Reprogrammed SimCells for Antimicrobial Therapy

Antimicrobial resistance (AMR) is a critical global health challenge. In this study, we developed a novel platform based on chromosome-free and non-replicating simple cells (SimCells, size 1-2 {micro}m) and mini-SimCells (size 100-400 nm) for targeted pathogen elimination. Engineered with surface-displayed nanobodies, SimCells and mini-SimCells selectively bind bacteria expressing specific antigens (e.g., OmpA in E. coli). 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 E. coli with high specificity and efficiency. Multi-dose reprogrammed mini-SimCell treatment led to a 103-fold selective reduction of targeted bacteria in mixed microbial communities, with minimal disruption to non-target bacteria. We demonstrate that reprogrammed mini-SimCells, engineered with nanobody targeting outer membrane protein OmpA of the clinically relevant multidrug resistant pathogen E. coli ST131, achieved elimination efficiencies over 97% at 24 and 48 hrs. This modularised plug-and-play antimicrobial platform provides a highly specific, efficient and adaptable solution for combating diverse AMR pathogens. Significance StatementAntimicrobial-resistant (AMR) bacteria cause millions of deaths worldwide annually, representing a critical global health challenge. In this study, we developed a novel therapeutic platform using two types of chromosome-free, non-replicating engineered bacterial cells: SimCells (1-2 {micro}m) and mini-SimCells (100-400 nm). These SimCells were engineered to selectively bind to targeted E. coli, facilitating precise delivery of toxic proteins via a Type VI secretion system (T6SS) and localised generation of hydrogen peroxide from aspirin. We demonstrate that mini-SimCells eliminated over 97% of a targeted AMR strain within 48 hours. Moreover, multiple-dose administration achieved a selective 103-fold reduction of targeted E. coli in mixed microbial communities. This modular plug-and-play platform offers an adaptable solution against diverse multi-drug-resistant pathogens.