation when no compound was present. Bars in b and c represent s.d. of the mean of four experiments, where controls with no compound were included in each experiment on each plate. No effects on bacterial growth rate were observed at these compound concentrations (Supplementary Fig. 2).

Figure 5

FN075 attenuation of virulence in vivo. (a) Curli mutants, UTI89ΔcsgBG and UTI89ΔcsgA, were significantly attenuated in bladder colonization compared to UTI89, as determined by CFU counts (P < 0.007 and P < 0.011, respectively, as determined by a two-tailed Mann Whitney for each mutant–wild-type pair). Geometric means are indicated by solid lines. (b) FN075 (2) reduced virulence of UTI89 in the mouse model of urinary tract infection. Bacteria were grown in the presence of 250 μM FN075 and then introduced into bladders of 7- to 8-week-old female C3H/HeN mice by a single transurethral inoculation of 1–2 × 10⁷ CFU. FN075-treated cells exhibited more than a 10-fold decrease in CFU/bladder at 6 hpi (P < 0.0001, two-tailed Mann-Whitney test) and produced significantly fewer IBCs than nontreated bacteria (P < 0.0001).