Emergence of hospital-associated drug-resistant bacterial infections represents one of the major challenges for the US healthcare system. There is an urgent need for development of new and more effective anti-bacterial agents targeting previously unexplored cellular processes. The goal of this proposal is to find a novel class of biological drugs active against Staphylococcus aureus and pathogenic Enterococci. The bacterial cell wall biogenesis is an established drug target for many existing and novel antibacterial agents. In recent years, lipoteichoic acid biosynthesis pathways emerged as an attractive target for development of new therapeutics. Published data indicate that lipoteichoic acid is required for the growth and survival of Gram-positive bacteria in the host. The experiments in this proposal are designed to generate single domain camelid antibodies (nanobodies) that bind and inactivate extracellular domains of enzymes involved in synthesis and modification of lipoteichoic acid. Upon successful completion of this project we will obtain a number of nanobodies active against S. aureus and Enterococci that would be further validated in in vivo studies. The strategies developed in the proposed study will be also applicable to other medically relevant bacterial pathogens.