Reprogrammed SimCells for antimicrobial therapy

Antimicrobial resistance (AMR) is a critical global health challenge. In this study, we developed a platform based on chromosome-free and nonreplicating simple cells (SimCells, size 1 to 2 µm) and mini-SimCells (size 100 to 400 nm) for targeted pathogen elimination. Engineered with surface-displayed nanobodies, SimCells and mini-SimCells selectively bind bacteria expressing specific antigens (e.g., OmpA in <i>Escherichia coli</i>). The selective interactions facilitate close SimCell-pathogen proximity, enabling two antimicrobial mechanisms: direct injection of toxic effectors into bacterial cytoplasm via a heterologous expression of type VI secretion system (T6SS), and enzymatic conversion of aspirin into catechol by engineered salicylate hydroxylase, leading to sustained local production of hydrogen peroxide (H₂O₂). Our results demonstrate that both reprogrammed SimCells and mini-SimCells can eliminate target <i>E. coli</i> with high specificity and efficiency. Multidose reprogrammed mini-SimCell treatment led to a 10³-fold selective reduction of targeted bacteria in mixed microbial communities, with minimal disruption to nontarget bacteria. We demonstrate that reprogrammed mini-SimCells, engineered with nanobody targeting outer membrane protein OmpA of the clinically relevant multidrug-resistant pathogen <i>E. coli</i> ST131, achieved elimination efficiencies over 97% at 24 and 48 h. This modularized "plug-and-play" antimicrobial platform provides a highly specific, efficient, and adaptable solution for combating diverse AMR pathogens.