Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli

Abstract

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.

Keywords: ATR-FTIR; FT-Raman; aggregation; biofilm; curli; functional amyloids.

Figure 1

Pairwise sequence alignment between CsgA fragments of E. coli and S. enterica bacteria. The differences in the amino acid compositions are highlighted with light purple.

Figure 2

Far-UV CD spectra of CsgA fragments on the day of the dissolving. (A) Spectra for E. coli fragments, (B) Spectra for S. enterica fragments on the day of the dissolving (final peptide concentration 500 μM).

Figure 3

Normalized ATR-FTIR spectra of E. coli fragments in the wavenumber range of 1725–1590 cm⁻¹ (Amide I), smoothed SG 35 (see Methods). (A) on the day of the dissolving (B) after one month of incubation at 37 °C. Peptide concentration was 500 μM.

Figure 4

Normalized ATR-FTIR spectra of S. enterica fragments in the wavenumber range of 1725–1590 cm⁻¹ (Amide I), smoothed with SG 35 (see Methods). (A) on the day of the dissolving (B) after month incubation at 37 °C. Peptide concentration was 500 μM.

Figure 5

PCA plot for E. coli and S. enterica. Samples on the day of the dissolving and after 30 days of incubation at 37 °C. Points on the left side correspond to aggregates, on the right side to random structures.

Figure 6

Normalized FT-Raman spectra of CsgA protein fragments, smoothed with SG 35 (see Methods), in the wavenumber range of 1725–1575 cm⁻¹ (Amide I). (A) Spectra for E. coli fragments after 30 days of incubation at 37 °C, (B) Spectra for S. enterica fragments after 30 days of incubation at 37 °C. Peptide concentration was 500 μM.

Figure 7

Electron micrographs of S. enterica fragments after one to seven days of incubation in 37 °C. Images registered at the magnification of 200 nm. Peptide concentration was 0.5 μM.

Figure 8

Time-dependent ThT fluorescence curves for R4 fragments. Here, grey dots represent E. coli, blue ones S. enterica. Peptide concentration was 500 μM.

Figure 9

Comparison R4 fragments. (A) E. coli on the day of dissolving (B) S. enterica on day of dissolving (C) E. coli after 7 days of incubation in 37 °C. Images registered at the magnification of 200 nm. Peptide concentration was 0.5 μM.