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Secondary bacterial infection (superinfection) post influenza is a serious clinical complication often leading to pneumonia and death. Eicosanoids are bioactive lipid mediators that play critical roles in the induction and resolution of inflammation. CYP450 lipid metabolites are anti-inflammatory lipid mediators that are produced at an excessive level during superinfection potentiating the vulnerability to secondary bacterial infection. Using Nanostring nCounter technology, we have defined the targeted transcriptional response where CYP450 metabolites dampen the Toll-like receptor signaling in macrophages. CYP450 metabolites are endogenous ligands for the nuclear receptor and transcription factor, PPARα. Activation of PPARα hinders NFκB p65 activities by altering its phosphorylation and nuclear translocation during TLR stimulation. Additionally, activation of PPARα inhibited anti-bacterial activities and enhanced macrophage polarization to an anti-inflammatory subtype (M2b). Lastly, Ppara–/– mice, which are partially protected in superinfection compared to C57BL/6 mice, have increased lipidomic responses and decreased M2-like macrophages during superinfection.

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.

Introduction Self-directed learning as a form of active learning is student centered rather than the faculty-centered model employed in traditional lectures or large-group teaching. In order to highlight the content taught during a microbiology course we developed a 2-hour workshop to help teach second-year medical students about pneumonia. Methods One week prior to session, the class is divided into groups of 30 and then further divided into groups of five, each of which is assigned one of six cases to review. The student then attend the session and perform a high-fidelity simulation matching the case they were assigned. After a debrief with a faculty facilitator each group is asked to diagnose their patient and present it to the other 25 learners. Results The workshop has received positive evaluations from our students and has been reported to achieve the learning objectives. Most second-year medical students (97.1%, n = 171) reported that the self-directed learning component and simulation complemented each other and enhanced the learning experience. Discussion Most students view working as a team as a positive experience and favor a student-facilitated session. Because of the success of this workshop, we have modified others to follow a similar format.

Infections can be pathogenic in systemic lupus erythematosus (SLE). Curli is a bacterial amyloid part of enteric bacterial biofilms, like those of E. coli and Salmonella. We have recently found that curli fibers form highly immunogenic composites with DNA within the biofilm. Here we studied the immune response to curli-DNA complexes in the context of murine SLE. For in vitro studies, we stimulated bone marrow-derived dendritic cells (cDCs) from WT and lupus-prone mice with curli amyloid from S. Typhimurium biofilms. A dose titration of curli amyloid induced robust proinflammatory cytokine and type I-IFN responsive gene expression in cDCs. Using synthetic curli peptide, we found that curli and nucleic acids synergize to activate cDCs. We injected in vivo pre-diseased lupus-prone NZB/W-F1 mice i.p. with curli-DNA complexes and found that they accelerate disease, inducing high titers of anti-dsDNA and anti-chromatin autoantibodies. Curli-DNA complexes induced autoantibodies also in non-autoimmune C57BL/6 mice. Finally, we infected NZB/W-F1 mice i.p. with curli-expressing or curli mutant E. coli or Salmonella. Curli-competent bacteria triggered far higher autoantibody titers than curli mutant strains. In conclusion, biofilm-derived curli-DNA complexes are potent activators of DCs and can rapidly break tolerance and accelerate autoimmunity in mice, suggesting that bacterial amyloids and biofilm-based bacterial infections are important environmental triggers in lupus.