Characterization of Borrelia-Derived Extracellular Vesicles: Implications for Pathogenesis and Diagnostics

The cause of chronic neurological effects associated with Lyme disease (LD) remains unclear. We propose that bacterial extracellular vesicles (BEVs) released by <i>Borrelia burgdorferi</i>, the causative agent of LD, exacerbate spirochete-induced damage and serve as a persistent source of antigenic stimulation. We showed that, over a 10-day period, in vitro cultures of <i>B. burgdorferi</i> B31 produced 38,000 BEVs per spirochete with a distinctive double-membrane structure and median diameter of 143.3 nm. BEVs contained known immunogenic and immunomodulatory molecules such as peptidoglycan, p66, flagellar filament protein (FlaB), basic membrane proteins A/B/D, BdrV, GroEL, CRASP-1, ErpA8, glycerophosphodiester phosphodiesterase, p37, OMS28, p13, OspA/B/C, VlsE, and outer membrane glycolipids (e.g., cholesteryl 6-O acyl beta D galactopyranoside). Chromosome-encoded <i>16S ribosomal RNA</i> and cp32 plasmid-encoded <i>OspE</i> and <i>terminase</i> genes were also detected in the BEVs. Of the 45 <i>Borrelia</i> proteins identified in the urine of a C3H/HeJ murine model of Lyme disease, 14 were associated with BEVs. In human urine samples, 31 of 289 spirochete proteins detected in patients with either acute Lyme disease or persistent <i>borreliosis</i> post-treatment symptoms, including p66 and FlaB, were also BEV-associated. BEV treatment of HMC3 human microglial cells reduced phagocytic activity and triggered aberrant activation of inflammatory and immunometabolic pathways, including upregulation of interferon-alpha (IFN-α), aconitate decarboxylase 1 (Acod1), and Toll-like receptor 2 (TLR2) gene expression. BEVs also induced NRF2 nuclear translocation. In conclusion, these findings support that BEVs can amplify spirochete-induced damage and act as antigenic debris, driving dampened phagocytic activity and dysregulated inflammation, with implications for diagnostics and therapeutics targeting vesicle-mediated pathology.