Targeted and Enzyme-Activated Self-Assembling Peptide Nanofibers for Intracellular Bacterial Clearance and Immune Restoration.

Intracellular bacterial persistence drives chronic and recurrent infections by exploiting host cells as shelters that shield pathogens from antibiotics and by secreting effector proteins that suppress host immune clearance. Conventional direct bactericidal strategies often inflict collateral damage on host cells and fail to counteract this effector-mediated immune evasion, ultimately leading to treatment failure and infection relapse. Here, we developed a targeted and enzyme-activated peptide self-assembling nanofiber system (TESAN) to address these challenges. We used orthogonal design to screen functional modules, balancing safety with efficacy to identify an optimal formulation. TESAN maintains charge masking during delivery to minimize host toxicity. Upon targeting bacteria within the cytoplasm, bacteria-secreted enzymes selectively activate the peptides, simultaneously releasing antimicrobial moiety for bacterial killing and inducing self-assembly into a dense nanofiber network on the bacterial surface. The assembled nanofibers restrict secretion of immunosuppressive effector proteins, which reverses local immune suppression and restores macrophage clearance function. TESAN eliminated 99.98% of intracellular persisters without compromising host cell viability. In a murine pulmonary infection model, TESAN achieved a 4.64 log10 reduction in bacterial burden and increased seven-day survival from 10% to 70%. Studies using alveolar macrophages from patient bronchoalveolar lavage fluid confirmed intracellular targeting and achieved up to 99.91% bacterial elimination. These results establish enzyme-responsive in situ self-assembly as an effective strategy for treating persistent intracellular infections.