On-Site Serine Delivery Drives Fermentation Pathway Reprogramming to Reverse Intracellular Staphylococcus aureus Persistence

Intracellular Staphylococcus aureus bacteria that survive high-dose antibiotic treatment are recognized as persisters, serving as reservoirs for recurrent infections. While enhancing bacterial metabolism has restored antibiotic efficacy against planktonic persisters, it is ineffective against intracellular forms due to host-imposed nutrient deprivation. To address this, we developed FAlsBm@Rif, a poly(amino acid)-based nanodrug designed to sensitize intracellular persisters to antibiotics via on-site serine delivery. FAlsBm@Rif is constructed by encapsulating rifampicin in serine- and mannose-functionalized copolymers, FAlsBm. Upon uptake of FAlsBm@Rif by macrophages through mannose-mediated endocytosis, the mannose ligand dissociates within the host cell, exposing phenylboronic acid groups to the nanodrug. This enables FAlsBm@Rif to specifically target the peptidoglycan of intracellular persisters. In this way, FAlsBm@Rif employs a cascade-targeting mechanism to precisely navigate both host cells and intracellular Staphylococcus aureus persisters, ensuring the localized release of serine and rifampicin at bacterial loci, thus counteracting host-imposed nutrient deprivation. On-site serine delivery shifts persisters to a fermentation pathway under host-induced stress, boosting ATP production and membrane potential. This metabolic shift reverses persistence by alleviating the stringent response and reducing cell wall stress. Consequently, FAlsBm@Rif eradicated 99.78% of intracellular persisters in vivo, significantly outperforming Rif alone (63.41%). This strategy offers a promising approach to combating intracellular persisters.