Fig 1.
Overall structure of the CsgG-CsgF complex.
(A) Overall structure of the nonameric CsgG-CsgF complex shown in ribbon representation from different perspectives: side view (left) and extracellular view (right). The CsgG-CsgF complex contains 9 CsgG-CsgF subcomplexes. CsgG (one CsgG monomer is highlighted in blue) and CsgF are colored in green and violet, respectively. In the complex structure, the C-terminal 5 residues of CsgG and 85 residues of CsgF are invisible. (B) Structure of the CsgG-bound CsgF20–53 fragment. Cryo-EM densities of CsgF20–53 fragment are shown on the left (blue), and its secondary structures in ribbon are shown on the right (violet). (C) Close-up view of the constriction regions of the CsgG-CsgF channel. Slab view of the electrostatic surface representation of the CsgG-CsgF channel showing the 2 constriction regions (left) and the lining residues at the 2 eyelets (middle). The CsgG constriction region and the CsgF constriction region have pore dimensions of approximately 12 Å and 14 Å, respectively, in diameter (shortest atom-to-atom distance). Map densities and side chains of the 9 Asn36 of CsgF that consist of the CsgF constriction region are shown on the right. Csg, curli-specific gene product; OM, outer membrane.
Fig 2.
Characterization of the interactions between CsgG and CsgF.
(A) Close-up view interactions between CsgG and CsgF in the CsgG-CsgF complex. Two adjacent CsgG molecules are colored in green and cyan. The most N-terminal loop (including residues 20–31, in yellow) of CsgF primarily binds at the joint area of 2 adjacent CsgG monomers; the α helix (including residues 37–46, in violet) of CsgF interacts with one CsgG monomer, and the connecting loop (including residues 32–36, in grey) of CsgF has no interaction with CsgG. (B) ITC measurement of the affinity between CsgG and the full-length CsgF. The underlying numerical data for the figure are shown in S1 Data. (C) GST pull-down experiments (upper panel) and western blot to detect CsgGHis (bottom panel). CsgG nonamers and the nonameric CsgG-CsgF20–53 complexes were unable to be dissociated to monomers on the 12% SDS-PAGE unless the samples were boiled at 95°C for 10 minutes before loading. CsgF20–53-GST has a close molecular mass (approximately 28.9 kDa) to CsgGHis. (D) Immunofluorescence assays showing that surface exposure of CsgF requires CsgG. DAPI is a fluorescent stain that binds strongly to adenine–thymine rich regions in DNA to show the location of the bacterial cells. Csg, curli-specific gene product; DAPI, 4′,6-diamidino-2-phenylindole; GST, glutathione s-transferase; ITC, isothermal titration calorimetry; WT, wild-type.
Fig 3.
Mutational analysis of the three lumen-facing residues of CsgF.
(A) CR staining assays showing curli production of the WT- and the ΔcsgF-E. coli K-12 BW25113 strains over time. Pictures were taken at 5 different incubation time points (12, 24, 36, 48, and 60 hours, respectively). (B) Positions of CsgF residues selected for mutational studies in the CsgG-CsgF complex. (C) Residues of CsgF (N36, A39, and N43) point to the lumen of the nonameric CsgG-CsgF channel (extracellular view). Residues are shown in stick mode. (D) Complementary assay using CR staining. Mutants N36R, A39R, and N43R displayed significantly reduced curli production. CR, Congo red; Csg, curli-specific gene product; WT, wild-type.
Fig 4.
The CsgF-derived peptides inhibit curli production.
(A) Amino acid sequences of 4 synthesized peptides. (B) Effects of peptides G20-D53_CsgF (left) and A23-N47_CsgB (right) on curli production of both the WT- and the ΔcsgF-E. coli strains. (C) Effects of peptides G20-N36_CsgF (left) and G20-R36_CsgF (right) on curli production of both the WT- and the ΔcsgF-E. coli strains. (D) Cell growth curves of the WT-E. coli (left) and the ΔcsgF-E. coli (right) stains in presence or absence of 3 CsgF-derived peptides. The underlying numerical data for the figure can be found in S1 Data. Csg, curli-specific gene product; O.D., optical density; WT, wild-type.