Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - BAS-SBBT (Bacterial Amyloid Secretion: Structural Biology and Biotechnology.)

Teaser

Curli are functional amyloid fibers that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria. Unlike the protein misfolding and aggregation events seen in pathological amyloid diseases such as...

Summary

Curli are functional amyloid fibers that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria. Unlike the protein misfolding and aggregation events seen in pathological amyloid diseases such as Alzheimer’s and Parkinson’s disease, curli are the product of a dedicated protein secretion machinery (Type VIII secretion). Curli formation requires a specialised and mechanistically unique transport/assembly machinery in the bacterial outer membrane, as well as two soluble accessory proteins thought to facilitate the safe guidance of the curli subunits across the periplasm and to coordinate their self-assembly at cell surface.

In the BAS-SBBT research program we will study the structural and molecular biology of E. coli curli biosynthesis and address the fundamental questions concerning the molecular processes that allow the spatially and temporally controlled transport and deposition of these amyloidogenic polypeptides. We will structurally unravel the secretion machinery, trap and analyse critical secretion intermediates and through in vitro reconstitution, assemble a minimal, self-sufficient peptide transport and fiber assembly system.

The new insights gained will set the stage for targeted interventions in curli -mediated biofilm formation and this research project will develop a new framework to harness the unique properties found in curli structure and biosynthesis for biotechnological applications as in patterned functionalized nanowires and directed, selective peptide carriers.
The research proposal encompasses an integrated use of Structural Biology, Molecular Biology, Biochemistry and Biophysics to address the objectives set out in the 4 principal questions:
Q1: What is the mechanism employed by the Type VIII secretion transporter CsgG?
Q2: How is premature curlin aggregation and cytotoxicity controlled prior to secretion?
Q3: What is the structure of the curli fibers and how is assembly coordinated with secretion?
Q4: Can we use the unique properties of the Type VIII secretion pathway for biotechnological applications?

Work performed

A major question in functional amyloid assembly is how bacterial cells avoid the build-up or activity of possibly toxic intermediates known to accompany pathological amyloid formation in human wasting diseases. Contrary to pathological amyloids, the fiber confirmation in functional amyloids is not the result from protein misfolding, but represents the native, functional state of the protein. It is unknown whether the nucleation and elongation of the functional amyloid fibrils take a similar pathways than what has been described for pathological amyloids.

We addressed these questions by means of high speed atomic force microscopy (AFM) imaging of the fibrillation pathway of curli subunit CsgA, starting from pure solution of CsgA monomers. When purified under denaturing conditions, CsgA monomers can be maintained in a monomeric state. Upon rapid desalting, CsgA monomers are in an intrinsically unfolded, but assembly-component conformational assemble that will transition to amyloid fibers over a matter of minutes to hours. Using high speed AFM, we show that curli display polar growth, and detect two kinetic regimes of fiber elongation. At high concentration, fibers exhibit steady-state kinetics with a constant instantaneous rate of growth. At low subunit concentration, however, single fibers exhibit stop-and-go dynamics characterized by bursts of steady growth, alternated with periods of stagnation. Strikingly, curli follow a one-step nucleation process, where monomeric species contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to the mature fibrils. This is in contrast to most pathological amyloids where nucleation is a multistep process involving amorphous aggregates and distinct oligomeric intermediates. The latter our thought form the main cytotoxic species involved in pathological amyloidogenesis. If we consider the biological context in which curli are formed, one-step direct nucleation can be considered a logical route. Although more examples are needed, the absence of a lengthy induction time could be a defining trait of functional amyloids. The reduction of the kinetic and energetic barriers for amyloid formation increase the efficiency gain of the aggregation process which fits the rationale from an evolutionary perspective. This stands in sharp contrast to the amyloid transformation of natively folded proteins where the un- and refolding steps into cross structures is the molecular origin of the induction time. For these cases, amyloid structures are an unwanted aberration and the large activation barriers associated with their formation are no evolutionary accident.

Apart from on optimized fiber nucleation and elongation process, the curli pathway includes an additional safeguard to amyloid toxicity in the form of the periplasmic protein CsgC. Molecular studies by the M. Chapman laboratory (U Michigan) have revealed that CsgC acts as an inhibitor to the amyloid transition of curli subunits in strong substoichiometric concentrations by an unknown mechanism. By following single fiber nucleation and elongation using AFM in presence and absence of CsgC we find that the natural inhibitor CsgC binds curli fibers rather than CsgA monomers, and predominantly acts at the level of fiber elongation, likely by capping the active growth poles of the fibers.

A first paper reporting these new findings has been published: Sleutel M, Van den Broeck I, Van Gerven N, Feuillie C, Jonckheere W, Valotteau C, Dufrêne YF, Remaut H. (2017) Nucleation and growth of a bacterial functional amyloid at single-fiber resolution. Nat Chem Biol. 13(8):902-908.

Final results

Published data from the first reporting period provide unprecedented insights in curli fibrillation at single fiber resolution. Hitherto, curli formation and inhibition had only been studied using bulk solvent techniques, where direct observation of rare and short-lived events such as nucleation are not possible. Our results can contribute to the production of future therapeutic agents that can prevent or combat host colonization and persistence in biofilm-associated bacterial amyloids, but also offer promising prospects in nanobiotechnology, where there is an increasing interest in harnessing the physical properties and self-assembling nature of amyloids for new biomaterials and nanotechnological purposes. Using our new insights, we are now working on methods and CsgA mutants where we can separate nucleation and elongation in space and time, in order to better control the self-assembly process of functionalized curli.

The unravelling of the transport mechanism employed by the curli translocation channel CsgG is ongoing. We anticipate that in the second half of the project, we will obtain atomic structures of CsgG bound to its various secretion partners and map out the interaction pathway of the curli subunits en route to the cell surface.