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Mathematical models are necessary to understand how the material composition of biofilms can influence their physical properties. Here, we developed a 4D computational toolchain for the analysis of bead trajectories, which laid the groundwork for establishing critical parameters for mathematical models of particle movement in biofilms. Using this open-source trajectory analyzer, we determined that the presence of bacterial amyloid curli changes the material properties of a biofilm, making the biofilm matrix rigid. This software is a powerful tool to analyze treatment- and environment-induced changes in biofilm structure and cell movement in biofilms. The open-source analyzer is fully adaptable and extendable in a modular fashion using VRL-Studio to further enhance and extend its functions., Biofilms exist in complex environments, including the intestinal tract, as a part of the gastrointestinal microbiota. The interaction of planktonic bacteria with biofilms can be influenced by material properties of the biofilm. During previous confocal studies, we observed that amyloid curli-containing Salmonella enterica serotype Typhimurium and Escherichia coli biofilms appeared rigid. In these studies, Enterococcus faecalis, which lacks curli-like protein, showed more fluid movement. To better characterize the material properties of the biofilms, a four-dimensional (4D) model was designed to track the movement of 1-μm glyoxylate beads in 10- to 20-μm-thick biofilms over approximately 20 min using laser-scanning confocal microscopy. Software was developed to analyze the bead trajectories, the amount of time they could be followed (trajectory life span), the velocity of movement, the surface area covered (bounding boxes), and cellular density around each bead. Bead movement was found to be predominantly Brownian motion. Curli-containing biofilms had very little bead movement throughout the low- and high-density regions of the biofilm compared to E. faecalis and isogenic curli mutants. Curli-containing biofilms tended to have more stable bead interactions (longer trajectory life spans) than biofilms lacking curli. In biofilms lacking curli, neither the velocity of bead movement nor the bounding box volume was strictly dependent on cell density, suggesting that other material properties of the biofilms were influencing the movement of the beads and flexibility of the material. Taken together, these studies present a 4D method to analyze bead movement over time in a 3D biofilm and suggest curli confers rigidity to the extracellular matrix of biofilms. IMPORTANCE Mathematical models are necessary to understand how the material composition of biofilms can influence their physical properties. Here, we developed a 4D computational toolchain for the analysis of bead trajectories, which laid the groundwork for establishing critical parameters for mathematical models of particle movement in biofilms. Using this open-source trajectory analyzer, we determined that the presence of bacterial amyloid curli changes the material properties of a biofilm, making the biofilm matrix rigid. This software is a powerful tool to analyze treatment- and environment-induced changes in biofilm structure and cell movement in biofilms. The open-source analyzer is fully adaptable and extendable in a modular fashion using VRL-Studio to further enhance and extend its functions.

Pathological self-assembly is a concept that is classically associated with amyloids, such as amyloid-β (Aβ) in Alzheimer's disease and α-synuclein in Parkinson's disease. In prokaryotic organisms, amyloids are assembled extracellularly in a similar fashion to human amyloids. Pathogenicity of amyloids is attributed to their ability to transform into several distinct structural states that reflect their downstream biological consequences. While the oligomeric forms of amyloids are thought to be responsible for their cytotoxicity via membrane permeation, their fibrillar conformations are known to interact with the innate immune system to induce inflammation. Furthermore, both eukaryotic and prokaryotic amyloids can self-assemble into molecular chaperones to bind nucleic acids, enabling amplification of Toll-like receptor (TLR) signaling. Recent work has shown that antimicrobial peptides (AMPs) follow a strikingly similar paradigm. Previously, AMPs were thought of as peptides with the primary function of permeating microbial membranes. Consistent with this, many AMPs are facially amphiphilic and can facilitate membrane remodeling processes such as pore formation and fusion. We show that various AMPs and chemokines can also chaperone and organize immune ligands into amyloid-like ordered supramolecular structures that are geometrically optimized for binding to TLRs, thereby amplifying immune signaling. The ability of amphiphilic AMPs to self-assemble cooperatively into superhelical protofibrils that form structural scaffolds for the ordered presentation of immune ligands like DNA and dsRNA is central to inflammation. It is interesting to explore the notion that the assembly of AMP protofibrils may be analogous to that of amyloid aggregates. Coming full circle, recent work has suggested that Aβ and other amyloids also have AMP-like antimicrobial functions. The emerging perspective is one in which assembly affords a more finely calibrated system of recognition and response: the detection of single immune ligands, immune ligands bound to AMPs, and immune ligands spatially organized to varying degrees by AMPs, result in different immunologic outcomes. In this framework, not all ordered structures generated during multi-stepped AMP (or amyloid) assembly are pathological in origin. Supramolecular structures formed during this process serve as signatures to the innate immune system to orchestrate immune amplification in a proportional, situation-dependent manner.

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host., Author summary Our manuscript focuses on curli, a ‘functional amyloid’ produced by Salmonella as well as other enteric bacteria. We present the first biochemical evidence that these fibers are produced in the gastrointestinal tract of mice after oral infection, the natural route for Salmonella infections. This finding is significant because of the immune impacts on the host; we show that curli cause an increase in autoimmunity and inflammation in the knee joints of infected mice. Reactive arthritis is a known autoimmune complication after enteric infections and our results indicate that presence of curli in the gut provides a novel linchpin of pathogenesis. As curli or curli-like amyloids are also produced by the commensal bacteria, it is possible that the unintended release of amyloids produced by the microbiota could trigger similar autoimmune reactions. Finally, our work provides conceptual evidence for the possibility of cross-seeding between bacterial amyloids like curli and human amyloids involved in amyloid-associated diseases such as Alzheimer’s Disease via the gut microbiome or infections.

Sepsis is a leading cause of morbidity and mortality in intensive care units. Previously, we identified Protein Kinase C-delta (PKCδ) as an important regulator of the inflammatory response in sepsis. An important issue in development of anti-inflammatory therapeutics is the risk of immunosuppression and inability to effectively clear pathogens. In this study, we investigated whether PKCδ inhibition prevented organ dysfunction and improved survival without compromising pathogen clearance. Sprague Dawley rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Post-surgery, PBS or a PKCδ inhibitor (200µg/kg) was administered intra-tracheally (IT). At 24 hours post-CLP, there was evidence of lung and kidney dysfunction. PKCδ inhibition decreased leukocyte influx in these organs, decreased endothelial permeability, improved gas exchange, and reduced blood urea nitrogen/creatinine ratios indicating organ protection. PKCδ inhibition significantly decreased bacterial levels in the peritoneal cavity, spleen and blood but did not exhibit direct bactericidal properties. Peritoneal chemokine levels, neutrophil numbers, or macrophage phenotypes were not altered by PKCδ inhibition. Peritoneal macrophages isolated from PKCδ inhibitor-treated septic rats demonstrated increased bacterial phagocytosis. Importantly, PKCδ inhibition increased survival. Thus, PKCδ inhibition improved survival and improved survival was associated with increased phagocytic activity, enhanced pathogen clearance, and decreased organ injury.

The cytokine IFN-γ has well-established antibacterial properties against the bacterium Salmonella enterica in phagocytes, but less is known about the effects of IFN-γ on Salmonella-infected nonphagocytic cells, such as intestinal epithelial cells (IECs) and fibroblasts. In this article, we show that exposing human and murine IECs and fibroblasts to IFN-γ following infection with Salmonella triggers a novel form of cell death that is neither pyroptosis nor any of the major known forms of programmed cell death. Cell death required IFN-γ-signaling via STAT1-IRF1–mediated induction of guanylate binding proteins and the presence of live Salmonella in the cytosol. In vivo, ablating IFN-γ signaling selectively in murine IECs led to higher bacterial burden in colon contents and increased inflammation in the intestine of infected mice. Together, these results demonstrate that IFN-γ signaling triggers release of Salmonella from the Salmonella-containing vacuole into the cytosol of infected nonphagocytic cells, resulting in a form of nonpyroptotic cell death that prevents bacterial spread in the gut.

Objective Infections contribute to morbidity and mortality in systemic lupus erythematosus (SLE). Uropathogenic Escherichia coli (UPEC) are known to trigger urinary tract infections (UTIs) and form biofilms, which are multicellular communities of bacteria that are strengthened by amyloids such as curli. We previously reported that curli naturally form complexes with bacterial extracellular DNA (eDNA), and these curli/eDNA complexes induce hallmark features of lupus in mouse models. The present study was undertaken to investigate whether anti-curli/eDNA complex antibodies play a role in the pathogenesis of SLE or development of flares in SLE. Methods In total, 96 SLE patients who met at least 4 Systemic Lupus International Collaborating Clinics disease criteria were investigated. Anti-curli/eDNA complex antibodies in the plasma were tested for both IgG and IgA subclasses. Results were compared to that in 54 age-, sex-, and race/ethnicity-matched healthy controls. Correlations of the levels of anti-curli/eDNA antibodies with clinical parameters, lupus disease status, and frequency of bacteriuria were assessed. Results Anti-curli/eDNA antibodies were detected in the plasma of SLE patients and healthy controls, and their levels correlated with the presence of asymptomatic persistent bacteriuria and occurrence of disease flares in lupus patients. Persistent bacteriuria contained curli-producing UPEC, and this was associated with an inflammatory phenotype. Finally, curli/eDNA complexes cross-reacted with lupus autoantigens, such as double-stranded DNA, in binding autoantibodies. Conclusion These results suggest that UTIs and persistent bacteriuria are environmental triggers of lupus and its flares. Antibodies against curli/eDNA could serve as a sign of systemic exposure to bacterial products in SLE.

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.

Molecular mimicry is a common mechanism used by many bacteria to evade immune responses. In recent years, it has become evident that bacteria also decorate the extracellular matrix (ECM) of their biofilms with molecules that resemble those of the host. These molecules include amyloids and other proteins, polysaccharides, and extracellular DNA. Bacterial amyloids, like curli, and extracellular DNA are found in the biofilms of many species. Recent work demonstrated that curli and DNA form unique molecular structures that are recognized by the immune system, causing activation of autoimmune pathways. Although a variety of mechanisms have been suggested as the means by which infections initiate and/or exacerbate autoimmune diseases, the mechanism remains unknown. In this article, we discuss recent work on biofilms that highlight the role of amyloids as a carrier for DNA and potentiator of autoimmune responses, and we propose a novel link between bacterial infections and autoimmune diseases.

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 pan-amyloid-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 antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms., Curli amyloid fibers are important components of bacterial biofilms formed by E. coli and Salmonella. Here, Tursi et al. show that a human monoclonal antibody with pan-amyloid binding activity can disrupt biofilms formed by Salmonella Typhimurium in vitro and in vivo.

Background Infections are a major contributor to lupus disease. We have previously demonstrated that bacterial amyloid curli, produced by E.coli, can accelerate disease in mouse models of lupus. Interestingly curli incorporates extracellular DNA, which in turn can be both adjuvant and a self-antigen in lupus. Finally, uropathogenic E. coli (UPEC) is responsible for the majority of urinary tract infections in SLE. Methods Based on our previous results, we hypothesize that exposure to UPEC triggers anti- curli/eDNA antibodies and curli/eDNA complexes can trigger the innate immune system. We investigated 98 lupus patients who met at least 4 SLICC criteria. Results were compared to 54 age, sex and race matched healthy controls. We tested the production of anti-curli/DNA complex for both IgG and IgA subclasses. We also correlated the levels of anti-curli/DNA antibodies with clinical parameters. Finally, we treated human neutrophils with curli/eDNA complexes. Results We found that curli/eDNA induces neutrophil extracellular traps in a ROS manner. Anti-curli/eDNA IgG levels were detected in lupus and controls plasma and the levels correlated with persistent bacteriuria (p Conclusions We conclude curli/eDNA complexes can activate the innate and adaptive immune system and could be a mechanism to sustaining disease in lupus. Funding Source(s): NIH/NIAMS and Lupus Research Alliance

Biofilms of enteric bacteria are highly complex, with multiple components that interact to fortify the biofilm matrix. Within biofilms of enteric bacteria such as Escherichia coli and Salmonella species, 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 of Salmonella enterica serovar 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.

Background The pathogenesis of Systemic Lupus Erythematosus (SLE) is multifactorial with genetic make-up and environmental triggers considered major players. Among the environmental triggers, infections are a major cause of morbidity and mortality in SLE patients. Bacteremia is often overlooked in SLE patients and soft tissue abscesses, bloodstream infections, and sepsis are more common in SLE patients, suggesting that frequent exposures to microbial products may trigger flares in lupus. We have recently shown that a bacterial amyloid termed curli, expressed in the multicellular communities (biofilms) by many bacteria including E. coli, plays a major role in triggering lupus autoimmunity during infection. Curli bind bacterial or eukaryotic DNA and form curli/DNA complexes that strongly activate innate immunity. When given systemically, curli/DNA complexes and infections with curli-expressing E. coli trigger production of anti-dsDNA and anti-chromatin autoAbs in lupus prone mice and in wild type mice. This affect was diminished in TLR2 or TLR9 deficient mice. Curli/DNA complexes activate dendritic cells and macrophages. We have now focused on the effects of curli/DNA complexes on B cells. Methods Young wild type C57BL/6 mice, lupus prone Sle1,2,3 mice and 3 H9 mice were used. Splenic B cells were stained by flow cytometry for in vivo experiments. For in vitro experiments, B cells were sorted by positive selection with CD45R (B220) Miltenyi Biotec micro bead or Easysep B cell isolation kit (Stem cell) supplemented with anti-CD43-Biotin. B cell purity (>98%), proliferation, viability, activation markers, surface antibodies and signaling were measured by CFSE dilution (Promokine), 7AAD and antibodies by Flow cytometry, Western Blot analysis and qRT-PCR. Results We found that curli/DNA complexes polyclonally activate B cells in vivo in wildtype mice, lupus-prone mice and 3 H9 mice, the latter expressing an anti-DNA Ig heavy chain and whose B cells are normally tolerized. Curli/DNA complexes can also activate B cells in vitro in the absence of T cell help. The induction of non-canonical NFκB in the absence of T cell help suggests that the fibrillar structure of curli/DNA complexes can cross-link BCRs, some recognizing DNA. Interestingly, curli/DNA complexes also induce isotype switching and aicda, the master regulator of class switch recombination, in the absence of T cells help in vitro. Conclusions Our results suggest that curli/DNA complexes may induce anti-DNA antibody production by simultaneous BCR/TLR signaling, which leads to B cell antibody production in the absence of T cell help. Acknowledgements We would like to thank the Lupus Research Alliance and the NIH NIAID (R21AI119947) for supporting our work.

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.

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., Author summary 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 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.

Summary Responses to host amyloids and curli amyloid fibrils of Escherichia coli and Salmonella enterica serotype Typhimurium are mediated through Toll-like receptor (TLR) 2. Here we show that TLR2 alone was not sufficient for mediating responses to curli. Instead, transfection experiments with human cervical cancer (HeLa) cells and antibody-mediated inhibition of TLR signalling in human macrophage-like (THP-1) cells suggested that TLR2 interacts with TLR1 to recognize curli amyloid fibrils. TLR1/TLR2 also serves as a receptor for tri-acylated lipoproteins, which are produced by E. coli and other Gram-negative bacteria. Despite the presence of multiple TLR1/TLR2 ligands on intact bacterial cells, an inability to produce curli amyloid fibrils markedly reduced the ability of E. coli to induce TLR2-dependent responses in vitro and in vivo. Collectively, our data suggest that curli amyloid fibrils from enterobacterial biofilms significantly contribute to TLR1/TLR2-mediated host responses against intact bacterial cells.

SummaryCurli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like β-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 β-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.

Salmonella enterica serotype Typhimurium elicits acute neutrophil influx in the human intestinal mucosa within 1 or 2 days after infection, resulting in inflammatory diarrhea. In contrast, no overt symptoms are observed within the first 1 or 2 weeks after infection with S. enterica serotype Typhi. Here we show that introduction of the capsule-encoding viaB locus of serotype Typhi reduced the ability of serotype Typhimurium to elicit acute intestinal inflammation in a streptomycin-pretreated mouse model. Serotype Typhimurium requires a functional invasion-associated type III secretion system (type III secretion system 1 [T3SS-1]) to elicit cecal inflammation within 48 h after infection of streptomycin-pretreated mice, and the presence of the viaB locus reduced its invasiveness for human intestinal epithelial cells in vitro. However, a reduced activity of T3SS-1 could not account for the ability of the viaB locus to attenuate cecal inflammation, because introduction of the viaB locus into an invasion-deficient serotype Typhimurium strain ( invA mutant) resulted in a significant reduction of pathology and inflammatory cytokine expression in the cecum 5 days after infection of mice. We conclude that a T3SS-1-independent mechanism contributes to the ability of the viaB locus to reduce intestinal inflammation.

The Salmonella enterica serotype Typhimurium (S. typhimurium) genome contains a large repertoire of putative fimbrial operons that remain poorly characterized because they are not expressed in vitro. In this study, insertions that induced expression of the putative stdABCD fimbrial operon were identified from a random bank of transposon mutants by screening with immuno-magnetic particles for ligand expression (SIMPLE). Transposon insertions upstream of csgC and lrhA or within dam, setB and STM4463 (renamed rosE) resulted in expression of StdA and its assembly into fimbrial filaments on the cell surface. RosE is a novel negative regulator of Std fimbrial expression as indicated by its repression of a std::lacZ reporter construct and by binding of the purified protein to a DNA region upstream of the stdA start codon. Expression of Std fimbriae in the rosE mutant resulted in increased attachment of S. typhimurium to human colonic epithelial cell lines (T-84 and CaCo-2). A rosE mutant exhibited a reduced ability to compete with virulent S. typhimurium for colonization of murine organs, while no defect was observed when both competing strains carried a stdAB deletion. These data suggest that a tight control of Std fimbrial expression mediated by RosE is required during host pathogen interaction.

Inflammatory diseases of the gastrointestinal tract are frequently associated with dysbiosis, characterized by changes in gut microbial communities that include an expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Here we show that a dysbiotic expansion of Enterobacteriaceae during gut inflammation could be prevented by tungstate treatment, which selectively inhibited molybdenum-cofactor-dependent microbial respiratory pathways that are operational only during episodes of inflammation. By contrast, we found that tungstate treatment caused minimal changes in the microbiota composition under homeostatic conditions. Notably, tungstate-mediated microbiota editing reduced the severity of intestinal inflammation in mouse models of colitis. We conclude that precision editing of the microbiota composition by tungstate treatment ameliorates the adverse effects of dysbiosis in the inflamed gut.

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. 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. 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. 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. Oral treatment with an amyloid protein found in bacterial biofilms helps to reduce inflammation in a mouse model of colitis. A research team led by Cagla Tukel from Temple University in Philadelphia, Pennsylvania, USA administered a single dose of curli—the protein secreted in the biofilms of both commensal and pathogenic bacteria—to mice with experimentally induced colitis. They observed elevated levels of interleukin-10 (IL-10), an anti-inflammatory immune molecule, and less severe symptoms of the gut disorder. The therapeutic benefit of curli was similar to that of an antibody therapy commonly used today in people with colitis. Using in vitro systems, the researchers showed that an immune receptor called TLR2 (toll-like receptor 2) recognized the curli and triggered the beneficial IL-10 response. The authors suggest that pills containing curli may hold potential for treating colitis in humans.

Knowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen-associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant (csgBA) was attenuated in its ability to elicit fluid accumulation and GROalpha mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL-8 production than the wild type in human macrophage-like cells. Purified thin curled fimbriae induced IL-8 expression in human embryonic kidney (HEK293) cells transfected with Toll-like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione-S-transferase (GST) elicited IL-8 production in HEK293 cells transfected with TLR2/CD14. Proteinase K treatment abrogated IL-8 production elicited in these cells by GST-CsgA, but not by synthetic lipoprotein. GST-CsgA elicited more IL-6 production than GST in bone marrow-derived macrophages from TLR2+/+ mice, while there was no difference in IL-6 secretion between GST-CsgA and GST in macrophages from TLR2-/- mice. These data suggested that CsgA is a PAMP that is recognized by TLR2.

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.

Capsular polysaccharides are important virulence factors of invasive bacterial pathogens. Here we studied the role of the virulence (Vi) capsular polysaccharide of Salmonella enterica serotype Typhi ( S. Typhi) in preventing innate immune recognition by complement. Comparison of capsulated S. Typhi with a noncapsulated mutant (Δ tviBCDE vexABCDE mutant) revealed that the Vi capsule interfered with complement component 3 (C3) deposition. Decreased complement fixation resulted in reduced bacterial binding to complement receptor 3 (CR3) on the surface of murine macrophages in vitro and decreased CR3-dependent clearance of Vi capsulated S. Typhi from the livers and spleens of mice. Opsonization of bacteria with immune serum prior to intraperitoneal infection increased clearance of capsulated S. Typhi from the liver. Our data suggest that the Vi capsule prevents CR3-dependent clearance, which can be overcome in part by a specific antibody response.

The lower gastrointestinal tract is densely populated with resident microbial communities (microbiota), which do not elicit overt host responses but rather provide benefit to the host, including niche protection from pathogens. However, introduction of bacteria into the underlying tissue evokes acute inflammation. Non-typhoidal Salmonella serotypes (NTS) elicit this stereotypic host response by actively penetrating the intestinal epithelium and surviving in tissue macrophages. Initial responses generated by bacterial host cell interaction are amplified in tissue through the interleukin (IL)-18/interferon-γ and IL-23/IL-17 axes, resulting in the activation of mucosal barrier functions against NTS dissemination. However, the pathogen is adapted to survive antimicrobial defenses encountered in the lumen of the inflamed intestine. This strategy enables NTS to exploit inflammation to outcompete the intestinal microbiota, and promotes the Salmonella transmission by the fecal/oral route.

The viaB locus enables Salmonella enterica serotype Typhi to reduce Toll-like receptor (TLR) dependent cytokine production in tissue culture models. This DNA region contains genes involved in the regulation (tviA), biosynthesis (tviBCDE) and export (vexABCDE) of the Vi capsule. Expression of the Vi capsule in S. Typhimurium, but not expression of the TviA regulatory protein, reduced tumour necrosis factor-alpha (TNF-alpha) and IL-6 production by murine bone-marrow derived macrophages. Production of TNF-alpha and IL-6 was dependent on expression of TLR4 as stimulation of macrophages from TLR4(-/-) mice with S. Typhimurium did not result in expression of these cytokines. Intraperitoneal infection of mice with S. Typhimurium induced expression of TNF-alpha and inducible nitric oxide synthase (iNOS) in the liver. Introduction of the cloned viaB region into S. Typhimurium reduced TNF-alpha and iNOS expression to levels observed after infection with a S. Typhimurium msbB mutant. In contrast, no differences in TNF-alpha expression between the S. Typhimurium wild type and strains expressing the Vi-capsule or carrying a mutation in msbB were observed after infection of TLR4(-/-) mice. We conclude that the Vi capsule prevents both in vitro and in vivo recognition of S. Typhimurium lipopolysaccharide by TLR4.

Salmonella Typhimurium is an intracellular pathogenic gram-negative bacterium that colonizes predominantly the intestinal lumen, activates Toll-like receptors, and accounts for most of food borne illnesses. The transcription factor Stat2 is vital in type I interferon activated antiviral and antitumor immunity and yet less is known about its role in bacterial infection. In a recent study, we reported that Stat2 was protective when tested in a mouse model of lipopolysaccharide-induced sepsis as mortality was accelerated inStat2 knockout (KO) mice. In this study, we evaluated the effect of Stat2 in a clinically relevant inflammation model of Salmonella-induced colitis. We found that when mice were pretreated with streptomycin and then orally infected with Salmonella, compared to wild type mice, Stat2KO mice showed altered intestinal responses by experiencing lower intestinal bacterial burdens. No differences were found in bacterial burdens at systemic sites. Stat2-dependent responses were lethal to wild type mice while Stat2KO mice were found to be more resistant. In contrast, Stat1KO mice were more susceptible to Salmonella infection than wild type mice suggesting that Stat2 and Stat1 play different roles during pathogenic bacterial infection. Our results indicate that disruption of the intestinal microbiota allows Stat2-dependent responses to play an adverse role in host defense against Salmonella-induced colitis. To our knowledge, this is the first evidence of Stat2 dependent responses playing a negative role in the host defense against pathogenic Salmonella bacteria. Future studies will reveal if this observation applies to other gram negative pathogens.

A massive neutrophil influx in the intestine is the histopathological hallmark of Salmonella enterica serovar Typhimurium-induced enterocolitis in humans. Two major hypotheses on the mechanism leading to neutrophil infiltration in the intestinal mucosa have emerged. One hypothesis suggests that S. enterica serovar Typhimurium takes an active role in triggering this host response by injecting proteins, termed effectors, into the host cell cytosol which induce a proinflammatory gene expression profile in the intestinal epithelium. The second hypothesis suggests a more passive role for the pathogen by proposing that bacterial invasion stimulates the innate pathways of inflammation because the pathogen-associated molecular patterns of S. enterica serovar Typhimurium are recognized by pathogen recognition receptors on cells in the lamina propria. A review of the current literature reveals that, while pathogen recognition receptors are clearly involved in eliciting neutrophil influx during S. enterica serovar Typhimurium infection, a direct contribution of effectors in triggering proinflammatory host cell responses cannot currently be ruled out.

The genus Salmonella contains a group of closely related organisms that are pathogenic for humans and other vertebrates. The human disease manifestations caused most frequently by Salmonella serotypes worldwide are typhoid fever and gastroenteritis (reviewed in reference [102][1]). Both illnesses

Amyloid fibers are β-sheet-rich protein polymers that are highly resistant to denaturation. The distinguishing amyloid fold can be adopted by a variety of proteins, without a shared primary structure, and is found in nearly all cell types. Despite the fact that amyloids have a richly informed scientific history, the diverse biology contributed by amyloids is only beginning to be appreciated. Initial amyloid studies focused on the intimate association of amyloid formation with cytotoxicity and neurodegenerative diseases like Alzheimer's, Huntington's, and the prion encephalopathies. Despite amyloid's somewhat sinister past, recent work on “functional” amyloids has revealed numerous ways that amyloids contribute to normal cellular biology [1]. Included among the activities in which amyloids participate are melanin production, the ability to act as non-Mendelian inheritable genetic elements, and as extracellular molecular scaffolds that hold bacterial communities together. The amyloid fold is tailor-made for the extracellular space, as amyloid polymers can self-assemble without requiring exogenous energy and the polymers are resistant to a slew of harsh denaturants that would devastate most protein folds. This article will address questions involving the various roles of bacterial amyloids in host, polymicrobial, and environmental interactions.

SummaryResearch 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.