Project Summary Antibiotic-resistant microbial infections are a critical healthcare problem in the U.S. and the world. Improved antimicrobial strategies – and an efficient mechanism for their ongoing generation to keep pace with microbial evolution – are sorely needed. Vancomycin-resistant Enterococcus faecium is particularly problematic and is in the CDC's “Serious Threat” category. Antimicrobial polypeptides (AMPs) offer a compelling class of therapeutics to provide potency, specificity, and hindrance to resistance. Engineered probiotic lactic acid bacteria can provide local delivery of AMPs to overcome barriers to traditional therapeutic delivery. The objective of the proposed studies is to engineer AMPs enterocin A and endolysin ORF9, via an innovative directed evolution platform, for enhanced potency and specificity to combat E. faecium infections. The AMPs will be engineered and evaluated via secretion from two probiotics: Lactococcus lactis and Lactobacillus johnsonii. The objective will be achieved via three aims. (1) Develop a platform for high throughput (~107/day), quantitative selection of potent AMPs as secreted from probiotics. This will be achieved via microdroplet co- encapsulation of probiotic and pathogen along with fluorescence-activated cell sorting. (2) Elucidate efficient evolutionary pathways for AMPs ORF9 lysin and entA to enhance potency and specificity, from a probiotic host, against E. faecium. Innovative combinatorial library designs, including some informed by bioinformatics, will be used and compared. (3) Evaluate engineered probiotic/AMP efficacy against E. faecium in a murine model while elucidating the impacts of potency, host, secretion efficiency, dose, and AMP diversity on E. faecium inhibition and resistance reduction.