Designed α-sheet peptides disrupt uropathogenic E. coli biofilms rendering bacteria susceptible to antibiotics and immune cells

Uropathogenic Escherichia coli account for the largest proportion of nosocomial infections in the United States. Nosocomial infections are a major source of increased costs and treatment complications. Many infections are biofilm associated, rendering antibiotic treatments ineffective or cause additional complications (e.g., microbiome depletion). This work presents a potentially complementary non-antibiotic strategy to fight nosocomial infections by inhibiting the formation of amyloid fibrils, a proteinaceous structural reinforcement known as curli in E. coli biofilms. Despite extensive characterization of the fibrils themselves and their associated secretion system, mechanistic details of curli assembly in vivo remain unclear. We hypothesized that, like other amyloid fibrils, curli polymerization involves a unique secondary structure termed “α-sheet”. Biophysical studies herein confirmed the presence of α-sheet structure in prefibrillar species of CsgA, the major component of curli, as it aggregated. Binding of synthetic α-sheet peptides to the soluble α-sheet prefibrillar species inhibited CsgA aggregation in vitro and suppressed amyloid fibril formation in biofilms. Application of synthetic α-sheet peptides also enhanced antibiotic susceptibility and dispersed biofilm-resident bacteria for improved uptake by phagocytic cells. The ability of synthetic α-sheet peptides to reduce biofilm formation, improve antibiotic susceptibility, and enhance clearance by macrophages has broad implications for combating biofilm-associated infections.

Peptide synthesis. Synthetic α-sheet peptide inhibitors were designed in silico as previously described (34), using backbone dihedral angle constraints derived from MD simulations (60, 61). Briefly, peptides contain two α-strands of seven residues each, with amino acids alternating sequentially between L-conformation and D-conformation in each of the strands.
The α-strands are connected by a five residue turn comprised of all L-amino acids, which gives the peptide a hairpin shape. Finally, the tail of each strand consists of a Gly and an Arg residue, followed by acetyl and amide caps at the N-and C-terminus, respectively. Peptides were assembled by solid phase peptide synthesis on Rink amide resin with Fmoc chemistry and HBTU activation. Peptides were cleaved from the resin and side chain deprotected by TFA/TIPS/H2O (95:2.5:2.5) and precipitated by cold ether. Crude peptides were purified to >95% by RP-HPLC using 5 μM C12 or C18 100 Å columns (Phenomenex; Torrance, CA) and atomic masses were confirmed by electrospray mass spectrometry on a Bruker Esquire Ion Trap (Bruker; Billerica, MA). Sequences for the three α-sheet designs described in this study (AP193, AP195, and AP5) as well as the unstructured control (P1) are listed in SI Table 1. In the case of AP193 and AP195, two monomers were linked together via their Cys residues to form a homodimer; an oxidation that was carried out by dissolving pure peptide monomer in isopropyl alcohol and diluting to 0.1 mg/mL in 100 mM ammonium carbonate buffer, pH ~10. Peptides were oxidized by air at room temperature with stirring for 24 h prior to a second round of purification and mass spectrometry.
The same procedure was applied for AP195/199 dimers, but in this case equimolar amounts of AP195 and AP199 monomer were oxidized to form a heterodimer product. Dimerization was confirmed by measuring the concentration of free thiols (indicative of monomeric peptide) in solution using the Ellman's reagent, high performance liquid chromatography (HPLC), and the mass of the monomeric and dimerized products was verified using mass spectrometry. All peptides were lyophilized after purification and stored at -20 or -80°C until use.
Biofilm culturing and assays. The E. coli strains used in this study are listed in SI Table   1. Overnight cultures were grown in LB medium (Thermo Scientific; Waltham, MA) for ~18 h.
Peptide stocks were dissolved in water and concentrations were determined by Nanodrop™ (Thermo Scientific; Waltham, MA). 20 μL of peptide stock and/or sterile ddH2O was added to each well of a sterile 48 well plate such that the final peptide concentration was 0, 2, 4, 10, or 16 μM (0, 1, 2, 5, or 8 μM for dimeric peptides), then 180 μL of diluted bacteria culture was added on top. Plates were covered, sealed in plastic bags, and incubated at 26°C for 48 h. After growth, planktonic cells and medium were removed and biofilms were rinsed once with 250 μL PBS.
Planktonic cells were spun down and resuspended in PBS, and the optical density of both planktonic and rinse samples was determined at 600 nm to estimate cell densities. The PBS solution was removed and biofilms were resuspended in 250 μL PBS + 20 μM ThT (Sigma-Aldrich; St. Louis, MO). Biofilms were homogenized by vigorous pipetting (30x per well), 3 min sonication, and 1 min on a plate shaker. 100 μL of each biofilm suspension was then transferred to a black-walled, clear-bottom 96 well plate for measurements in a plate reader (PerkinElmer; Waltham, MA). ThT fluorescence was measured at 438/495 nm as a proxy for amyloid formation, and biofilm absorbance was measured at 600 nm to estimate bacterial cell density. For UTI89 WT, biofilm ThT fluorescence values were normalized to the average value of peptide-free controls, and then the average fluorescence value of UTI89 ΔcsgA samples was subtracted to account for nonspecific binding. In the case of antibiotic susceptibility tests, biofilms were cultivated in the same manner, but 100 μL YESCA or 100 μL YESCA supplemented with 900 μg/mL gentamicin (Thermo Scientific; Waltham, MA) was added to wells 6 h before the end of incubation. After incubation, planktonic cells and medium were removed and biofilms were rinsed once in sterile PBS. Biofilms were then resuspended in sterile PBS, homogenized by ultrasonication for 5 s on ice, and then diluted in tenfold increments for CFU plate counts with the drop plate method (44).

CsgA expression and purification.
A synthetic gene corresponding to the E. coli CsgA protein, minus its sec signal sequence, was designed and synthesized by GenScript (Piscataway, NJ). The gene was cloned into the pET30a(+) vector, which added a C-terminal 6x His tag for purification. Plasmids were transformed into E. coli BL21 (DE3) cells and protein expression was carried out in 2 L shake flasks at 37°C. Cultures grew to an OD600nm of 0.6-0.8 prior to induction with 1 mM IPTG. After 3-4 hours of additional growth, cells were harvested by centrifugation and resuspended in 30 mL denaturing buffer (8M Gnd-HCl, 50 mM NaPi, pH 8.0) and lysed overnight with stirring at 4°C. Insoluble material was removed by centrifugation at 14,000 xg for 30 minutes and 15 mL of supernatant was incubated with 5 mL HisPur Ni-NTA beads (Thermo Fisher; Waltham, MA) for 2 h at room temperature with end-over-end rotation. The beads were then washed twice with denaturing buffer, twice again with denaturing buffer plus 15 mM imidazole, and twice again with denaturing buffer plus 30 mM imidazole. Finally, protein was eluted with denaturing buffer plus 400 mM imidazole. Samples from each step of the purification were precipitated from guanidinium hydrochloride by trichloroacetic acid(62) and analyzed by SDS-PAGE. Purified fractions were of high concentration and purity, so they were not purified further prior to aggregation assays.
Aggregation assays and analysis. Immediately prior to use, protein eluents were thawed and desalted according to the Zeba desalting column protocol (Thermo Fisher) into 50 mM potassium phosphate pH 6.2 and kept on ice. Protein concentration was determined by absorption at 280 nm, and the stock was diluted to a working concentration of 0.2 mg/mL (~14 μM) or 10 μM, depending on the assay. ThT stock was added to a concentration of 20 μM, and then CsgA was aliquotted into black-walled, clear-bottom 96 well plates at 100 μL per well. Plates were incubated in the dark at 25°C without shaking, and fluorescence measurements were taken every ~12 h on a plate reader (PerkinElmer) with soft motion robotics to avoid agitation. Every well in the plate was read at every timepoint, regardless of the number of wells containing sample, in order to maintain consistent agitation. In the case of peptide inhibition tests, synthetic α-sheet peptide AP193 was prepared as above and added to the incubation mixture at a concentration of Circular dichroism. At given timepoints, CsgA samples for CD were transferred directly from microtiter incubation plates to a 1 mm quartz cuvette. CD spectra were recorded on a J-720 spectropolarimeter (Jasco, Inc.; Easton, MD) with the following settings: wavelength 260-195 nm, 0.5 nm data pitch, 1 s integration, 2 nm bandwidth, 25°C, and 6 accumulations. Only data points with detector voltages below 600 V were used. All spectra were corrected by a blank spectrum of relevant buffer and smoothed with a Savitsky-Golay filter at window size 35 and polynomial order 2. The same protocol and settings were used for the designed peptides at 25 µM.

Microscopy. For adherence tests, UTI89 WT biofilms were grown in 8 well Chamber
Slides (Lab-Tek™, Thermo Fisher) for 48 h in YESCA broth + 4% DMSO. Planktonic cells and medium were removed, biofilms were rinsed once with PBS, and biofilms fixed with 4% paraformaldehyde and stained with SYTO 9 (Thermo Fisher) prior to imaging on a Zeiss Axioscope inverted fluorescence microscope using the GFP filter (Carl Zeiss AG; Oberkochen, Germany). For phagocytosis, samples were prepared as described below for flow cytometry. A portion of each sample was applied to a glass microscope slide using a Cytospin™ centrifuge

RT-qPCR.
For analysis of csgA expression in E. coli UTI89 WT, biofilms were cultivated in 48 well plates with or without 8 μM AP193, as described above. Biofilm-associated bacteria were collected and normalized to a concentration of 10 8 cells/mL. Bacterial mixtures were combined with RNAprotect Bacteria Reagent (Qiagen; Hilden, Germany) and lysed. For analysis of macrophage polarization markers, RAW 264.7 cells were seeded in 24 well plates at ~5 x 10 4 cells per well. After 24 h, the growth medium was aspirated and replaced with media supplemented with synthetic peptides (5 μM AP193, 5 μM AP195, 10 μM AP5, 10 μM P1), or sterile water in the case of controls. The plate was incubated a further 24 h, then cells were trypsinized to detach them from the plate, washed, and resuspended in FACS buffer. For all samples, total RNA was extracted and purified with the RNeasy Mini Kit (Qiagen) according to manufacturer instructions. cDNA was synthesized with iScript RT Supermix (BioRad; Hercules, CA) and quantified with the Qubit system (Thermo Fisher). Each RT-qPCR reaction utilized 10-20 ng of cDNA, 10 μL Power SYBR Green Master Mix (Thermo Fisher), and 0.4 μM each of forward and reverse primers; samples were prepared in duplicate with appropriate housekeeping gene controls and template-free controls (primers in SI Table 1). Thermal cycling was carried out SI Table 1. Peptide and primer sequences; bacterial strain descriptions. a "AP" refers to "Alternating Peptide", indicating alternating L-and D-amino acid templating. "P" refers to "Peptide", indicating a lack of templating. b L-amino acids are displayed in all upper case; D-amino acids are displayed in lower case and underlined. c F = forward primer; R = reverse primer. * Indicates housekeeping gene SI Figure S1. E. coli produce curli through a coordinated system of proteins. The major curli subunit, CsgA, begins as a soluble monomer in the cytoplasm prior to its export and cleavage by the general secretory (Sec) system. In the periplasm, CsgA interacts with chaperone CsgC and is then exported to the extracellular space by CsgE and CsgG. There, CsgB serves as a template for rapid amyloid fiber formation by CsgA, and CsgF anchors the fibril complex. See references 9 and 10 for further information.

CsgG CsgE Sec
CsgC SI Figure S2. Normalized ThT fluorescence values for E. coli UTI89 WT biofilms (gray bars) grown in the presence of increasing concentrations of the synthetic α-sheet peptide, AP5. Biofilms of UTI89 ΔcsgA (white bars) were cultivated in the same experiment to provide an estimate of non-specific ThT fluorescence. This non-specific signal was subtracted from UTI89 WT signals to produce the corrected fluorescence values shown Figure 1C,D. Error bars indicate the standard deviation from the mean of three replicates. ns = not significant; * p < 0.05; ** p < 0.01 according to a two-tailed, homoscedastic Student's t-Test.  Figure S4. Alternating backbone chirality generates uniquely featureless CD spectra (blue and orange lines for dimeric α-sheet peptides AP193 and AP195, respectively) compared to that for a peptide with random coil secondary structure (green line for random coil peptide P1).  Figure S5. Estimation of bacterial cell counts in biofilm ThT assays. Cells were collected and homogenized during each phase of the assay (planktonic, rinse, biofilm) and the number of cells was estimated according to the optical density of samples at 600 nm. Peptides did not affect growth; instead, they shifted bacteria from the biofilm-associated state to the planktonic state. Error bars indicate the standard deviation from the mean of three replicates. AP193 and AP195 are homodimers, AP193/AP195 is a heterodimer, and AP5 and P1 are monomers. Figure S6. AP401 has no effect on antibiotic susceptibility of E. coli in CsgA knockout. pvalue > 0.05.