Nucleation and growth of a bacterial functional amyloid at single-fiber resolution

Sleutel, Mike; Broeck, Imke Van den; Gerven, Nani Van; Feuillie, Cécile; Jonckheere, Wim; Valotteau, Claire; Dufrêne, Yves F.; Remaut, Han

doi:10.1038/nchembio.2413

Cited by 58 publications

(91 citation statements)

References 52 publications

(65 reference statements)

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“…Curli assembly involves fibrillization, which has been monitored using approaches similar to those used to characterize fibrillization of amyloid ␤ (e.g., binding to thioflavin T [ThT]), but structural intermediates in the curli fibrillization process had not been identified (28,29). We found that we could interfere with the formation of mature curli fibrils by increasing turbulence during the biofilm formation in liquid culture.…”

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confidence: 99%

Cytotoxic Curli Intermediates Form during Salmonella Biofilm Development

Nicastro

Tursi

et al. 2019

J Bacteriol

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Enterobacteriaceae produce amyloid proteins called curli that are the major proteinaceous component of biofilms. Amyloids are also produced by humans and are associated with diseases such as Alzheimer's. During the multistep process of amyloid formation, monomeric subunits form oligomers, protofibrils, and finally mature fibrils. Amyloid ␤ oligomers are more cytotoxic to cells than the mature amyloid fibrils. Oligomeric intermediates of curli had not been previously detected. We determined that turbulence inhibited biofilm formation and that, intriguingly, curli aggregates purified from cultures grown under high-turbulence conditions were structurally smaller and contained less DNA than curli preparations from cultures grown with less turbulence. Using flow cytometry analysis, we demonstrated that CsgA was expressed in cultures exposed to higher turbulence but that these cultures had lower levels of cell death than less-turbulent cultures. Our data suggest that the DNA released during cell death drives the formation of larger fibrillar structures. Consistent with this idea, addition of exogenous genomic DNA increased the size of the curli intermediates and led to binding to thioflavin T at levels observed with mature aggregates. Similar to the intermediate oligomers of amyloid ␤, intermediate curli aggregates were more cytotoxic than the mature curli fibrils when incubated with bone marrow-derived macrophages. The discovery of cytotoxic curli intermediates will enable research into the roles of amyloid intermediates in the pathogenesis of Salmonella and other bacteria that cause enteric infections. IMPORTANCE Amyloid proteins are the major proteinaceous components of biofilms, which are associated with up to 65% of human bacterial infections. Amyloids produced by human cells are also associated with diseases such as Alzheimer's. The amyloid monomeric subunits self-associate to form oligomers, protofibrils, and finally mature fibrils. Amyloid ␤ oligomers are more cytotoxic to cells than the mature amyloid fibrils. Here we detected oligomeric intermediates of curli for the first time. Like the oligomers of amyloid ␤, intermediate curli fibrils were more cytotoxic than the mature curli fibrillar aggregates when incubated with bone marrow-derived macrophages. The discovery of cytotoxic curli intermediates will enable research into the roles of amyloid intermediates in the pathogenesis of Salmonella and other bacteria that cause enteric infections.

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confidence: 99%

Cytotoxic Curli Intermediates Form during Salmonella Biofilm Development

Nicastro

Tursi

et al. 2019

J Bacteriol

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“…The sensitivity of quartz nanopipettes to both macromolecular and protein unfolding changes provide a promising outlook for studying the dynamic process of filament formation across diverse molecules using native proteins. This label-free method would work well with visualising protein aggregation, such as alphasynuclein in real-time, which may find clinical significance in understanding the mechanism of Parkinson's and Alzheimer's diseases 50 . Furthermore, the high sensitivity achieved suggests the technique may provide a…”

Section: Discussionmentioning

confidence: 99%

Single-molecule nanopore sensing of actin dynamics and drug binding

Wang

Wilkinson

Lin

et al. 2019

Preprint

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Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.through different modes of action. Finally, we demonstrate the ability of nanopipette-based nanopores in drug discovery by measuring real-time drug-binding at a single molecule level, calculating the observed rate constant and the saturation concentration of Swinholide A dimer formation. Using these measurements, we propose a positively cooperative actin-binding model for the Swinholide A drug's mode of action. The ability to perform such studies, label-free and with single-molecule resolution demonstrates the potential of using quartz nanopipettes in both the direct probing of the unfolding of complex biomolecules but also for future molecular diagnostics and drug discovery.6 Results Experimental configurationNanopipettes were fabricated using laser-assisted pulling as reported previously 28 . The pulled nanopipettes terminated with a single nanopore with an average diameter of 25 ± 4 nm (N = 5), as measured by Scanning Electron Microscopy (Fig. 1a and Fig. S1). Linear IV curves were obtained in the monomeric actin buffer, and the nanopore conductance was determined to be 37.0 ± 3.9 nS (Fig. S2). Given the high potassium chloride concentration (1 M), we confirmed that actin remained monomeric in the nanopore buffer (Fig. S3). In all experiments, the analyte was introduced into an external reservoir (CIS) along with a reference/ground Ag/AgCl electrode. The pipette was filled only with buffer solution, and a patch/working Ag/AgCl electrode was inserted ( Fig. 1a). When an external electric field is applied, protein transport through the nanopore is a result of cooperative and competitive contributions from diffusion, electrophoretic (EP) and electroosmotic (EO) flows 29 . At pH 8.0, both the surface of the nanopipettes and actin (isoelectric point ~5.3) are negatively charged, meaning we see no translocation events at a negative voltage (Fig. S4). We used rel...

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“…However, this approach seeks to primarily determine the binding energy and does not attempt to resolve the folding process, an approximation making it a more suitable measurement of separating dimers, rather than a growing fibril. Experimental evidence hints at concurrent folding and oligomerization, 27 but this has not been confirmed. Additionally, no method is currently known to the authors that could accurately evaluate the energetic cost of folding during binding, making this the most suitable approach available.…”

Section: Csgb-r1mentioning

confidence: 99%

“…Growing fibrils could also develop "scars" where structural perturbations appeared at the tip that could remain in the fiber or be resolved. 27 AFM has also been applied to study binding of CsgA monomers and curliated bacteria with fibronectin-functionalized cantilevers. This revealed the formation of multiple quantized bonds, requiring about~50 pN to unbind.…”

Section: Althoughmentioning

confidence: 99%

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Dimerization energetics of curli fiber subunits CsgA and CsgB

2019

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Curli fibers are functional amyloids that exhibit strong adhesion and robust self-assembly as biofilm structural components; however, the binding energetics and mechanical properties of wild-type curli are not well understood. To address this, we present dimer structures made up of the major and minor curli subunits (CsgA and CsgB), perform free energy calculations to obtain absolute binding energies, and estimate the Young's modulus and persistence length of curli fibers. Equilibrium molecular dynamics simulations are used to evaluate nonbonded interactions. Binding energies are most favorable for CsgB-CsgA, while CsgA-CsgA dimers have a higher binding energy than CsgB-CsgB despite possessing less favorable nonbonded interaction energies. Decomposing each potential of mean force of separation indicated that solvent effects positively impact CsgA-CsgA binding but not CsgB-CsgB and CsgB-CsgA. Charged residues and conserved polar residues were also notable contributors to attractive nonbonded interactions, underlining their importance in dimer assembly. Our findings elucidate sequence effects on binding energy contributions and establish theoretical limits for the elasticity, persistence length, and strength of curli fibers.npj Computational Materials (2019) 5:27 ; https://doi.

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Nucleation and growth of a bacterial functional amyloid at single-fiber resolution

Cited by 58 publications

References 52 publications

Cytotoxic Curli Intermediates Form during Salmonella Biofilm Development

Cytotoxic Curli Intermediates Form during Salmonella Biofilm Development

Single-molecule nanopore sensing of actin dynamics and drug binding

Dimerization energetics of curli fiber subunits CsgA and CsgB

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