Velez, A. Z.; Radin, J. N.; Kennedy, E. N.; Parsons, J. B.; Tong, H. M.; Jung, E.; Alam, E.; Radlinski, L. C.; Wagner, N. J.; Fowler, V. G.; Rowe, S. E.; Kehl-Fie, T.; Conlon, B. P.

{beta}-lactam antibiotics are widely used to treat bacterial infections, yet treatment failures frequently occur even without resistance. Here, we show that the innate immune protein calprotectin (CP), released by neutrophils and abundant at infection sites, induces tolerance to {beta}-lactam antibiotics in Staphylococcus aureus. CP is a potent zinc chelator and was found to inhibit the activity of S. aureus autolysins, zinc-dependent enzymes essential for bacterial lysis following {beta}-lactam-mediated inhibition of cell wall synthesis. This protection was independent of bacterial growth or metabolism and was specific to {beta}-lactam antibiotics. Mechanistically, CP inactivated the amidase activity of Atl, the major S. aureus autolysin, through zinc sequestration. In vivo, oxacillin was significantly more effective in CP-deficient mice, demonstrating that CP reduces {beta}-lactam efficacy during infection. These findings reveal a host-derived mechanism of antibiotic tolerance and suggest that zinc availability at infection sites may directly influence {beta}-lactam treatment outcomes.