Project SummaryAntibiotic resistant bacterial pathogens are a major threat to public health. Pseudomonas aeruginosa is aGram-negative opportunistic pathogen displaying high levels of antibiotic resistance even in the absence ofnovel acquired mutations. This resistance is mediated in part by the outer membrane barrier, which alsocontributes to resistance to host-derived antimicrobial peptides and is an essential structure for bacterialsurvival. Despite this, relatively few inhibitors targeting outer membrane biogenesis have been developed andmultiple elements of outer membrane biology are poorly understood. This project has used InsertionSequencing (InSeq) as a transposon mutagenesis strategy to identify genetic determinants of envelope-mediated intrinsic antibiotic resistance. Informed by InSeq results we used a novel resource, an arrayed libraryof P. aeruginosa strains expressing camelid intrabodies, to identify candidate inhibitors of P. aeruginosa outermembrane barrier function. This proposed work will explore the phenotypic impacts of the 14 most promisingcandidate inhibitors and identify binding partners of five selected intrabodies in order to propose mechanismsof action and gain new insights into major antibiotic resistance mechanisms in this opportunistic pathogen.