Curli Fibers Research Articles

Intrinsic aggregation propensity of the CsgB nucleator protein is crucial for curli fiber formation.

Several organisms exploit the extraordinary physical properties of amyloid fibrils forming natural protective amyloids, in an effort to support complex biological functions. Curli amyloid fibers are a major component of mature biofilms, which are produced by many Enterobacteriaceae species and are responsible, among other functions, for the initial adhesion of bacteria to surfaces or cells. The main axis of curli fibers is formed by a major structural subunit, known as CsgA. CsgA self-assembly is promoted by oligomeric nuclei formed by a minor curli subunit, known as the CsgB nucleator protein. Here, by implementing AMYLPRED2, a consensus prediction method for the identification of 'aggregation-prone' regions in protein sequences, developed in our laboratory, we have successfully identified potent amyloidogenic regions of the CsgB subunit. Peptide-analogues corresponding to the predicted 'aggregation-prone' segments of CsgB were chemically synthesized and studied, utilizing several biophysical techniques. Our experimental data indicate that these peptides self-assemble in solution, forming fibrils with characteristic amyloidogenic properties. Using comparative modeling techniques, we have developed three-dimensional models of both CsgA and CsgB subunits. Structural analysis revealed that the identified 'aggregation-prone' segments may promote gradual polymerization of CsgB. Briefly, our results indicate that the intrinsic self-aggregation propensity of the CsgB subunit, most probably has a pivotal role in initiating the formation of curli amyloid fibers by promoting the self-assembly process of the CsgB nucleator protein.

The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σ(E) -dependent sRNA RybB.

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self-produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ(E) cell envelope stress response and thereby reducing the expression of CsgD - a crucial activator of curli and cellulose biosynthesis - due to csgD mRNA targeting by the σ(E) -dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm-associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.

Rope coiling spinning of curled and meandering hollow-fiber membranes

Curved and structured membrane geometries are known to have the potential to reduce concentration polarization and fouling rates in membrane filtration and membrane contacting applications. However, their production is cumbersome and often comprises various successive processing steps. We present a new direct fabrication process of two new hollow fiber geometries: Curled and meandering hollow fibers. The phenomenon of liquid rope-coiling is adapted to a membrane spinning process resulting in mostly spiralling curly fiber geometries. Parameters influencing geometric properties are evaluated and experimentally obtained structures are presented. Fibers were prepared from a polyethersulfone-based polymer solution in an air gap spinning process. Prepared structured hollow fibers are investigated regarding geometrical measures and pore structures. A comprehensive analysis of the complex interplay between the influencing parameters is suggested.

Single Molecular Force Sensing Reveals Fibronectin-Specific Binding Epitopes of Bacterial Curli Fimbriae

Curli are functional microbial amyloids that are major components of the complex extracellular matrix produced by Enterobacteriaceae. They play important functions in biofilm formation, bacterial invasion into host cells, and pathological processes which lead to numerous infections and autoimmune diseases. Curli fibers interact with host cell multi-domain adhesive glycoproteins, such as laminin and fibronectin. Several groups have investigated the structure of curli fimbriae, as well as their mechanical properties and adhesion mechanisms. However, the specificity and exact mechanism of curli-mediated bacterial binding to cells remains obscure. Here, single-molecule force spectroscopy reveals dynamic and statistical information about the formation of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We show that a specific interaction between the bacterial curli protein CsgA and the RGD domain of fibronectin leads to tight binding. Via single-cell force spectroscopy we measured distinct interaction forces on the surface between the isolated CsgA protein or the bacteria-producing CsgA curli and full length fibronectin, fibronectin domain III, and RGD peptide, respectively. These experiments demonstrate that the forces required to break adhesion are quantized and reflect formation of a dense collective network. These networks comprise at least 20 specific molecular bonds that lead to tight bacterial binding. Our results underline the significance of amyloid formation on bacterial surfaces as multi-bond structural components in curli-mediated bacterial adhesion to fibronectin.

Engineering E. coli to enable electron transfer along curli protein nanofibers

Event Abstract Back to Event Engineering E. coli to enable electron transfer along curli protein nanofibers Noémie-Manuelle Dorval Courchesne1, Pei Kun R. Tay1, 2, Peter Q. Nguyen1 and Neel S. Joshi1, 2 1 Harvard University, Wyss Institute for Biologically Inspired Engineering, United States 2 Harvard University, School of Engineering and Applied Sciences, United States Protein-based materials capable of mediating long-range electron transport are attractive for incorporation in several devices including biosensing, light harvesting, and electrobiosynthetic systems, as well as microbial fuel cells and other electronic devices. These technologies can benefit from the flexibility, bio-compatibility, nano-size, and self-assembling properties of protein materials. Some conductive extracellular appendages are naturally produced by microorganisms like Shewanella and Geobacter, but it is difficult to improve upon their intrinsic conductivity via rational design because of the difficulty to genetically engineer these organisms, the limited variety of functional groups of natural amino acids, and the lack of structural information available. Here, we propose to use Escherichia coli as a more versatile platform for the production of conductive protein fibers. E. coli can be easily engineered, is routinely grown under aerobic conditions, and naturally produces curli fibers composed on repeated CsgA proteins. CsgA has an amyloid structure and spontaneously assembles into microns-long nanofibers. Engineering the CsgA gene allows for the addition of various active protein domains along the fibers, or for the selective mutation of residues that change surface chemistry. The curli nanofibers can thus be used as a scaffold for the creation of functional porous fibrous networks. In order to generate electron transfer, we aimed at creating pi-pi stacking along curli fibers. We identified residues in CsgA that can be mutated without altering its amyloid structure, and then selected series of residues to mutate aligned along the five repeats of the beta-helix structure of CsgA. We postulated that the conjugated side-chains of aligned aromatic residues, or their non-natural amino acid derivatives, could interact, form pi-stacks, and induce electron delocalization. Congo red binding assays and electron microscopy indicate that mutant CsgA proteins with various sets of aligned aromatic residues can still form amyloid curli fibers. The engineered bacteria can be used directly to grow biofilms, or alternatively His-tagged curli fibers are purified to investigate the properties of single nanofibers. The ability of the mutant fibers to transfer electrons is investigated via conductive AFM and electrochemical methods, comparing with naturally-occurring conductive nanowires. Overall, our engineered E. coli system provides a tunable platform for the synthesis of conductive protein networks. Diverse functional peptides could be further introduced in the curli scaffold, such as metalloproteins, redox enzymes, binding domains, and stimuli-responsive peptides, to add additional functionalities. This versatile biomaterial could be used in a variety of devices both for biomedical or energy applications. Keywords: Biomimetic, nanofiber, electric, engineered cell Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Nano-structured materials for unique functions Citation: Dorval Courchesne N, Tay PR, Nguyen PQ and Joshi NS (2016). Engineering E. coli to enable electron transfer along curli protein nanofibers. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01972 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Noémie-Manuelle Dorval Courchesne Pei Kun R Tay Peter Q Nguyen Neel S Joshi Google Noémie-Manuelle Dorval Courchesne Pei Kun R Tay Peter Q Nguyen Neel S Joshi Google Scholar Noémie-Manuelle Dorval Courchesne Pei Kun R Tay Peter Q Nguyen Neel S Joshi PubMed Noémie-Manuelle Dorval Courchesne Pei Kun R Tay Peter Q Nguyen Neel S Joshi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers.

Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.

Biofilm-associated bacterial amyloids dampen inflammation in the gut: oral treatment with curli fibres reduces the severity of hapten-induced colitis in mice.

Background/objectives:A disruption of epithelial barrier function can lead to intestinal inflammation. Toll-like receptor (TLR) 2 activation by microbial products promotes intestinal epithelial integrity and overall gut health. Several bacterial species, including enteric bacteria, actively produce amyloid proteins as a part of their biofilms. Recognition of amyloid fibres found in enteric biofilms, termed curli, by the Toll-like receptor (TLR)2/1 complex reinforces barrier function. Here, we investigated the effect of purified curli fibres on inflammation in a mouse model of acute colitis.Methods:Bone marrow–derived macrophages as well as lamina propria cells were treated with curli fibres of both pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli Nissle 1917 biofilms. Mice were given 0.1 or 0.4 mg of purified curli orally 1 day post administration of 1% 2,4,6-trinitrobenzene sulphonic acid (TNBS) enema. Histopathological analysis was performed on distal colonic tissue taken 6 days post TNBS enema. RNA extracted from colonic tissue was subjected to RT-PCR.Results:Here we show that curli fibres of both pathogenic and commensal bacteria are recognised by TLR2 leading to the production of IL-10, immunomodulatory cytokine of intestinal homeostasis. Treatment of mice with a single dose of curli heightens transcript levels of Il10 in the colon and ameliorates the disease pathology in TNBS-induced colitis. Curli treatment is comparable to the treatment with anti-tumour necrosis factor alpha (anti-TNFα) antibodies, a treatment known to reduce the severity of acute colitis in humans and mice.Conclusion:These results suggest that the bacterial amyloids had a role in helping to maintain immune homeostasis in the intestinal mucosa via the TLR2/IL-10 axis. Furthermore, bacterial amyloids may be a potential candidate therapeutic to treat intestinal inflammatory disorders owing to their remarkable immunomodulatory activity.

Comparison of hollow fiber module designs in membrane distillation process employed lumen-side and shell-side feed

In the present work, randomly packed, curly fiber, spacer scattered, and spacer knitted modules were fabricated and their performance in direct contact membrane distillation employed lumen-side feed and shell-side feed was investigated, respectively. Experimental results showed that the flux of different module configurations in shell-side feed operations were lower than that in lumen-side feed operations. Modified modules showed higher flux compared with randomly packed module and the spacer knitted module had the best performance with 51.8% enhancement at 328 K in lumen-side feed performance. The module flux increased as the feed flow rate increased, and all the modified modules showed relative high flux even at a low feed flow rate, which confirmed the fluid improvement caused by the spacers or wavy geometries. The overall heat transfer coefficient of modules ranged from 704 to 1,961 W/(m2 K) in lumen-side feed operations, while that was lower to be 425–645 W/(m2 K) in shell-side feed operations. In addition, higher heat transfer coefficients and temperature polarization coefficient were observed with modified module configurations. The sodium chloride tracer response technique was used to reveal the shell-side flow pattern and distribution for various module designs. Results showed that the modified module configurations can provide a better flow distribution in shell side with longer residence time and smaller variance, thus higher thermal efficiency and flux could be accomplished.

Vertical stratification of matrix production is essential for physical integrity and architecture of macrocolony biofilms of Escherichia coli.

SummaryBacterial macrocolony biofilms grow into intricate three‐dimensional structures that depend on self‐produced extracellular polymers conferring protection, cohesion and elasticity to the biofilm. In E scherichia coli, synthesis of this matrix – consisting of amyloid curli fibres and cellulose – requires CsgD, a transcription factor regulated by the stationary phase sigma factor RpoS, and occurs in the nutrient‐deprived cells of the upper layer of macrocolonies. Is this asymmetric matrix distribution functionally important or is it just a fortuitous by‐product of an unavoidable nutrient gradient? In order to address this question, the RpoS‐dependent csgD promoter was replaced by a vegetative promoter. This re‐wiring of csgD led to CsgD and matrix production in both strata of macrocolonies, with the lower layer transforming into a rigid ‘base plate’ of growing yet curli‐connected cells. As a result, the two strata broke apart followed by desiccation and exfoliation of the top layer. By contrast, matrix‐free cells at the bottom of wild‐type macrocolonies maintain colony contact with the humid agar support by flexibly filling the space that opens up under buckling areas of the macrocolony. Precisely regulated stratification in matrix‐free and matrix‐producing cell layers is thus essential for the physical integrity and architecture of E . coli macrocolony biofilms.

Bakterielle Megastädte

What makes a multicellular aggregate of bacterial cells a biofilm is the presence of an extracellular polymeric matrix. Besides being protective, matrix components such as amyloid curli fibres and cellulose in Escherichia coli also assemble into a complex architecture that is controlled by RpoS and c-di-GMP. This confers extreme cohesion and elasticity to macrocolony biofilms. As a consequence, these biofilms behave like tissues, i. e. they buckle up into intricate morphological structures during growth.

Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains.

The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP-stimulated production of amyloid curli fibers and cellulose. Thus, EAEC, which adhere in a "stacked brick" biofilm mode, have a potential for high c-di-GMP accumulation due to DgcX, a strongly expressed additional DGC. In contrast, EHEC and UPEC, which use alternative adherence mechanisms, tend to have extra PDEs, suggesting that low cellular c-di-GMP levels are crucial for these strains under specific conditions. Overall, our study also indicates that GGDEF/EAL domain proteins evolve rapidly and thereby contribute to adaptation to host-specific and environmental niches of various types of E. coli.

Logical-continuous modelling of post-translationally regulated bistability of curli fiber expression in Escherichia coli.

BackgroundBacteria have developed a repertoire of signalling mechanisms that enable adaptive responses to fluctuating environmental conditions. The formation of biofilm, for example, allows persisting in times of external stresses, e.g. induced by antibiotics or a lack of nutrients. Adhesive curli fibers, the major extracellular matrix components in Escherichia coli biofilms, exhibit heterogeneous expression in isogenic cells exposed to identical external conditions. The dynamical mechanisms underlying this heterogeneity remain poorly understood. In this work, we elucidate the potential role of post-translational bistability as a source for this heterogeneity.ResultsWe introduce a structured modelling workflow combining logical network topology analysis with time-continuous deterministic and stochastic modelling. The aim is to evaluate the topological structure of the underlying signalling network and to identify and analyse model parameterisations that satisfy observations from a set of genetic knockout experiments. Our work supports the hypothesis that the phenotypic heterogeneity of curli expression in biofilm cells is induced by bistable regulation at the post-translational level. Stochastic modelling suggests diverse noise-induced switching behaviours between the stable states, depending on the expression levels of the c-di-GMP-producing (diguanylate cyclases, DGCs) and -degrading (phosphodiesterases, PDEs) enzymes and reveals the quantitative difference in stable c-di-GMP levels between distinct phenotypes. The most dominant type of behaviour is characterised by a fast switching from curli-off to curli-on with a slow switching in the reverse direction and the second most dominant type is a long-term differentiation into curli-on or curli-off cells. This behaviour may implicate an intrinsic feature of the system allowing for a fast adaptive response (curli-on) versus a slow transition to the curli-off state, in line with experimental observations.ConclusionThe combination of logical and continuous modelling enables a thorough analysis of different determinants of bistable regulation, i.e. network topology and biochemical kinetics, and allows for an incorporation of experimental data from heterogeneous sources. Our approach yields a mechanistic explanation for the phenotypic heterogeneity of curli fiber expression. Furthermore, the presented work provides a detailed insight into the interactions between the multiple DGC- and PDE-type enzymes and the role of c-di-GMP in dynamical regulation of cellular decisions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0183-x) contains supplementary material, which is available to authorized users.

Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.

Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces.

Bacterial amyloids promote type I interferon production and accelerate autoimmunity (BA6P.124)

Abstract Infections can be pathogenic in systemic lupus erythematosus (SLE). Curli is a bacterial amyloid part of enteric bacterial biofilms, like those of E. coli and Salmonella. We have recently found that curli fibers form highly immunogenic composites with DNA within the biofilm. Here we studied the immune response to curli-DNA complexes in the context of murine SLE. For in vitro studies, we stimulated bone marrow-derived dendritic cells (cDCs) from WT and lupus-prone mice with curli amyloid from S. Typhimurium biofilms. A dose titration of curli amyloid induced robust proinflammatory cytokine and type I-IFN responsive gene expression in cDCs. Using synthetic curli peptide, we found that curli and nucleic acids synergize to activate cDCs. We injected in vivo pre-diseased lupus-prone NZB/W-F1 mice i.p. with curli-DNA complexes and found that they accelerate disease, inducing high titers of anti-dsDNA and anti-chromatin autoantibodies. Curli-DNA complexes induced autoantibodies also in non-autoimmune C57BL/6 mice. Finally, we infected NZB/W-F1 mice i.p. with curli-expressing or curli mutant E. coli or Salmonella. Curli-competent bacteria triggered far higher autoantibody titers than curli mutant strains. In conclusion, biofilm-derived curli-DNA complexes are potent activators of DCs and can rapidly break tolerance and accelerate autoimmunity in mice, suggesting that bacterial amyloids and biofilm-based bacterial infections are important environmental triggers in lupus.

The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Toll-like receptor 2 and NLRP3 cooperate to recognize a functional bacterial amyloid, curli.

Amyloids are proteins with cross-β-sheet structure that contribute to pathology and inflammation in complex human diseases, including Alzheimer's disease, Parkinson's disease, type II diabetes, and secondary amyloidosis. Bacteria also produce amyloids as a component of their extracellular matrix during biofilm formation. Recently, several human amyloids were shown to activate the NLRP3 inflammasome, leading to the activation of caspase 1 and production of interleukin 1β (IL-1β). In this study, we investigated the activation of the NLRP3 inflammasome by bacterial amyloids using curli fibers, produced by Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we show that curli fibers activate the NLRP3 inflammasome, leading to the production of IL-1β via caspase 1 activation. Investigation of the underlying mechanism revealed that activation of Toll-like receptor 2 (TLR2) by curli fibers is critical in the generation of IL-1β. Interestingly, activation of the NLRP3 inflammasome by curli fibers or by amyloid β of Alzheimer's disease does not cause cell death in macrophages. Overall, these data identify a cross talk between TLR2 and NLRP3 in response to the bacterial amyloid curli and generation of IL-1β as a product of this interaction.

Cyclic‐di‐GMP signalling and biofilm‐related properties of the Shiga toxin‐producing 2011 German outbreak Escherichia coli O104:H4

In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)—producing the biofilm-promoting second messenger c-di-GMP—that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.

Strong underwater adhesives made by self-assembling multi-protein nanofibres.

Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly, and structure-function relationship of those natural amyloid fibers remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibers. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibers have an underwater adhesion energy approaching 20.9 mJ/m2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibers taken on their own at all pHs and exhibit better tolerance to auto-oxidation than Mfps at pH ≥7.0. This work establishes a platform for engineering multi-component self-assembling materials inspired by nature.

Sulfate assimilation pathway intermediate phosphoadenosine 59-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli.

The enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 59-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 59-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene downregulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with a distinct mechanism and role: whilst APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to remodulation of the cell surface, as part of a more global effect on cell physiology.

The Influence of Hot Pressing Temperatures in Needle Punching Nonwoven

Non-woven textile industry in an emerging field, with the process short, high yield, low cost and wide source of raw materials, but also has excellent performance of many functions on, making non-woven over the past half century gained textiles attention and consumers of all ages. The proportion of the world of non-woven fiber material used in the product, 85% in rayon ,and the other 15% in natural fibers, polyester fibers which accounted for the largest proportion of use. The experiment uses a low melting point polyester fiber (LPET) 20%, three-dimensional hollow curly polyester fiber (TPET) and recycled far infrared fiber (REPET) 40% each as the basic conditions change pressing temperature 100 °C-140 °C, in order to observe and compare the effects of temperature on the non-woven fabric, this experiment tests including air permeability, tensile strength testing, infrared testing and SEM, respectively in different hot pressing temperature, each of the non-woven hot pressing temperatures sample go through microscopic to analysis for non-woven with the hot temperatures strong reason to improve or decline with hot temperature of air permeability.