Biofilms 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.

The human microbiota is the community of microorganisms that live upon or within their human host. The microbiota consists of various microorganisms including bacteria, fungi, viruses, and archaea; the gut microbiota is comprised mostly of bacteria. Many bacterial species within the gut microbiome grow as biofilms, which are multicellular communities embedded in an extracellular matrix. Studies have shown that the relative abundances of bacterial species, and therefore biofilms and bacterial byproducts, change during progression of a variety of human diseases including gastrointestinal, autoimmune, neurodegenerative, and cancer. Studies have shown the location and proximity of the biofilms within the gastrointestinal tract might impact disease outcome. Gram-negative enteric bacteria secrete the amyloid curli, which makes up as much as 85% of the extracellular matrix of enteric biofilms. Curli mediates cell-cell attachment and attachment to various surfaces including extracellular matrix components such as fibronectin and laminin. Structurally, curli is strikingly similar to pathological and immunomodulatory human amyloids such as amyloid-β, which has been implicated in Alzheimer's disease, α-synuclein, which is involved in Parkinson's disease, and serum amyloid A, which is secreted during the acute phase of inflammation. The immune system recognizes both bacterial amyloid curli and human amyloids utilizing the same receptors, so curli also induces inflammation. Moreover, recent work indicates that curli can participate in the self-assembly process of pathological human amyloids. Curli is found within biofilms of commensal enteric bacteria as well as invasive pathogens; therefore, evidence suggests that curli contributes to complex human diseases. In this review, we summarize the recent findings on how bacterial biofilms containing curli participate in the pathological and immunological processes in gastrointestinal diseases, systemic autoimmune diseases, and neurodegenerative diseases.

Amyloids are proteins with cross β-sheet structure, that contribute to pathology and inflammation in complex human diseases including Alzheimers Disease, Parkinsons 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 NLRP3 inflammasome leading to the activation of caspase-1 and production of IL-1β. In this study, we investigated the activation of NLRP3 inflammasome by bacterial amyloids using curli fibers, produced by Salmonella enterica serovar Typhimurium and Escherichia coli. Here, we show that curli fibers activate 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 (TLR) 2 by curli fibers is critical in the generation of IL-1β. Interestingly, activation of NLRP3 inflammasome neither by curli fibers nor by amyloid β of Alzheimer's Disease causes cell death in macrophages. Overall, these data identify a crosstalk between TLR2 and NLRP3 in response to bacterial amyloid curli and generation of IL-1β as a product of this interaction.

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. Bacterial amyloids are conserved proteins expressed by many bacteria in biofilms. Bacterial amyloid curli and DNA form highly immunogenic complexes that stimulate autoimmunity and accelerate the progression of systemic lupus erythematosus. Here, we show that the innate immune receptors TLR2 and TLR9 are critical for shaping the autoimmune adaptive immune response to curli-DNA complexes. Mice deficient in these receptors show attenuated production of anti-double-stranded DNA autoantibodies and type I IFNs. The cross beta-sheet structure of curli is recognized by TLR2, leading to endosomal internalization of the curli-DNA complex and subsequent binding to TLR9. Synchrotron diffraction studies suggest that curli-DNA immune complexes present double-stranded DNA rods at an inter-DNA spacing that matches well to the steric size of TLR9, thus promote multivalent amplification of binding and TLR9 activation. Overall, our results identify a novel series of events pivotal to induction of autoimmunity by amyloid-expressing bacteria.

The Toll-like receptor 2 (TLR2)/TLR1 receptor complex responds to amyloid fibrils, a common component of biofilm material produced by members of the phylaFirmicutes,Bacteroidetes, andProteobacteria. 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 pathogenSalmonella entericaserotype Typhimurium contributes to T helper 1 and T helper 17 responses by measuring cytokine production in the mouse colitis model. AcsgBAmutant, deficient in curli production, elicited decreased expression of interleukin 17A (IL-17A) and IL-22 in the cecal mucosa compared to theS. Typhimurium wild type. In TLR2-deficient mice, IL-17A and IL-22 expression was blunted duringS. 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 mannerin 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 duringS. 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.