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Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro . The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to 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. 

ABSTRACT During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro , providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy. 

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models ofStaphylococcus aureusorCandida albicansinfection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.