Role of ZBP1 in pathogenesis of Salmonella biofilms

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PROJECT SUMMARY Infections with enteric pathogens such as Salmonella, Campylobacter, Shigella, or Yersinia are leading causes of morbidity and mortality worldwide. Although in most individuals the infection resolves, approximately 5% of patients subsequently develop a painful chronic inflammatory condition known as Reactive Arthritis (ReA). How Salmonella infections trigger ReA is not well understood. Using Salmonella enterica serovar Typhimurium (STm) as a model organism, we discovered that a Salmonella protein, curli, is a dominant instigator of inflammation following Salmonella infection. Curli is a secreted protein and major component of the STm biofilm in the gastrointestinal tract. Curli fibrils bind extruded bacterial DNA within the biofilm. It is these curli:DNA complexes, rather than curli alone, that are potent triggers of type I interferon, IL-17, and anti-double stranded DNA autoantibody production, leading to ReA. Unknown, however, is why curli:DNA complexes are so inflammatory. We report in this proposal the remarkable discovery that the DNA present within curli:DNA complexes is not solely B-DNA, the classic right-handed (Watson-Crick) double-helix, but includes copious amounts of left-handed Z-DNA as well. Z-form nucleic acids, such as Z-DNA and Z-RNA, were thought not to readily occur in nature, until we showed last year that Z-RNA is indeed produced during virus infections and is a ligand for the necroptosis-activating host sensor protein ZBP1. Our preliminary results now show that the Z-DNA within curli:DNA complexes activates ZBP1 in intestinal epithelial cells (IECs) and fibroblasts, resulting in RIPK3- dependent necroptosis of these cells. These findings allow us to put forward the hypothesis that Z-DNA within curli:DNA fibrils in Salmonella biofilms activates ZBP1 to instigate RIPK3-dependent necroptosis in intestinal epithelial cells (IECs) and other cell types. Necroptosis, in turn, causes cell loss and disrupts gut barrier integrity, releasing inflammatory mediators that eventually result in autoimmunity and ReA. As necroptosis is a highly inflammatory mode of cell death, these findings, at long last, supply a plausible mechanism for why curli:DNA complexes are hyperinflammatory, and, therefore, for how Salmonella triggers ReA. They also identify Z-DNA as a new PAMP, implicate ZBP1 – until now considered an antiviral protein – as a sensor of bacterial infections, and position RIPK3 inhibitors as unanticipated new therapeutics for the treatment of ReA. In aim 1, we will determine how Z-DNA forms within curli:DNA complexes, and how Z-DNA activates ZBP1. In aim 2, we will identify the ZBP1-driven immune pathways that promote curli:DNA induced inflammation and ReA, and determine the cell types in which ZBP1 signaling is important for pathogenesis. In aim 3, we will evaluate whether necroptosis blockade with RIPK3 kinase inhibitors will have preventive or therapeutic benefit in ReA. A successful outcome to these studies will outline an entirely new mechanism of Salmonella-triggered inflammation. They also stand to open up exciting new therapeutic avenues for Salmonella-induced ReA, with potentially game-changing ramifications for this currently incurable disease.