The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

Abstract

Curli are functional amyloid fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In E. coli, the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares approximately 30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus (CsgB(trunc)) was not outer membrane-associated, but secreted away from the cell. CsgB(trunc) was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble CsgB(trunc) assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. CsgB(trunc) fibers were able to seed soluble CsgA polymerization in vitro. CsgB(trunc) displayed modest nucleator activity in vivo, as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, CsgB(trunc) was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for how E. coli guides efficient amyloid fiber formation on the cell surface.

Fig. 1.

Biochemical characterization of CsgB and CsgB_trunc. (A) The imperfect repeating units in CsgA and CsgB (R1–R5) are predicted to form β-strand-loop-β-strand structures. Amino acids comprising the β-strand are located below the arrows, and amino acids predicted to comprise the loops are italicized. Bolded letters represent amino acids conserved in CsgB and CsgA at each position relative to the start of each repeating unit. Boxed letters represent amino acids conserved throughout the repeating units in both proteins. Positively charged amino acids in the fifth repeating unit of CsgB are marked with asterisks. (B) Western blot analysis of WT CsgB (*) and CsgB_trunc (**) expressed in the Δcsg strain LSR12. The csgB constructs were coexpressed with the isopropyl β-d-thiogalactoside-inducible csgG containing plasmid pMC1 or the corresponding empty vector, pTrc99A. Cultures were grown in LB to an OD₆₀₀ of 0.9 and induced with 250 μM isopropyl β-d-thiogalactoside. Postinduction cell suspensions were normalized by OD₆₀₀ and separated by centrifugation into cell (C) and supernatant fractions (S). The blot was probed by using αCsgB peptide antisera. Lane 1 contains LSR12/pTrc99A/pNH3 cells, and lane 2 contains corresponding supernatant; lanes 3 (C) and 5 (S) contain LSR12/pTrc99A/pNH4; lanes 4 (C) and 6 (S) contain LSR12/pMC1/pNH4; lanes 7 (C) and 9 (S) contain LSR12/pTrc99A/pNH2; and lanes 8 (C) and 10 (S) contain LSR12/pMC1/pNH2. (C) Western blot of purified CsgB_trunc probed with αCsgB peptide antibody. Two samples containing equal concentrations of purified soluble CsgB_trunc were incubated at room temperature overnight before centrifugation for 1 min in an Eppendorf microcentrifuge (Eppendorf-5 Prime, Boulder, CO). The supernatant was decanted and the pellet was resuspended in 2× SDS loading buffer (lane 1) or pretreated with 90% formic acid (FA) and dried before resuspension in 2× SDS loading buffer (lane 2).

Fig. 2.

Amyloid-like properties of CsgB_trunc aggregates. (A) TEM analysis of soluble CsgB_trunc incubated at room temperature overnight reveals fibrous aggregates. (Scale bars: 500 nm.) (B) Freshly purified CsgB_trunc was mixed with the amyloid-specific dye thioflavin T and incubated at room temperature for 600 min at the following concentrations: 43 μM (open diamonds), 33 μM (open circles), 22 μM (filled diamonds), 16 μM (filled circles), 11 μM (open triangles), 8 μM (“x”s), 5 μM (filled triangles), 0 μM (solid line). Relative florescence emitted at 495 nm was measured every 10 min after excitation at 438 nm. (C) Far-UV spectral analysis of 22 μM CsgB_trunc immediately after purification (open circles), after incubation for 24 h (open squares) at room temperature, or after incubation at room temperature for 15 days (filled squares). The potassium-phosphate-buffer (KPi)-only control is represented by a solid black line.

Fig. 3.

CsgB_trunc can nucleate CsgA in vitro. (A) Thioflavin T kinetic plot monitoring the polymerization of 30 μM CsgA (filled circles) and 30 μM CsgA in the presence of 10% wt/wt (open squares) or 6% wt/wt (open circles) CsgB_trunc aggregates. (B) TEM of 30 μM CsgA. (C) TEM of the fibers produced by polymerization of 30 μM CsgA seeded by 10% CsgB_trunc. (Scale bars: 500 nm.)

Fig. 4.

In vivo nucleation properties of CsgB_trunc. (A) Congo red-containing yeast extract casamino acids (YESCA) plates comparing the Congo red binding phenotypes of csgB and csgBF harboring an empty vector control (pLR2), WT csgB on a plasmid (pLR8), or csgB_trunc (pNH1). Cells: area 1, WT/pLR2; area 2, csgB/pLR2; area 3, csgB/pLR8; area 4, csgB/pNH1; area 5, csgBF/pNH1; area 6, csgBF/pNH1; area 7, csgBF/pLR8; area 8, csgBF/pLR2. All strains contain pTrc99A except area 6, which contains pCsgF. (B Upper) WC lysate samples probed with αCsgB peptide antiserum reveal WT CsgB (*) and CsgB_trunc (**). Samples were treated with (+) or without (−) formic acid (FA). (B Lower) WCs and the underlying agar (plugs) were collected from YESCA plates and probed for CsgB. (C Upper) Western blots of WCs probed with αCsgA antiserum. (C Lower) Western blots of WCs and underlying agar (plug) samples probing for CsgA. The WT cells in lanes 1 and 2 contain empty vectors pLR2 and pTrc99A. All other strains in A and B also contain the empty vector pTrc99A in addition to either pLR2 (pEmpty) or pNH1 (pCsgB_trunc).

Fig. 5.

Characterization of fibers nucleated by CsgB_trunc in vivo. (A) TEM of csgB/pLR8. (B) TEM of csgBF/pNH1. (Scale bars: 500 nm.) (C) Cells were mechanically scraped off of a Congo red-containing YESCA plate by using disposable inoculating loops to compare the Congo red-binding phenotypes of WT/pLR2 (empty vector; labeled “1”), csgBF/pNH1 (csgB_trunc; labeled “2”), csgFBA/pNH1 (csgB_trunc; labeled “3”), or csgFBA/pLR2 (empty vector; labeled “4”). (D) The Congo red-containing YESCA plate used to cultivate cells in Fig. 4C after cells have been removed. In D, the numbers indicate the region of the Congo red plate where the following strains had grown: 1, WT/pLR2; 2, csgBF/pNH1; 3, csgFBA/pNH1; and 4, csgFBA/pLR2.