Curli Fibrils Research Articles

Structural insight into Escherichia coli CsgA amyloid fibril assembly.

The discovery of functional amyloids in bacteria dates back several decades, and our understanding of the Escherichia coli curli biogenesis system has gradually expanded over time. However, due to its high aggregation propensity and intrinsically disordered nature, CsgA, the main structural component of curli fibrils, has eluded comprehensive structural characterization. Recent advancements in cryo-electron microscopy (cryo-EM) offer a promising tool to achieve high-resolution structural insights into E. coli CsgA fibrils. In this study, we outline an approach to addressing the colloidal instability challenges associated with CsgA, achieved through engineering and electrostatic repulsion. Then, we present the cryo-EM structure of CsgA fibrils at 3.62 Å resolution. This structure provides new insights into the cross-β structure of E. coli CsgA. Additionally, our study identifies two distinct spatial arrangements within several CsgA fibrils, a 2-CsgA-fibril pair and a 3-CsgA-fibril bundle, shedding light on the intricate hierarchy of the biofilm extracellular matrix and laying the foundation for precise manipulation of CsgA-derived biomaterials.IMPORTANCEThe visualization of the architecture of Escherichia coli CsgA amyloid fibril has been a longstanding research question, for which a high-resolution structure is still unavailable. CsgA serves as a major subunit of curli, the primary component of the extracellular matrix generated by bacteria. The support provided by this extracellular matrix enables bacterial biofilms to resist antibiotic treatment, significantly impacting human health. CsgA has been identified in members of Enterobacteriaceae, with pathogenic E. coli being the most well-known model system. Our novel insights into the structure of E. coli CsgA protofilaments form the basis for drug design targeting diseases associated with biofilms. Additionally, CsgA is widely researched in biomaterials due to its self-assembly characteristics. The resolved spatial arrangements of CsgA amyloids revealed in our study will further enhance the precision design of functional biomaterials. Therefore, our study uniquely contributes to the understanding of CsgA amyloids for both microbiology and material science.

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses.

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro. The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.

Bacterial Amyloid Curli Associated Gut Epithelial Neuroendocrine Activation Predominantly Observed in Alzheimer's Disease Mice with Central Amyloid-β Pathology.

Substantial evidence from recent research suggests an influential and underappreciated force in Alzheimer's disease (AD) pathogenesis: the pathological signals originate from outside the brain. Pathogenic bacteria produce amyloid-like proteins "curli" that form biofilms and show functional similarities to human amyloid-β (Aβ). These proteins may contribute to neurological disease progression via signaling cascade from the gut to the brain. We propose that curli causes neuroendocrine activation from the gut to brain that promotes central Aβ pathology. PGP9.5 and TLR2 levels in response to curli in the lumen of Tg2576 AD mice were analyzed by immunohistochemical and qRT-PCR analysis. Western blot and human 3D in vitro enteroids culture systems were also used. 16S rRNA gene sequencing was used to investigate bacterial dysbiosis. We found significant increase in bacterial-amyloid curli with elevated TLR2 at the mRNA level in the pre- and symptomatic Tg-AD gut compared to littermate WT controls. This data associates with increased gram-positive bacterial colonization in the ileum of the symptomatic AD mice. We found fundamental evidence for vagus nerve activation in response to bacterial curli. Neuroendocrine marker PGP9.5 was significantly elevated in the gut epithelium of symptomatic AD mice, and this was colocalized with increased TLR2 expression. Enteroids, 3D-human ileal mini-gut monolayer in vitro model system also revealed increase levels of TLR2 upon stimulation with purified bacterial curli fibrils. These findings reveal the importance of pathological changes within the gut-vagus-brain signaling in response to luminal bacterial amyloid that might play a vital role in central Aβ pathogenesis seen in the AD brain.

Purification of the Bacterial Amyloid "Curli" from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues.

Microbiologists are learning to appreciate the importance of "functional amyloids" that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

Immunogenic characterization and protective efficacy of recombinant CsgA, major subunit of curli fibers, against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is one of the major pathogens responsible for vibriosis and zoonotic infections in teleosts, marine invertebrates, and also humans through consumption of contaminated or unprocessed seafood. Emergence of resistance against current accessible antibiotics and spread to the food chain and environment necessitate the development of safe and effective subunit vaccine against this bacterium. Many bacteria including V. parahaemolyticus produce extracellular curli fibrils, heteropolymeric filaments of major and minor subunit, which have been implicated in adhesion, biofilm formation, and virulence. Adhesins are the primary contact points with the host which help in establishing infection and colonization. CsgA, an adhesin, is the major structural component of the curli fiber that forms homopolymers of several hundred units. Due to their exposure on the cell surface, the curli fibers are recognized by the host's immune system, would generate high immune response, and therefore can serve as effective vaccine candidate. In the present study, we describe characterization of the csgA gene, and preparation of recombinant soluble CsgA of V. parahaemolyticus (rVpCsgA), and evaluation of its vaccine potential. Immunization of BALB/c mice with the rVpCsgA mounted a strong immune response. Cellular immune assays such as antibody isotyping, in vitro splenocyte proliferation analysis, and cytokine profiling revealed mixed T-helper cell immune response. The anti-rVpCsgA antiserum was agglutination positive and specifically cross-reacted with the curli CsgA present on the outer membrane of V. parahaemolyticus cells, thus demonstrating its neutralization potential. One hundred percent survival of the immunized mice upon challenge with the lethal dosage of the bacterium established that the rVpCsgA could serve as an effective vaccine against the bacterium. KEY POINTS: • Recombinant histidine-tagged CsgA of V. parahaemolyticus, rVpCsgA, was purified. • The rVpCsgA immunization produced mixed immune response and agglutinating antibodies. • Immunization with the rVpCsgA protected mice against V. parahaemolyticus challenge.

Cytotoxic Curli Intermediates Form during Salmonella Biofilm Development.

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 β 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 β, 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 β 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 β, 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.

Effect of amyloid curli fibrils and curli CsgA monomers from Escherichia coli on in vitro model of intestinal epithelial barrier stimulated with cytokines

Amyloid curli fibrils produced by Escherichia coli are well-known virulence factor influencing E. coli adhesion and biofilm formation. However, the impact of curli on intestinal epithelial barrier stimulated with proinflammatory cytokines is unknown. In the study, we examined the effect of curli produced by nonpathogenic E. coli K-12 and wild-type E. coli EC32 strains, and purified CsgA proteins on differentiated Caco-2 cell monolayers stimulated with a mixture of IL-1β, TNF-α, and INFγ cytokines as a model of ‘inflamed intestinal epithelial barrier’ in vitro. The results of the study indicated that curliated E. coli adhered better to polarized Caco-2 cells than their curli-deficient mutants and the adherence was further augmented by stimulation of epithelial cells with proinflammatory cytokines. Interestingly, curli reduced internalization but enhanced intracellular survival of the wild-type E. coli strain EC32 within intestinal epithelial cells. Curli-expressing E. coli, as well as purified CsgA proteins, attenuated IL-8 secretion by unstimulated Caco-2 cells, although the effect was barely observed on cytokine-stimulated cells. The findings of the study revealed that curli fibrils are an important virulence factor enabling curliated E. coli to effectively colonize intestinal epithelium especially in individuals with inflammatory intestinal disorders.

Mechanical unfolding of alpha- and beta-helical protein motifs.

Alpha-helices and beta-sheets are the two most common secondary structure motifs in proteins. Beta-helical structures merge features of the two motifs, containing two or three beta-sheet faces connected by loops or turns in a single protein. Beta-helical structures form the basis of proteins with diverse mechanical functions such as bacterial adhesins, phage cell-puncture devices, antifreeze proteins, and extracellular matrices. Alpha-helices are commonly found in cellular and extracellular matrix components, whereas beta-helices such as curli fibrils are more common as bacterial and biofilm matrix components. It is currently not known whether it may be advantageous to use one helical motif over the other for different structural and mechanical functions. To better understand the mechanical implications of using different helix motifs in networks, here we use Steered Molecular Dynamics (SMD) simulations to mechanically unfold multiple alpha- and beta-helical proteins at constant velocity at the single molecule scale. We focus on the energy dissipated during unfolding as a means of comparison between proteins and work normalized by protein characteristics (initial and final length, # H-bonds, # residues, etc.). We find that although alpha-helices such as keratin and beta-helices CsgA and CsgB can require similar amounts of work to unfold, the normalized work per hydrogen bond, initial end to end length, and number of residues is greater for beta-helices at the same pulling rate. To explain this, we analyze the orientation of the backbone alpha carbons and backbone hydrogen bonds during unfolding. We find that the larger width and shorter height of beta-helices results in smaller angles between the protein backbone and the pulling direction during unfolding. As subsequent strands are separated from the beta-helix core, the angle between the backbone and the pulling direction diminishes. This marks a transition where beta-sheet hydrogen bonds become loaded predominantly in a collective shearing mode, which requires a larger rupture force. This finding underlines the importance of geometry in optimizing resistance to mechanical unfolding in proteins. The helix radius is identified here as an important parameter that governs how much sacrificial energy dissipation capacity can be stored in protein networks, where beta-helices offer unique properties.

Modulation of innate immunity by amyloidogenic peptides: Systematic review of pattern recognition receptors and peptide aggregation state

Amyloid formation contributes to the development and progression of multiple aging-associated neurodegenerative disorders. Local amyloidoses are characterized by activation of monocytes and macrophages, phagocytic peptide clearance, and upregulation of pro-inflammatory cytokines. Increasing evidence suggests a role for amyloid-producing gut microbiota – as well as amyloid associated with comorbid type 2 diabetes – in regulation of brain amyloidogenesis. However, it is unclear whether these diverse peptides share common mechanisms of innate immune activation, and which aggregate forms induce a pro-inflammatory response. To evaluate the evidence for specific amyloidogenic peptide species and their associated pattern recognition receptors, we systematically searched Embase, PubMed, and Medline for empiric studies through February 2017. We identified 150 studies of diverse peptides – including amyloid-beta, tau, alpha-synuclein, cellular prion protein, serum amyloid A, islet amyloid polypeptide, and bacterial Curli fibrils – demonstrating scavenger receptor-mediated phagocytosis and templating, Toll-like receptor-mediated inflammasome priming, or inflammasome activation. Activation of TLR2 and NLRP3 was common to most peptides identified. An additional 76 studies implicated soluble species (34%), insoluble fibrils (24%), or both (35%). These reports suggest that both early and late aggregates of diverse amyloid-forming peptides contribute to distinct, evolutionarily conserved pattern recognition events with functional roles in pathogen recognition and antimicrobial defense. Further characterization of the interaction between amyloidogenic peptides and innate immune cells will aid in the development of anti-amyloid and immune-modulating therapies.

Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9

Abstract Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is amyloid curli which forms immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation type I interferons (IFNs) and anti-double stranded (ds) DNA autoantibodies. DNA complexed with curli stimulates Toll-like receptor (TLR) 9 through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface TLR2, enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to TLR9, inducing production of type I IFNs. Upon stimulation of macrophages with curli-DNA complexes, only 5% of TLR2−/− macrophages harbored intracellular curli, suggesting that TLR2 drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization led to a significant decrease in Ifnβ expression. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils results in the formation of ordered curli-DNA immune complexes resulting in optimal spacing to TLR9. Production of anti-dsDNA autoantibodies and type I IFNS in response to curli-DNA fibers was attenuated in the TLR2−/−, TLR9 mutant, and TLR2−/− - TLR9 mutant mice compared to wildtype mice, suggesting that TLR2 and TLR9 are critical for the autoimmune immune response. Overall, our results identify a pivotal series of events that leads to the severe pro-autoimmune effects of amyloid-expressing bacteria.

Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9.

Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is the amyloid curli, which forms highly immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation of type I interferons (IFNs) and anti-double-stranded DNA antibodies. Here, we show that DNA complexed with amyloid curli powerfully stimulates Toll-like receptor 9 (TLR9) through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface Toll-like receptor 2 (TLR2), enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to endosomal TLR9, inducing production of type I IFNs. Analysis of wild-type and TLR2-deficient macrophages showed that TLR2 is the major receptor that drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization via endocytosis inhibitors led to a significant decrease in Ifnβ expression. Confocal microscopy analysis confirmed that the TLR2-bound curli was required for shuttling of DNA to endosomal TLR9. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils resulted in the formation of ordered curli-DNA immune complexes. Curli organizes parallel, double-stranded DNA rods at an inter-DNA spacing that matches up well with the steric size of TLR9. We also found that production of anti-double-stranded DNA autoantibodies in response to curli-DNA was attenuated in TLR2- and TLR9-deficient mice and in mice deficient in both TLR2 and TLR9 compared to wild-type mice, suggesting that both innate immune receptors are critical for shaping the autoimmune adaptive immune response. We also detected significantly lower levels of interferon-stimulated gene expression in response to purified curli-DNA in TLR2 and TLR9 deficient mice compared to wild-type mice, confirming that TLR2 and TLR9 are required for the induction of type I IFNs. Finally, we showed that curli-DNA complexes, but not cellulose, were responsible elicitation of the immune responses to bacterial biofilms. This study defines the series of events that lead to the severe pro-autoimmune effects of amyloid-expressing bacteria and suggest a mechanism by which amyloid curli acts as a carrier to break immune tolerance to DNA, leading to the activation of TLR9, production of type I IFNs, and subsequent production of autoantibodies.

Structural predictions for curli amyloid fibril subunits CsgA and CsgB

CsgA are the building blocks of curli fibrils.

Detection of Curli Biogenesis Genes Among Enterobacter cloacae Isolated From Blood Cultures

Background: Enterobacter cloacae bacteremia infection is an significant cause of morbidity and mortality in both developing and developed countries. Curli fibrils are considered the most important virulence factor in these bacteria. Objectives: The purpose of this research was to detect curli biogenesis genes among Enterobacter cloacae isolated from blood cultures. Materials and Methods: Nine E. cloacae isolates were collected from blood cultures of inpatients admitted to three hospitals in the Tehran, Iran during December 2012 to November 2013. Confirmation of identity of the infecting organism as E. cloacae was performed by API20E system and the presence of a csgA and csgD genes by PCR using csgA and csgD specific primers. Results: All of E. cloacae bloodstream strains (100%) harbored csgD gene (curli biogenesis activator) and seven (77.75%) carried csgA gene (curli major subunit) which is indicative of wide distribution of this virulence factor among our isolates. Conclusions: Extensive presence of curli biogenesis genes (csgD and csgA) among E.cloacae strains and probably expression of those crucial role of this virulence factor in E. cloacae pathogenesis and invasiveness.

Synthesis and patterning of tunable multiscale materials with engineered cells.

Many natural biological systems - such as biofilms, shells and skeletal tissues - are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.

Curli Functional Amyloid Systems Are Phylogenetically Widespread and Display Large Diversity in Operon and Protein Structure

Escherichia coli and a few other members of the Enterobacteriales can produce functional amyloids known as curli. These extracellular fibrils are involved in biofilm formation and studies have shown that they may act as virulence factors during infections. It is not known whether curli fibrils are restricted to the Enterobacteriales or if they are phylogenetically widespread. The growing number of genome-sequenced bacteria spanning many phylogenetic groups allows a reliable bioinformatic investigation of the phylogenetic diversity of the curli system. Here we show that the curli system is phylogenetically much more widespread than initially assumed, spanning at least four phyla. Curli fibrils may consequently be encountered frequently in environmental as well as pathogenic biofilms, which was supported by identification of curli genes in public metagenomes from a diverse range of habitats. Identification and comparison of curli subunit (CsgA/B) homologs show that these proteins allow a high degree of freedom in their primary protein structure, although a modular structure of tightly spaced repeat regions containing conserved glutamine, asparagine and glycine residues has to be preserved. In addition, a high degree of variability within the operon structure of curli subunits between bacterial taxa suggests that the curli fibrils might have evolved to fulfill specific functions. Variations in the genetic organization of curli genes are also seen among different bacterial genera. This suggests that some genera may utilize alternative regulatory pathways for curli expression. Comparison of phylogenetic trees of Csg proteins and the 16S rRNA genes of the corresponding bacteria showed remarkably similar overall topography, suggesting that horizontal gene transfer is a minor player in the spreading of the curli system.

Epithelial Cells Augment Barrier Function via Activation of the Toll-Like Receptor 2/Phosphatidylinositol 3-Kinase Pathway upon Recognition of Salmonella enterica Serovar Typhimurium Curli Fibrils in the Gut

Curli fibrils, the best-characterized functional bacterial amyloids, are an important component of enterobacterial biofilms. We have previously shown that curli fibrils are recognized by the Toll-like receptor 2 (TLR2)/TLR1 heterodimer complex. Utilizing polarized T-84 cells, an intestinal epithelial cell line derived from colon carcinoma grown on semipermeable tissue culture inserts, we determined that infection with a Salmonella enterica serovar Typhimurium csgBA mutant, which does not express curli, resulted in an increase in intestinal barrier permeability and an increase in bacterial translocation compared to infection with curliated wild-type S. Typhimurium. When the TLR2 downstream signaling molecule phosphatidylinositol 3-kinase (PI3K) was blocked using wortmannin or LY294002, the difference in disruption of the intestinal epithelium and bacterial translocation was no longer observed. Additionally, disruption of polarized T-84 cells treated basolaterally with the TLR5 ligand flagellin was prevented when the polarized cells were simultaneously treated with the synthetic TLR2/TLR1 ligand Pam(3)CSK(4) or with purified curli fibrils in the apical compartment. Similar to in vitro observations, C57BL/6 mice infected with the csgBA mutant suffered increased disruption of the intestinal epithelium and therefore greater dissemination of the bacteria to the mesenteric lymph nodes than mice infected with wild-type S. Typhimurium. The differences in disruption of the intestinal epithelium and bacterial dissemination in the mice infected with csgBA mutant or wild-type S. Typhimurium were not apparent in TLR2-deficient mice. Overall, these studies report for the first time that activation of the TLR2/PI3K pathway by microbial amyloids plays a critical role in regulating the intestinal epithelial barrier as well as monitoring bacterial translocation during infection.

Microbial amyloids induce interleukin 17A (IL-17A) and IL-22 responses via Toll-like receptor 2 activation in the intestinal mucosa.

The Toll-like receptor 2 (TLR2)/TLR1 receptor complex responds to amyloid fibrils, a common component of biofilm material produced by members of the phyla Firmicutes, Bacteroidetes, and Proteobacteria. To determine whether this TLR2/TLR1 ligand stimulates inflammatory responses when bacteria enter intestinal tissue, we investigated whether expression of curli amyloid fibrils by the invasive enteric pathogen Salmonella enterica serotype Typhimurium contributes to T helper 1 and T helper 17 responses by measuring cytokine production in the mouse colitis model. A csgBA mutant, deficient in curli production, elicited decreased expression of interleukin 17A (IL-17A) and IL-22 in the cecal mucosa compared to the S. Typhimurium wild type. In TLR2-deficient mice, IL-17A and IL-22 expression was blunted during S. Typhimurium infection, suggesting that activation of the TLR2 signaling pathway contributes to the expression of these cytokines. T cells incubated with supernatants from bone marrow-derived dendritic cells (BMDCs) treated with curli fibrils released IL-17A in a TLR2-dependent manner in vitro. Lower levels of IL-6 and IL-23 production were detected in the supernatants of the TLR2-deficient BMDCs treated with curli fibrils. Consistent with this, three distinct T-cell populations-CD4(+) T helper cells, cytotoxic CD8(+) T cells, and γδ T cells-produced IL-17A in response to curli fibrils in the intestinal mucosa during S. Typhimurium infection. Notably, decreased IL-6 expression by the dendritic cells and decreased IL-23 expression by the dendritic cells and macrophages were observed in the cecal mucosa of mice infected with the curli mutant. We conclude that TLR2 recognition of bacterial amyloid fibrils in the intestinal mucosa represents a novel mechanism of immunoregulation, which contributes to the generation of inflammatory responses, including production of IL-17A and IL-22, in response to bacterial entry into the intestinal mucosa.

A statistical mechanical approach to protein aggregation

We develop a theory of aggregation using statistical mechanical methods. An example of a complicated aggregation system with several levels of structures is peptide/protein self-assembly. The problem of protein aggregation is important for the understanding and treatment of neurodegenerative diseases and also for the development of bio-macromolecules as new materials. We write the effective Hamiltonian in terms of interaction energies between protein monomers, protein and solvent, as well as between protein filaments. The grand partition function can be expressed in terms of a Zimm-Bragg-like transfer matrix, which is calculated exactly and all thermodynamic properties can be obtained. We start with two-state and three-state descriptions of protein monomers using Potts models that can be generalized to include q-states, for which the exactly solvable feature of the model remains. We focus on n × N lattice systems, corresponding to the ordered structures observed in some real fibrils. We have obtained results on nucleation processes and phase diagrams, in which a protein property such as the sheet content of aggregates is expressed as a function of the number of proteins on the lattice and inter-protein or interfacial interaction energies. We have applied our methods to Aβ(1-40) and Curli fibrils and obtained results in good agreement with experiments.

The Functional Curli Amyloid Is Not Based on In-register Parallel β-Sheet Structure

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-beta structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel beta-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a beta-helix-like structure but are not sufficient to establish such a structure definitively.

Responses to Amyloids of Microbial and Host Origin Are Mediated through Toll-like Receptor 2

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimer's disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.