SUMMARYBiofilms of enteric bacteria are highly complex, with multiple components that interact to fortify the biofilm matrix. Within biofilms of enteric bacteria such asEscherichia coliandSalmonellaspecies, the main component of the biofilm is amyloid curli. Other constituents include cellulose, extracellular DNA, O antigen, and various surface proteins, including BapA. Only recently, the roles of these components in the formation of the enteric biofilm individually and in consortium have been evaluated. In addition to enhancing the stability and strength of the matrix, the components of the enteric biofilm influence bacterial virulence and transmission. Most notably, certain components of the matrix are recognized as pathogen-associated molecular patterns. Systemic recognition of enteric biofilms leads to the activation of several proinflammatory innate immune receptors, including the Toll-like receptor 2 (TLR2)/TLR1/CD14 heterocomplex, TLR9, and NLRP3. In the model ofSalmonella entericaserovar Typhimurium, the immune response to curli is site specific. Although a proinflammatory response is generated upon systemic presentation of curli, oral administration of curli ameliorates the damaged intestinal epithelial barrier and reduces the severity of colitis. Furthermore, curli (and extracellular DNA) of enteric biofilms potentiate the autoimmune disease systemic lupus erythematosus (SLE) and promote the fibrillization of the pathogenic amyloid α-synuclein, which is implicated in Parkinson's disease. Homologues of curli-encoding genes are found in four additional bacterial phyla, suggesting that the biomedical implications involved with enteric biofilms are applicable to numerous bacterial species.

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.

bAmyloids are proteins with cross-␤-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 1␤ (IL-1␤). 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-1␤ 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-1␤. Interestingly, activation of the NLRP3 inflammasome by curli fibers or by amyloid ␤ 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-1␤ as a product of this interaction.A myloid proteins are produced by both bacteria and humans. In humans, more than 60 amyloidogenic proteins are produced throughout the body (1). Amyloids accumulate, forming deposits, during several complex diseases, such as Alzheimer's disease (AD), Parkinson's disease, type II diabetes, and secondary amyloidosis. Although it was initially thought that amyloids were only misfolded proteins causing disease pathology, it is becoming more apparent that the proteins have a function in the human body (2, 3). For instance, Pmel17, involved in melanin production, prevents melanocyte cytotoxicity (4-6), and secretory hormones in the endocrine system are stored in a cross-beta-sheetrich structure in secretory granules (7). Furthermore, it has recently been proposed that the amyloid ␤ peptide, found in the senile plaques of Alzheimer's disease patients, binds specific DNA regions and participates in gene regulation (8).Bacteria produce amyloids as a component of their extracellular matrix (ECM) to build multicellular communities termed biofilms (9). Biofilms are characterized by their resistant nature in response to environmental insults, including chemical treatments, antibiotics, and the immune system (10). It is thought that the amyloids act as a shield to protect bacteria in biofilms due to their highly resistant nature against chemicals and proteolytic enzymes. Although it is estimated that up to 40% of bacterial species produce amyloids in their biofilms (11), most of these proteins remain uncharacterized. Curli fibers, amyloids produced in the biofilms of Escherichia coli and Salmonella enterica serovar Typhimurium, are the most studied bacterial amyloid to date. Curli fibers are encoded by the csg gene cluster formed by two operons, csgBAC and csgDEFG (12-14). CsgA, the ...

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with panamyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibioticmediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.

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.