Project Summary The Mycobacterium bovis BCG vaccine has variable efficacy against adult pulmonary tuberculosis (TB) but protects children against both TB and unrelated infections and is used in bladder cancer treatment. Recent epidemiological studies have associated BCG vaccination with lowered COVID-19 mortality. While multiple, randomized controlled trials are now underway to test causality, molecular studies are urgently needed to address the proposed `trained [innate] immunity' mechanism of BCG cross-protection. Trained immunity may also contribute to BCG's specific protection against TB, a disease that afflicts 10 million people each year. The innate immune response to live mycobacteria or inactivated components (e.g. complete Freund's adjuvant [CFA]) has long thought to originate with the mycobacterial cell envelope, specifically the muramic acid moiety of cell wall peptidoglycan and trehalose dimycolates (TDM) in the outer `myco' membrane. During trained immunity, for example, epigenetic reprograming of monocytes depends on the innate immune receptor Nod2. Nod2, in turn, is potently stimulated by the N-glycolyl muramic acid found in mycobacteria and their relatives. Determining how fragments of the BCG envelope interact with the innate immune system may enhance our understanding of both heterologous and TB-specific protection. The molecular identities of the fragments recognized in vivo are unknown and the list of mammalian binding partners is likely incomplete. We will address these long-standing questions by synthesizing, validating and deploying clickable photoaffinity probes that either mimic envelope fragments or that incorporate into the peptidoglycan or TDM of live BCG. In proof- of-concept experiments, we will identify host proteins that interact with BCG peptidoglycan and TDM. Probes that label the envelope of live bacteria will permit host delivery of microbe-associated molecular patterns (MAMPs) in a more physiologically-relevant context. In parallel, synthesis of functionalized peptidoglycan fragments and TDM will allow head-to-head comparison of host proteins that interact with synthesized or purified MAMPs and those that interact with MAMPs released by live bacteria during infection. These experiments will reveal the earliest host–BCG interactions that may shape protection against TB and unrelated pathologies like COVID-19. Further, they will provide enabling tools to the community for studying the innate immune response to non-protein adjuvants, vaccines, and pathogens. Our approach is extendable to other glycan- and lipid-containing MAMPs (e.g., LPS).