The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation

Abstract

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β-strand-loop-β-strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the first, second, or third repeating units were able to convert CsgA into fibers. However, mutants missing either the fourth or fifth repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2, and 4 self-assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.

Figure 1. Biochemical and physiological properties of WT CsgB

(A) Coomassie stained SDS-PAGE of OD600 normalized cell lysates pre-induction (IPTG −) or post-induction (IPTG +) lanes 1 through 3. Cells were resuspended in SDS loading buffer (FA −) or pretreated with formic acid (FA+). CsgB was solubilized post purification by incubation in 8 M guanidine HCl (Lane 4). (B) Representative ThT kinetic plot of 70 μM (○), 35 μM (□), or 17.5 μM (◇) purified CsgB and 27 μM purified CsgA (X). Relative fluorescent units (RFUs) emitted at 495 nm were recorded every 10 min. after excitation at 438 nm. (C) TEM of 65 μM CsgB incubated at room temperature for 24 hours. The scale bar represents 500 nm. (D) Coomassie stained SDS-PAGE of 65 μM CsgB polymerized into fibers, centrifuged and resuspended in SDS loading buffer with (+) or without (−) prior formic acid (FA) treatment. (E) ThT kinetic plot of 27 μM CsgA (X), 27 μM CsgA and +5% w/w WT CsgB seeds (●) and 27μM CsgA + 12% w/w WT CsgB seeds (■). (F) Surface plasmon resonance (SPR) sensorgrams of interactions between monomeric CsgA and CsgA seeds or CsgB seeds. 0.25μM fresh monomeric CsgA was flow over the CM5 chip immobilized with 3uM CsgA seeds, 3.5μM CsgB seeds or no seeds. The interaction was recorded in resonance units.

Figure 2. Exogenously added CsgB functions as a nucleator

(A) 10 ul of 37 μM purified CsgB was overlaid on csgA and csgB mutants that had been grown for 24 hours, and then stained with Congo red dye after an additional 24-hour incubation at 26°C. WT cells are shown as a positive staining control. (B) TEM of csgB cells that were incubated with 37 μM purified CsgB. The scale bar represents 500nm. (C) CsgA western blot analysis of a whole cell (WC) lysate of the csgB cells in (A) that were incubated in the absence of CsgB (Lanes 1 and 2) or presence of CsgB (lanes 3 and 4). Samples were resuspended in SDS loading buffer with (+) or without (−) formic acid (FA) pretreatment.

Figure 3. Contribution of each repeating unit to CsgB function in vivo

(A) Amino acid sequence alignment of the CsgA and CsgB repeating units. Each repeating unit contains two predicted β-sheets (arrows). (B) Congo red binding phenotype of MC4100 (WT), or csgB harboring a vector control plasmid (vector), a plasmid vector containing WT csgB, csgB^Δr1 (Δr1), csgB^Δr2 (Δr2), csgB^Δr3 (Δr3), csgB^Δr4 (Δr4) or csgB^Δr5 (Δr5). (C) Western blot analysis of a csgB mutant strain harboring vector control (vector lanes 1 and 2), a plasmid vector containing WT csgB (lanes 3 and 4), csgB^Δr1 (Δr1 lanes 5 and 6), csgB^Δr2 (Δr2 lanes 7 and 8), csgB^Δr3 (Δr3 lanes 9 and 10), csgB^Δr4 (Δr4 lanes 11 and 12) or csgB^Δr5 (Δr5 lanes 13 and 14). Samples were resuspended in SDS loading buffer with (+) or without (−) formic acid (FA) pretreatment. The top two panels are blots probed with anti-CsgB antibody. The bottom panel is a blot probed with anti-CsgA antibody. Whole cells (WC) samples are represented in the first and third panel, while samples containing whole cells and the underlying agar are represented in the second panel. (D) TEM of csgB mutants harboring CsgB^Δr1 (Δr1), CsgB^Δr2 (Δr2), CsgB^Δr3 (Δr3), or CsgB^Δr4 (Δr4) grown under curli-inducing conditions. The scale bars represent 500 nm.

Figure 4. In vitro characterization of the repeating unit deletions

(A) Coomassie stained SDS-PAGE of OD₆₀₀ normalized cell lysates of cells harboring the empty vector (EV) pET11d (EV lanes 1 and 2), WT CsgB (lanes 3 and 4), CsgB^Δr1 (Δr1 lanes 5 and 6), CsgB^Δr2 (Δr2 lanes 7 and 8), CsgB^Δr3 (Δr3 lanes 9 and 10), CsgB^Δr4 (Δr4 lanes 11 and 12), or CsgB^Δr5 (Δr5 lanes 13 and 14) after 1 hour induction with 0.5 mM IPTG. Samples were resuspended in SDS loading buffer with (+) or without (−) formic acid (FA) pretreatment. (B) Representative ThT kinetic plot of purified WT CsgB (○), CsgB^Δr2 (Δr2□), CsgB^Δr3 (Δr3△), CsgB^Δr4 (Δr4 X), and CsgB^Δr5 (Δr5 ◇). The inset is a representative bar graph of the RFUs for each protein after 12 hours of incubation at room temperature with ThT. (C) TEM of purified CsgB^Δr2 (Δr2 top panel) or purified CsgB^Δr4 (Δr4 bottom panel) after 24 hours of incubation at room temperature. The scale bars represent 500nm. (D) Representative ThT kinetic plot monitoring the polymerization of 14 μM CsgA (X), μM seeded with 9% w/w WT CsgB (●), seeded with 3.5% w/w WT CsgB (○), seeded with 9% w/w CsgB^Δr2 (Δr2 ■), seeded with 3.5% w/w CsgB^Δr2 (Δr2 □), seeded with 8% w/w CsgB^Δr4 (Δr4 ▲), or seeded with 3.5% w/w CsgB^Δr4 (Δr4 △). (E) Relative fluorescent units (RFUs) produced by synthetic peptides composed of the amino acids in r1 (145 μM), r2 (102 μM), r3 (138 μM), r4 (176 μM), and r5 (167 μM ) after incubation at room temperature for 24 hours in the presence of ThT.