N-Formylation modifies membrane damage associated with PSMα3 interfacial fibrillation.

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

Controlling differentiation of stem cells via bioactive disordered cues.

Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Biomimetic scaffolds imitate the native extracellular matrix (ECM) and are often utilized in vitro as analogues of the natural ECM to facilitate investigations of cell-ECM interactions and processes. In vivo, the cellular microenvironment has a crucial impact on regulating cell behavior and functions. A PET surface was activated and then functionalized with mimetic peptides to promote human mesenchymal stem cell (hMSC) adhesion and differentiation into an osteogenic lineage. Spray technology was used to randomly micropattern peptides (RGD and BMP-2 mimetic peptides) on the PET surface. The distribution of the peptides grafted on the surface, the roughness of the surfaces and the chemistry of the surfaces in each step of the treatment were ascertained by atomic force microscopy, fluorescence microscopy, time-of-flight secondary ion mass spectrometry, Toluidine Blue O assay, and X-ray photoelectron spectroscopy. Subsequently, cell lineage differentiation was evaluated by quantifying the expression of immunofluorescence markers: osteoblast markers (Runx-2, OPN) and osteocyte markers (E11, DMP1, and SOST). In this article, we hypothesized that a unique combination of bioactive micro/nanopatterns on a polymer surface improves the rate of morphology change and enhances hMSC differentiation. In DMEM, after 14 days, disordered micropatterned surfaces with RGD and BMP-2 led to a higher osteoblast marker expression than surfaces with a homogeneous dual peptide conjugation. Finally, hMSCs cultured in osteogenic differentiation medium (ODM) showed accelerated cell differentiation. In ODM, our results highlighted the expression of osteocyte markers when hMSCs were seeded on PET surfaces with random micropatterns.

Aptamer-based nanotrains and nanoflowers as quinine delivery systems.

In this study, we designed aptamer-based self-assemblies for the delivery of quinine. Two different architectures were designed by hybridizing quinine binding aptamers and aptamers targeting Plasmodium falciparum lactate dehydrogenase (PfLDH): nanotrains and nanoflowers. Nanotrains consisted in controlled assembly of quinine binding aptamers through base-pairing linkers. Nanoflowers were larger assemblies obtained by Rolling Cycle Amplification of a quinine binding aptamer template. Self-assembly was confirmed by PAGE, AFM and cryoSEM. The nanotrains preserved their affinity for quinine and exhibited a higher drug selectivity than nanoflowers. Both demonstrated serum stability, hemocompatibility, low cytotoxicity or caspase activity but nanotrains were better tolerated than nanoflowers in the presence of quinine. Flanked with locomotive aptamers, the nanotrains maintained their targeting ability to the protein PfLDH as analyzed by EMSA and SPR experiments. To summarize, nanoflowers were large assemblies with high drug loading ability, but their gelating and aggregating properties prevent from precise characterization and impaired the cell viability in the presence of quinine. On the other hand, nanotrains were assembled in a selective way. They retain their affinity and specificity for the drug quinine, and their safety profile as well as their targeting ability hold promise for their use as drug delivery systems.

WITHDRAWN: Aptamer-based nanotrains and nanoflowers as quinine delivery systems.

This article has been withdrawn: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been withdrawn at the request of the author, editor and publisher. The publisher regrets that an error occurred during the publication of this paper, which was intended to be published in International Journal of Pharmaceutics: X (not International Journal of Pharmaceutics). This error bears no reflection on the scientific content of this article or its authors. The publisher apologizes to the readers for this unfortunate error.

The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes.

The lipid composition of cellular membranes and the balance between the different lipid components can be impacted by aging, certain pathologies, specific diets and other factors. This is the case in a subgroup of individuals with psychiatric disorders, such as schizophrenia, where cell membranes of patients have been shown to be deprived in polyunsaturated fatty acids (PUFAs), not only in brain areas where the target receptors are expressed but also in peripheral tissues. This PUFA deprivation thus represents a biomarker of such disorders that might impact not only the interaction of antipsychotic medications with these membranes but also the activation and signaling of the targeted receptors embedded in the lipid membrane. Therefore, it is crucial to understand how PUFAs levels alterations modulate the different physical properties of membranes. In this paper, several biophysical approaches were combined (Laurdan fluorescence spectroscopy, atomic force microscopy, differential scanning calorimetry, molecular modeling) to characterize membrane properties such as fluidity, elasticity and thickness in PUFA-enriched cell membranes and lipid model systems reflecting the PUFA imbalance observed in some diseases. The impact of both the number of unsaturations and their position along the chain on the above properties was investigated. Briefly, data revealed that PUFA presence in membranes increases membrane fluidity, elasticity and flexibility and decreases its thickness and order parameter. Both the level of unsaturation and their position affect these membrane properties.

Interplay of matrix stiffness and stress relaxation in directing osteogenic differentiation of mesenchymal stem cells.

The aim of this study is to investigate the impact of the stiffness and stress relaxation of poly(acrylamide-co-acrylic acid) hydrogels on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Varying the amount of the crosslinker and the ratio between the monomers enabled the obtainment of hydrogels with controlled mechanical properties, as characterized using unconfined compression and atomic force microscopy (AFM). Subsequently, the surface of the hydrogels was functionalized with a mimetic peptide of the BMP-2 protein, in order to favor the osteogenic differentiation of hMSCs. Finally, hMSCs were cultured on the hydrogels with different stiffness and stress relaxation: 15 kPa - 15%, 60 kPa - 15%, 140 kPa - 15%, 100 kPa - 30%, and 140 kPa - 70%. The cells on hydrogels with stiffnesses from 60 kPa to 140 kPa presented a star-like shape, typical of osteocytes, which has only been reported by our group for two-dimensional substrates. Then, the extent of hMSC differentiation was evaluated by using immunofluorescence and by quantifying the expression of both osteoblast markers (Runx-2 and osteopontin) and osteocyte markers (E11, DMP1, and sclerostin). It was found that a stiffness of 60 kPa led to a higher expression of osteocyte markers as compared to stiffnesses of 15 and 140 kPa. Finally, the strongest expression of osteoblast and osteocyte differentiation markers was observed for the hydrogel with a high relaxation of 70% and a stiffness of 140 kPa.

Synthesis and Characterization of Conjugated Hyaluronic Acids. Application to Stability Studies of Chitosan-Hyaluronic Acid Nanogels Based on Fluorescence Resonance Energy Transfer.

Hyaluronic acid (HA) was functionalized with a series of amino synthons (octylamine, polyethylene glycol amine, trifluoropropyl amine, rhodamine). Sodium hyaluronate (HAs) was first converted into its protonated form (HAp) and the reaction was conducted in DMSO by varying the initial ratio (-NH2 (synthon)/COOH (HAp)). HA derivatives were characterized by a combination of techniques (FTIR, 1H NMR, 1D diffusion-filtered 19F NMR, DOSY experiments), and degrees of substitution (DSHA) varying from 0.3% to 47% were determined, according to the grafted synthon. Nanohydrogels were then obtained by ionic gelation between functionalized hyaluronic acids and chitosan (CS) and tripolyphosphate (TPP) as a cross-linker. Nanohydrogels for which HA and CS were respectively labeled by rhodamine and fluorescein which are a fluorescent donor-acceptor pair were subjected to FRET experiments to evaluate the stability of these nano-assemblies.

Actin Bundle Nanomechanics and Organization Are Modulated by Macromolecular Crowding and Electrostatic Interactions.

The structural and mechanical properties of actin bundles are essential to eukaryotic cells, aiding in cell motility and mechanical support of the plasma membrane. Bundle formation occurs in crowded intracellular environments composed of various ions and macromolecules. Although the roles of cations and macromolecular crowding in the mechanics and organization of actin bundles have been independently established, how changing both intracellular environmental conditions influence bundle mechanics at the nanoscale has yet to be established. Here we investigate how electrostatics and depletion interactions modulate the relative Young's modulus and height of actin bundles using atomic force microscopy. Our results demonstrate that cation- and depletion-induced bundles display an overall reduction of relative Young's modulus depending on either cation or crowding concentrations. Furthermore, we directly measure changes to cation- and depletion-induced bundle height, indicating that bundles experience alterations to filament packing supporting the reduction to relative Young's modulus. Taken together, our work suggests that electrostatic and depletion interactions may act counteractively, impacting actin bundle nanomechanics and organization.

Interaction of Tau construct K18 with model lipid membranes.

One of the hallmarks of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, resulting from the aggregation of the tubulin associated unit protein (Tau), which holds a vital role in maintaining neuron integrity in a healthy brain. The development of such aggregates and their deposition in the brain seem to correlate with the onset of neurodegeneration processes. The misfolding and subsequent aggregation of the protein into paired helical filaments that further form the tangles, lead to dysfunction of the protein with neuronal loss and cognitive decline. The aggregation of the protein then seems to be a causative factor of the neurodegeneration associated with AD. The hypothesis of an involvement of the membrane in modulating the misfolding and assembly of Tau into paired helical filaments attracts increasing interests. To provide more insight about how lipids can modulate the interactions with Tau, we have conducted a comprehensive Atomic Force Microscopy (AFM) study involving supported lipid bilayers of controlled compositions with the Tau microtubule-binding construct K18. Particularly, the effects of zwitterionic and negatively charged phospholipids on the interaction have been investigated. Deleterious solubilization effects have been evidenced on fluid zwitterionic membranes as well as an inability of K18 to fragment gel phases. The role of negative lipids in the aggregation of the peptide and the particular ability of phosphatidylinositol-4,5-bisphosphate (PIP2) in inducing K18 fibrillization on membranes are also reported.

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

The microbial adhesive arsenal deciphered by atomic force microscopy.

Microbes employ a variety of strategies to adhere to abiotic and biotic surfaces, as well as host cells. In addition to their surface physicochemical properties (e.g. charge, hydrophobic balance), microbes produce appendages (e.g. pili, fimbriae, flagella) and express adhesion proteins embedded in the cell wall or cell membrane, with adhesive domains targeting specific ligands or chemical properties. Atomic force microscopy (AFM) is perfectly suited to deciphering the adhesive properties of microbial cells. Notably, AFM imaging has revealed the cell wall topographical organization of live cells at unprecedented resolution, and AFM has a dual capability to probe adhesion at the single-cell and single-molecule levels. AFM is thus a powerful tool for unravelling the molecular mechanisms of microbial adhesion at scales ranging from individual molecular interactions to the behaviours of entire cells. In this review, we cover some of the major breakthroughs facilitated by AFM in deciphering the microbial adhesive arsenal, including the exciting development of anti-adhesive strategies.

High Speed AFM and NanoInfrared Spectroscopy Investigation of Aß1-42 Peptide Variants and Their Interaction With POPC/SM/Chol/GM1 Model Membranes.

Due to an aging population, neurodegenerative diseases such as Alzheimer's disease (AD) have become a major health issue. In the case of AD, Aß1 - 42 peptides have been identified as one of the markers of the disease with the formation of senile plaques via their aggregation, and could play a role in memory impairment and other tragic syndromes associated with the disease. Many studies have shown that not only the morphology and structure of Aß1 - 42 peptide assembly are playing an important role in the formation of amyloid plaques, but also the interactions between Aß1 - 42 and the cellular membrane are crucial regarding the aggregation processes and toxicity of the amyloid peptides. Despite the increasing amount of information on AD associated amyloids and their toxicity, the molecular mechanisms involved still remain unclear and require in-depth investigation at the local scale to clearly decipher the role of the sequence of the amyloid peptides, of their secondary structures, of their oligomeric states, and of their interactions with lipid membranes. In this original study, through the use of Atomic Force Microscopy (AFM) related-techniques, high-speed AFM and nanoInfrared AFM, we tried to unravel at the nanoscale the link between aggregation state, structure and interaction with membranes in the amyloid/membrane interaction. Using three mutants of Aß peptides, L34T, oG37C, and WT Aß1 - 42 peptides, with differences in morphology, structure and assembly process, as well as model lipidic membranes whose composition and structure allow interactions with the peptides, our AFM study coupling high spatial and temporal resolution and nanoscale structure information clearly evidences a local correlation between the secondary structure of the peptides, their fibrillization kinetics and their interactions with model membranes. Membrane disruption is associated to small transient oligomeric entities in the early stages of aggregation that strongly interact with the membrane, and present an antiparallel ß-sheet secondary structure. The strong effect on membrane integrity that exists when these oligomeric Aß1 - 42 peptides interact with membranes of a particular composition could be a lead for therapeutic studies.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Bacterial Sexuality at the Nanoscale.

Understanding the basic mechanisms of bacterial sexuality is an important topic in current microbiology and biotechnology. While classical methods used to study gene transfer provide information on whole cell populations, nanotechnologies offer new opportunities for analyzing the behavior of individual mating partners. We introduce an innovative atomic force microscopy (AFM) platform to study and mechanically control DNA transfer between single bacteria, focusing on the large conjugative pXO16 plasmid of the Gram-positive bacterium Bacillus thuringiensis. We demonstrate that the adhesion forces between single donor and recipient cells are very strong (∼2 nN). Using a mutant plasmid, we find that these high forces are mediated by a pXO16 aggregation locus that contains two large surface protein genes. Notably, we also show that AFM can be used to mechanically induce plasmid transfer between single partners, revealing that transfer is very fast (<15 min) and triggers major cell surface changes in transconjugant cells. We anticipate that the single-cell technology developed here will enable researchers to mechanically control gene transfer among a wide range of Gram-positive and Gram-negative bacterial species and to understand the molecular forces involved. Also, the method could be useful in nanomedicine for the design of antiadhesion compounds capable of preventing intimate cell-cell contacts, therefore providing a means to control the resistance and virulence of bacterial pathogens.

Adhesion of Staphylococcus aureus to Corneocytes from Atopic Dermatitis Patients Is Controlled by Natural Moisturizing Factor Levels.

The bacterial pathogen Staphylococcus aureus plays an important role in atopic dermatitis (AD), a chronic disorder that mostly affects children. Colonization of the skin of AD patients by S. aureus exacerbates the disease, but the molecular determinants of the bacterium-skin adhesive interactions are poorly understood. Specifically, reduced levels of natural moisturizing factor (NMF) in the stratum corneum have been shown to be associated with more severe AD symptoms, but whether this is directly related to S. aureus adhesion is still an open question. Here, we demonstrate a novel relationship between NMF expression in AD skin and strength of bacterial adhesion. Low-NMF corneocytes, unlike high-NMF ones, are covered by a dense layer of nanoscale villus protrusions. S. aureus bacteria isolated from AD skin bind much more strongly to corneocytes when the NMF level is reduced. Strong binding forces originate from a specific interaction between the bacterial adhesion clumping factor B (ClfB) and skin ligands. Remarkably, mechanical tension dramatically strengthens ClfB-mediated adhesion, as observed with catch bonds, demonstrating that physical stress plays a role in promoting colonization of AD skin by S. aureus Collectively, our findings demonstrate that patient NMF levels regulate the strength of S. aureus-corneocyte adhesion, the first step in skin colonization, and suggest that the ClfB binding mechanism could represent a potential target for new therapeutic treatments.IMPORTANCE Bacterium-skin interactions play important roles in skin disorders, yet their molecular details are poorly understood. In this study, we decipher the molecular forces at play during adhesion of Staphylococcus aureus to skin corneocytes in the clinically important context of atopic dermatitis (AD), also known as eczema. We identify a unique relationship between the level of natural moisturizing factor (NMF) in the skin and the strength of bacterium-corneocyte adhesion. Bacterial adhesion is primarily mediated by the surface protein clumping factor B (ClfB) and is enhanced by physical stress, highlighting the role of protein mechanobiology in skin colonization. Similar to a catch bond behavior, this mechanism represents a promising target for the development of novel antistaphylococcal agents.

Binding of Nucleic Acid Components to the Serpentinite-Hosted Hydrothermal Mineral Brucite.

The adsorption of nucleic acid components onto the serpentinite-hosted hydrothermal mineral brucite has been investigated experimentally by determining the equilibrium adsorption isotherms in aqueous solution. Thermodynamic characterization of the adsorption data has been performed using the extended triple-layer model (ETLM) to establish a model for the stoichiometry and equilibrium constants of surface complexes. Infrared characterization of the molecule-mineral complexes has helped gain insight into the molecular functional groups directly interacting with the mineral surface. Quantum mechanical calculations have been carried out to identify the possible complexes formed on surfaces by nucleic acid components and their binding configurations on mineral surfaces, both in the presence of water molecules and in water-free conditions. The results indicate that brucite favors adsorption of nucleotides with respect to nucleosides and nucleobases from dilute aqueous environments. The surface of this mineral is able to induce well-defined orientations of the molecules through specific molecule-mineral interactions. This result suggests plausible roles of the mineral brucite in assisting prebiotic molecular self-organization. Furthermore, the detection of the infrared spectroscopic features of such building blocks of life adsorbed on brucite at very low degrees of coverage provides important support to life detection investigations.

Mechanical Forces Guiding Staphylococcus aureus Cellular Invasion.

Staphylococcus aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. The current model for cellular invasion involves the interaction between the bacterial cell surface located fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5ß1 integrin in the host cell membrane. While it is believed that the extracellular matrix protein Fn serves as a bridging molecule between FnBPs and integrins, the fundamental forces involved are not known. Using single-cell and single-molecule experiments, we unravel the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA-Fn-integrin interaction. We show that FnBPA mediates bacterial adhesion to soluble Fn via strong forces (∼1500 pN), consistent with a high-affinity tandem ß-zipper, and that the FnBPA-Fn complex further binds to immobilized α5ß1 integrins with a strength much higher than that of the classical Fn-integrin bond (∼100 pN). The high mechanical stability of the Fn bridge favors an invasion model in which Fn binding by FnBPA leads to the exposure of cryptic integrin-binding sites via allosteric activation, which in turn engage in a strong interaction with integrins. This activation mechanism emphasizes the importance of protein mechanobiology in regulating bacterial-host adhesion. We also find that Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes. Collectively, our results provide a molecular foundation for the ability of FnBPA to trigger host cell invasion by S. aureus and offer promising prospects for the development of therapeutic approaches against intracellular pathogens.

Force-Induced Strengthening of the Interaction between Staphylococcus aureus Clumping Factor B and Loricrin.

Bacterial pathogens that colonize host surfaces are subjected to physical stresses such as fluid flow and cell surface contacts. How bacteria respond to such mechanical cues is an important yet poorly understood issue. Staphylococcus aureus uses a repertoire of surface proteins to resist shear stress during the colonization of host tissues, but whether their adhesive functions can be modulated by physical forces is not known. Here, we show that the interaction of S. aureus clumping factor B (ClfB) with the squamous epithelial cell envelope protein loricrin is enhanced by mechanical force. We find that ClfB mediates S. aureus adhesion to loricrin through weak and strong molecular interactions both in a laboratory strain and in a clinical isolate. Strong forces (~1,500 pN), among the strongest measured for a receptor-ligand bond, are consistent with a high-affinity "dock, lock, and latch" binding mechanism involving dynamic conformational changes in the adhesin. Notably, we demonstrate that the strength of the ClfB-loricrin bond increases as mechanical force is applied. These findings favor a two-state model whereby bacterial adhesion to loricrin is enhanced through force-induced conformational changes in the ClfB molecule, from a weakly binding folded state to a strongly binding extended state. This force-sensitive mechanism may provide S. aureus with a means to finely tune its adhesive properties during the colonization of host surfaces, helping cells to attach firmly under high shear stress and to detach and spread under low shear stress.IMPORTANCEStaphylococcus aureus colonizes the human skin and the nose and can cause various disorders, including superficial skin lesions and invasive infections. During nasal colonization, the S. aureus surface protein clumping factor B (ClfB) binds to the squamous epithelial cell envelope protein loricrin, but the molecular interactions involved are poorly understood. Here, we unravel the molecular mechanism guiding the ClfB-loricrin interaction. We show that the ClfB-loricrin bond is remarkably strong, consistent with a high-affinity "dock, lock, and latch" binding mechanism. We discover that the ClfB-loricrin interaction is enhanced under tensile loading, thus providing evidence that the function of an S. aureus surface protein can be activated by physical stress.

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

Curli are functional amyloids produced by proteobacteria like Escherichia coli as part of the extracellular matrix that holds cells together into biofilms. The molecular events that occur during curli nucleation and fiber extension remain largely unknown. Combining observations from curli amyloidogenesis in bulk solutions with real-time in situ nanoscopic imaging at the single-fiber level, we show that curli display polar growth, and we detect two kinetic regimes of fiber elongation. Single fibers exhibit stop-and-go dynamics characterized by bursts of steady-state growth alternated with periods of stagnation. At high subunit concentrations, fibers show constant, unperturbed burst growth. Curli follow a one-step nucleation process in which monomers contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to those of the mature fibrils. Kinetic data and interaction studies of curli fibrillation in the presence of the natural inhibitor CsgC show that the inhibitor binds curli fibers and predominantly acts at the level of fiber elongation.