Phenol-Soluble Modulins From Staphylococcus aureus Biofilms Form Complexes With DNA to Drive Autoimmunity

The bacterial amyloid curli, produced by Enterobacteriales including Salmonella species and Escherichia coli, is implicated in the pathogenesis of several complex autoimmune diseases. Curli binds to extracellular DNA, and these complexes drive autoimmunity via production of anti-double-stranded DNA autoantibodies. Here, we investigated immune activation by phenol-soluble modulins (PSMs), the amyloid proteins expressed by Staphylococcus species. We confirmed the amyloid nature of PSMs expressed by S. aureus using a novel specific amyloid stain, (E,E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (FSB). Direct interaction of one of the S. aureus PSMs, PSMα3, with oligonucleotides promotes fibrillization of PSM amyloids and complex formation with bacterial DNA. Finally, utilizing a mouse model with an implanted mesh-associated S. aureus biofilm, we demonstrated that exposure to S. aureus biofilms for six weeks caused anti-double-stranded DNA autoantibody production in a PSM-dependent manner. Taken together, these results highlight how the presence of PSM-DNA complexes in S. aureus biofilms can induce autoimmune responses, and suggest an explanation for how bacterial infections trigger autoimmunity

STAT2 dependent Type I Interferon response promotes dysbiosis and luminal expansion of the enteric pathogen Salmonella Typhimurium.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2-/- mice, we observed decreased hypoxia in the gut tissue of Stat2-/- mice. Neutrophil numbers were similar in wild-type and Stat2-/- mice, yet Stat2-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium

STAT2 dependent Type I Interferon response promotes dysbiosis and luminal expansion of the enteric pathogen Salmonella Typhimurium.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2-/- mice, we observed decreased hypoxia in the gut tissue of Stat2-/- mice. Neutrophil numbers were similar in wild-type and Stat2-/- mice, yet Stat2-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium

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

<div><p>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 <i>Ifnβ</i> 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.</p></div

The structure of CsgA_R4-5-DNA complexes explains their ability to induce type I interferon responses via TLR9.

<p><b>A</b>. SAXS diffraction profiles for CsgA_R4-5 amyloid fibrillized in the absence (black curve) and presence (red curve) of CpG dsDNA. No diffraction peaks indicating columnar structure are observed for the CsgA_R4-5 fibrils in absence of DNA, but fibrillization of CsgA_R4-5 in the presence of DNA results in a sharp diffraction peak at <i>q</i>d = 0.151 Å^-1 (inset). This indicates that CsgA_R4-5 organizes the CpG DNA into an ordered columnar lattice with inter-DNA spacing <i>d</i> = 2π/<i>q</i>_d = 4.16 nm. <b>B</b>. Schematic depicting CpG DNA binding to TLR9 from two orthogonal views. The anionic DNA binds electrostatically to the cationic paddles of TLR9. <b>C</b>. Closest approach of adjacent TLR9 receptors on the endosomal membrane does not allow size-matching and multivalent binding to HIV-TAT/DNA complexes (top). This data was adopted from Schmidt et al [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006315#ppat.1006315.ref046" target="_blank">46</a>]. In contrast, the inter-DNA spacing for CsgA_R4-5-DNA complexes (bottom) allows for amplification of binding from multivalent and electrostatic effects.</p

Curli-DNA complex leads to a delayed autoantibody response in TLR deficient mice.

<p><b>A</b>) wild-type, (<b>B</b>) TLR2^-/- mice, (<b>C</b>) TLR9 mutant or (<b>D</b>) TLR2/TLR9-deficient mice were intraperitoneally injected with sterile PBS (circles and gray lines) or 50 μg of curli fibers isolated from <i>S</i>. Typhimurium IR715 <i>msbB</i> dissolved in sterile PBS (open triangles and black lines) twice weekly for 6 weeks. Each week serum was collected, and anti-dsDNA autoantibody quantified by ELISA. Serum collected from a naïve wild-type mouse was used as a negative control (dotted line). Mean and SE were calculated by averaging results from three independent experiments. Significance was calculated using Two-way Anova analysis followed by Tukey post hoc test. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Curli-DNA complexes elicit a type I interferon response in innate immune cells in a TLR2/TLR9 dependent fashion.

<p><b>A</b>. Wild-type IMMs (1x10⁵ cells) were stimulated with increasing concentrations of curli-DNA complexes purified from <i>S</i>. Typhimurium IR715 <i>msbB</i> (0, 0.5, 1.0, 1.5, and 2.5 μg/mL) for 3 hours, and levels of <i>Ifnβ (</i>left panel), <i>Irf7</i> (middle panel), and <i>Isg15</i> (right panel) were determined by qPCR. Wild-type (WT), TLR2^-/-, TLR9^-/-, and TLR2^-/-/TLR9^-/- IMMs (1x10⁵ cells) were stimulated with 2.5 μg/ml of curli-DNA complexes for 3 hours. (<b>B</b>) <i>Ifnβ</i>, <i>Isg15</i>, <i>Irf7</i>, and <i>Cxcl10</i> levels were determined by qPCR. (<b>C</b>) <i>Tnfα</i> mRNA levels were determined by qPCR. <b>D</b>. IL-10 production was quantified by ELISA in the supernatants. Mean and SE were calculated from results from at least three independent experiments. * p <0.05 as determined by Students t-test.</p

TLR9 activation by curli-DNA complex leads to a type I interferon response <i>in vivo</i>.

<p>Wild-type, TLR2^-/- mice, TLR9 mutant or TLR2/TLR9-deficient mice were intraperitoneally injected with sterile PBS or 50 μg of purified curli-DNA dissolved in sterile PBS. Mice were euthanized 4 hours after injection, and peritoneal lavage fluid was collected. (<b>A</b>) <i>Ifnβ</i>, (<b>B</b>) <i>Irf7</i>, (<b>C</b>) <i>Isg15</i>, and (<b>D</b>) <i>Tnfα</i> mRNA expression were quantified by qPCR. Mean and SE were calculated by averaging results from three independent experiments. Significance was calculated using students t-test. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Curli and DNA contribute to the IFN response, but cellulose does not.

<p><b>A</b>. Biofilms of <i>S</i>. Typhimurium wild-type IR715, <i>msbB</i> (LPS mutant), and <i>bscE</i> (cellulose mutant) were grown on glass coverslips in 24-well tissue culture dishes for 72 hours at 28°C. Biofilms were stained with 3 μg/ml Syto9 and visualized by microscopy (63x). 3D reconstructions were made using ImageJ software. <b>B</b>. Wild-type BMDCs were layered on top of established biofilms of wild-type <i>S</i>. Typhimurium, <i>msbB</i> and <i>bscE</i>. After 3 hours, <i>Ifnβ</i> was quantified in BMDCs by qPCR. As indicated, 2.5 μg/ml of curli-DNA complexes were layered on top of the biofilms prior to adding BMDCs. <b>C</b>. Wild-type IMMs (1x10⁵ cells per well) were stimulated with 2.5 μg/mL of curli-DNA complexes purified from <i>S</i>. Typhimurium, <i>E</i>. <i>coli</i> MC4100, or <i>S</i>. Typhimurium <i>bsc</i> for 3 hours, and <i>Ifnβ</i> was quantified by qPCR. <b>D</b>. Wild-type IMMs (1x10⁵ cells per well) were stimulated with 2.5 μg/ml of His-CsgA, His-CsgA polymerized in the presence of 10 ng/ml CpG DNA, synthetic peptide CsgA_R4-5, or CsgA_R4-5 polymerized in the presence of 10 ng/ml CpG DNA. After 3 hours, <i>Ifnβ</i> was quantified by qPCR. <b>E</b>. Wild-type IMMs (1x10⁵ cells per well) were stimulated with 2.5 μg/mL curli-DNA complexes, 10 ng/mL CpG DNA, or 2.5 μg/ml curli-DNA complexes and 10 ng/ml CpG. After 3 hours, <i>Ifnβ</i> was quantified by qPCR. Error bars indicate means ± S.E.M. from three independent experiments, * p <0.05 by students t-test. <b>F</b>. Wild-type, TLR2^-/-, TLR9 mutant, and TLR2/TLR9-deficient mice were intraperitoneal injected with 1x10⁵ curli-expressing <i>E</i>. <i>coli</i> MC4100 (closed circles) or <i>E</i>. <i>coli</i> MC4100 <i>csgBA</i> (LSR13) (open circles) once weekly for 6 weeks. Serum collected from a naïve wild-type mouse was used as a negative control (dotted line). dsDNA autoantibodies were quantified by ELISA after 6 weeks of injections. Mean and SE were calculated by averaging results from two independent experiments. Significance was calculated using Two-way Anova analysis followed by Tukey post hoc test. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p