Borrelia burgdorferi c-di-AMP is a key extracellular pathogen-associated molecular pattern to elicit type I interferon responses in mammalian hosts.

Borrelia burgdorferi (or Borreliella burgdorferi), the extracellular spirochete responsible for Lyme disease, elicits a type I interferon (IFN-I) response critical for the development of Lyme arthritis. However, the specific pathogen-associated molecular pattern (PAMP) driving this response remains unidentified. Previous studies have reported that B. burgdorferi culture supernatants significantly stimulate macrophage IFN-I responses, but the responsible component was unknown. In this study, we identified cyclic-di-adenosine monophosphate (c-di-AMP) as the critical PAMP for the induction of IFN response. Inactivation of cdaA, which encodes diadenylate cyclase for c-di-AMP synthesis, significantly reduced IFN-β production in murine macrophage cell lines (RAW264.7) and bone marrow-derived macrophages. Conversely, the deletion of dhhP, which encodes c-di-AMP phosphodiesterase, dramatically increased IFN-β production. We further demonstrated that B. burgdorferi releases c-di-AMP, which is the major component in the culture supernatant responsible for stimulating the IFN-I response in macrophages. Furthermore, B. burgdorferi c-di-AMP-induced IFN-I response depends on STING, as inactivation or inhibition of STING signaling markedly reduced IFN-I induction. These findings establish c-di-AMP as a key PAMP of B. burgdorferi that activates host STING signaling to induce IFN-I responses, highlighting its potential as a therapeutic target for Lyme arthritis.