SUMMARY Research on the human microbiome has established that commensal and pathogenic bacteria can influence obesity, cancer, and autoimmunity through mechanisms mostly unknown. We found that a component of bacterial biofilms, the amyloid protein curli, irreversibly formed fibers with bacterial DNA during biofilm formation. This interaction accelerated amyloid polymerization and created potent immunogenic complexes that activated immune cells, including dendritic cells, to produce cytokines such as Type I interferons, which are pathogenic in systemic lupus erythematosus (SLE). When given systemically, curli-DNA composites triggered immune activation and production of autoantibodies in lupus-prone and wild-type mice. We also found that the infection of lupus-prone mice with curli-producing bacteria triggered higher autoantibody titers compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity.

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 ...

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 ␥␦ 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.

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.

Background: Curli fibers are recognized by the TLR2/TLR1 complex. Results: CD14 binds curli fibers and enhances TLR2/TLR1-dependent NF-B activation as well as cytokine and nitrite production. Conclusion: CD14 is an adaptor protein for the TLR2/TLR1 complex binding the fibrillar structure of curli fibers. Significance: Understanding the immune receptor complexes that recognize amyloids will have implications for amyloidassociated diseases.