Fueling the Fire with Fibers: Bacterial Amyloids Promote Inflammatory Disorders

Abstract

Bacterial infection is associated with increased morbidity in patients with systematic lupus erythematosus. In a recent Immunity paper, Gallo et al. (2015) report that extracellular DNA is bound tightly by bacterial amyloid fibrils during biofilm formation and that amyloid/DNA composites are immune stimulators when injected into mice, leading to autoimmunity.

Amyloid proteins, which are characterized by the formation of a highly organized cross-β sheet quaternary structure, are primarily linked with neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and prion diseases. These diseases are associated with protein misfolding and amyloid fiber formation, which can be cytotoxic and contribute to the disease state. However, not all amyloid fibers are a detriment to the organism that produces them. Some bacterial species produce so-called “functional” amyloids that perform physiological roles that ultimately benefit the organism. One such example is curli, an extracellular amyloid produced by Escherichia coli and Salmonella typhimurium. Curli amyloid fibers are the major proteinaceous component of the extracellular matrix (ECM), which is required for floating pellicle biofilm formation. The main curli subunit protein, CsgA, is transported to the cell surface and polymerized into an amyloid fiber by the corresponding csgBAC and csgDEFG operons (Evans and Chapman, 2014). In addition to their role in biofilm formation, curli fibrils contribute to bacterial pathogenesis during numerous infections, including urinary tract infections and sepsis (Kai-Larsen et al., 2010; Cegelski et al., 2009).

Mammalian amyloids have been shown to be capable of inducing autoimmunity when complexed with cofactors such as host DNA (Di Domizio et al., 2012b). Autoimmunity describes abnormal circumstances where the host immune system erroneously identifies normal host-derived substances or tissues as invading foreign matter, ultimately resulting in self-inflicted damage to the host. This improper anti-self response may be restricted to certain organs, such as is the case with autoimmune thyroiditis, may target a specific type of tissue that is present throughout the body, as occurs in Goodpasture syndrome, or may result in the breakdown of tolerance to nuclear antigens, which is observed in patients with systematic lupus erythematosus (lupus). Environmental cofactors, such as DNA, RNA, or glyocloaminoglycans, promote the conversion of mammalian amyloid precursor proteins into insoluble amyloid fibrils by binding to the precursor proteins and increasing the rate of amyloid polymerization (Di Domizio et al., 2012a). Amyloid/DNA complexes are recognized and phagocytosed by circulating plasmacytoid dendritic cells (pDCs) (Di Domizio et al., 2012b). Like other debris taken up by pDCs, the amyloid/DNA complex enters the endocytic compartment for processing and eventual degradation. However, in some cases, amyloid fibrils are able to attach to receptors present on the cell membrane, thus preventing the shuttling of the complex to the lysosome for degradation (Di Domizio et al., 2012b). The retention of the amyloid/DNA complex in the early endosome triggers the intracellular DNA sensor, toll-like receptor (TLR) 9, stimulating an immune cascade that results in the transcription of type 1 IFNs and, eventually, the production of antinuclear antibodies, which can lead to autoimmune diseases like lupus (Di Domizio et al., 2012b). Depletion of pDCs in mice injected with amyloid/DNA complex abolished type 1 IFN production and dramatically reduced the number of antinuclear antibodies (Di Domizio et al., 2012b).

The ability of mammalian amyloid/DNA complexes to stimulate autoimmunity led Gallo et al. (2015) to postulate that functional amyloids, like curli, may also be able to stimulate a lupus-like autoimmunity within a host. A link between bacterial infection and autoimmunity is supported by the clinical observations that bacterial infections often trigger disease flairs and represent a major cause of morbidity and mortality in patients with lupus (Petri, 1998). Further support for the hypothesis that amyloid/DNA composites are involved in triggering autoimmunity is the fact that one of the major infectious agents associated with morbidity in patients with lupus is the curli-producing organism Salmonella. Also, it has been reported that curli are recognized by the host immune system through interactions with TLR2/TLR-1 heterocomplex (Tükel et al., 2010). This interaction between curli and the TLR1/2 heterocomplex is thought to reinforce gut membrane permeability, suggesting a tissue- or cell-specific response to the amyloid proteins. Using a S. typhimurium strain whose curli promoter drives expression of GFP, Gallo et al. (2015) were able to demonstrate that the majority of bacteria within a biofilm express curli. Furthermore, the authors detected extracellular DNA (eDNA) in these biofilms that was intimately associated with the curli amyloid fibers. Like DNA bound to mammalian amyloid fibrils, eDNA bound to curli was resistant to DNase treatment and was retained in the curli fibrils even after the curli was purified from the biofilm. Therefore, eDNA that had been incorporated into the biofilm was protected from cellular degradation.

The formation of biofilms is a defining step for many bacterial invaders as it provides protection from the mounting immune response. Gallo et al. (2015) showed that the addition of DNA to unpolymerized curli subunits resulted in rapid curli polymerization, indicating that the incorporation of DNA in vivo likely increases the rate of biofilm formation. However, bacteria within a biofilm were not completely protected from the immune response as conventional dendritic cells (cDCs) incubated with the Salmonella biofilms were seen to send dendrites into the biofilm community and phagocytose bacteria and eDNA (Figure 1). Interestingly, exposing cDCs isolated from lupus-prone mice to curli/DNA composites stimulated high levels of pro-inflammatory cytokines, including IL-6, IL-12 indicating the potent immunogenicity of the curli/DNA composites. Curli/DNA composites also resulted in the production of type 1 IFNs and IFN-stimulated genes in exposed cDCs. Injection of young lupus-prone and non-lupus prone mice with curli/DNA composites resulted in the induction of anti-DNA and anti-chromatin antinuclear antibodies, with mice testing positive for anti-dsDNA antibodies within 2 weeks of the initial injection. The majority of induced antinuclear antibodies were of immunoglobulin G (IgG) subclass 2a and 2b, which represent the two antibody subclasses generally associated with systemic autoimmunity in patients (Clynes et al., 1998). Further, mice injected with the curli/DNA complex tested positive for lupus while mock-treated mice of the same age did not, indicating that exposure to curli/DNA complexes can stimulate or exacerbate lupus in mice.

Figure 1. Schematic Overview of Autoimmunity Stimulated by Bacterial Amyloid Biofilm Formation.

eDNA is integrated into the curli biofilm formed by Salmonella typhimurium, increasing the rate of amyloid polymerization and biofilm formation. Subsequently, dendritic cells (DCs) can recognize and phagocytose curli/eDNA composites, stimulating an immunogenic response that leads to the induction of inflammatory cytokines and the production of antinuclear antibodies. The presence of antinuclear antibodies can lead to autoimmune diseases, like lupus.

The discovery by Gallo et al. (2015) that the curli/DNA complexes from the intestinal pathogen S. typhimurium can influence the development or progression of lupus in their mouse model has significant implications because it raises the possibility that an infection could play a role in initiating or exacerbating autoimmunity (Figure 1). However, the mechanism by which auto-immunity is induced is largely unknown, though it may mirror what occurs during uptake of mammalian amyloid/DNA complexes by pDCs (Di Domizio et al., 2012b). In a more general sense, this study provokes questions regarding a potential role for curli-expressing members of the gut microbiota, like E. coli, to influence gastrointestinal inflammation. E. coli is a small but prevalent component of the healthy human gut microbiota that undergoes significant population expansion during periods of intestinal disorder (Kotlowski et al., 2007). If curli-producing E. coli strains are able to integrate eDNA into their curli biofilms in vivo, then these organisms might also be capable of stimulating inflammation and/or anti-self antibodies, similar to what the authors have shown occurs during S. typhimurium infection. Humans harbor trillions of microbes as part of their gastrointestinal microbiota. The complex function of these communities in promoting human health and the consequences of disease when the microbiota becomes dysbiotic are the subject of intense research. Unraveling the intersection of bacterial amyloid production, DNA secretion, and host susceptibility to inflammation and autoimmune disease will undoubtedly provide fascinating insights into mechanisms by which the host prevents inappropriate anti-self immune responses and the basis for a mechanism by which commensal bacteria influence inflammation and autoimmunity in the host.