The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete.

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon-exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.

HLA-DR-Expressing Fibroblast-Like Synoviocytes Are Inducible Antigen Presenting Cells That Present Autoantigens in Lyme Arthritis.

OBJECTIVE: HLA-DR-expressing fibroblast-like synoviocytes (FLS) are a prominent cell type in synovial tissue in chronic inflammatory forms of arthritis. FLS-derived extracellular matrix (ECM) proteins, including fibronectin-1 (FN1), contain immunogenic CD4+ T cell epitopes in patients with postinfectious Lyme arthritis (LA). However, the role of FLS in presentation of these T cell epitopes remains uncertain. METHODS: Primary LA FLS and primary murine FLS stimulated with interferon gamma (IFNγ), Borrelia burgdorferi, and/or B burgdorferi peptidoglycan (PG) were assessed for properties associated with antigen presentation. HLA-DR-presented peptides from stimulated LA FLS were identified by immunopeptidomics analysis. OT-II T cells were co-cultured with stimulated murine FLS in the presence of cognate ovalbumin antigen to determine the potential of FLS to act as inducible antigen presenting cells (APCs). RESULTS: FLS expressed HLA-DR molecules within inflamed synovial tissue and tendons from patients with postinfectious LA in situ. Major histocompatibility complex (MHC) class II and co-stimulatory molecules were expressed by FLS following in vitro stimulation with IFNγ and B burgdorferi and presented both foreign and self-MHC-II peptides, including an immunogenic T cell epitope derived from Lyme autoantigen FN1. Stimulated FLS induced proliferation of naive OT-II CD4+ T cells that were dependent on OT-II antigen and CD40. Stimulation with B burgdorferi PG enhanced FLS-mediated T cell activation. CONCLUSION: MHC-II+ FLS are inducible APCs that can induce CD4+ T cell activation in an antigen- and CD40-dependent manner. Activated FLS can also present ECM-derived Lyme autoantigens, implicating FLS in amplifying tissue-localized autoimmunity in LA.

Peptidoglycan in osteoarthritis synovial tissue is associated with joint inflammation.

OBJECTIVES: Peptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes. METHODS: Intraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC) and immunofluorescence microscopy (IFM). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response. RESULTS: A total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, and PG staining colocalized with markers of synovial macrophages and fibroblasts by IFM. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels. CONCLUSION: Peptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.

Human leukocyte antigen HLA-DR-expressing fibroblast-like synoviocytes are inducible antigen presenting cells that present autoantigens in Lyme arthritis.

Background: HLA-DR-expressing fibroblast-like synoviocytes (FLS) are a prominent cell type in synovial tissue in chronic inflammatory forms of arthritis. We recently showed that peptides from several extracellular matrix (ECM) proteins, including fibronectin-1 (FN1), contained immunogenic CD4+ T cell epitopes in patients with postinfectious Lyme arthritis (LA). However, the role of FLS in presentation of these T cell epitopes remains uncertain. Methods: Primary LA FLS and primary murine FLS stimulated with interferon gamma (IFNγ), Borrelia burgdorferi, and/or B. burgdorferi peptidoglycan (PG) were assessed for properties associated with antigen presentation. HLA-DR-presented peptides from stimulated LA FLS were identified by immunopeptidomics analysis. OT-II T cells were cocultured with stimulated murine FLS in the presence of cognate ovalbumin antigen to determine the potential of FLS to act as inducible antigen presenting cells (APC). Results: FLS expressed HLA-DR molecules within inflamed synovial tissue and tendons from patients with post-infectious LA patients in situ. MHC class II and costimulatory molecules were expressed by FLS following in vitro stimulation with IFNγ and B. burgdorferi and presented both foreign and self MHC-II peptides, including T cell epitopes derived from two Lyme autoantigens fibronectin-1 (FN1) and endothelial cell growth factor (ECGF). Stimulated murine FLS induced proliferation of naïve OT-II CD4+ T cells, particularly when FLS were stimulated with both IFNγ and PG. Conclusions: MHC-II+ FLS are inducible APCs that can induce CD4+ T cell activation and can present Lyme autoantigens derived from ECM proteins, thereby amplifying tissue-localized autoimmune CD4+ T cell responses in LA.

Recent Progress in Lyme Disease and Remaining Challenges.

Lyme disease (also known as Lyme borreliosis) is the most common vector-borne disease in the United States with an estimated 476,000 cases per year. While historically, the long-term impact of Lyme disease on patients has been controversial, mounting evidence supports the idea that a substantial number of patients experience persistent symptoms following treatment. The research community has largely lacked the necessary funding to properly advance the scientific and clinical understanding of the disease, or to develop and evaluate innovative approaches for prevention, diagnosis, and treatment. Given the many outstanding questions raised into the diagnosis, clinical presentation and treatment of Lyme disease, and the underlying molecular mechanisms that trigger persistent disease, there is an urgent need for more support. This review article summarizes progress over the past 5 years in our understanding of Lyme and tick-borne diseases in the United States and highlights remaining challenges.