ABSTRACT Accounting for ten times more cases than all of vector-borne diseases combined in the United States, Lyme disease is an escalating and pervasive problem. Upon infection with the Lyme disease agent—Borrelia burgdorferi— patients experience mild, flu-like symptoms that can go on to become more severe. In later stages, even after antibiotic therapy, patients can experience heart, joint, and neurological problems. The reason(s) for persistent symptoms is poorly understood and the most debated topic in the field. The lead investigator of this proposal discovered that B. burgdorferi sheds ~45% of its peptidoglycan (PG)—the essential component of the bacterial cell-wall—from inside the cell, into its environment. B. burgdorferi PG can be detected in the synovial fluid of Lyme arthritis patients' months after oral and/or intravenous antibiotics. When injected into a mouse, B. burgdorferi PG alone, is capable of causing arthritis. Virtually all bacteria have PG but, as it turns out, B. burgdorferi PG is chemically unique and unlike any previously studied. Real-timing PG tracking experiments in live animals suggest that the unique features of B. burgdorferi muropeptides contributes to both half-life and discrete tissue localization. These findings led us to hypothesize that shed B. burgdorferi PG fragments contain unique chemical signature(s) that are required for persistence, Lyme disease pathogenesis, and arthritis. This proposal will critically test these theories and provide a comprehensive understanding of 1) how and 2) where PG is able to persist; 3) what molecular features contribute to PG persistence; 4) the mechanistic details that underlie PG-induced Lyme arthritis; and 5) the importance of muropeptide shedding in the natural life cycle of B. burgdorferi. Our proposed studies challenge conventional thinking and may lead to a paradigm shift in how we understand Lyme disease pathogenesis. They may also lead to novel ways to treat patients suffering with Lyme disease and post-treatment Lyme disease syndrome, even after conventional therapies have failed.