PROJECT SUMMARY Project 3: Defining and defeating the mechanisms of outer membrane biogenesis in Gram-negative bacteria Gram-negative bacteria are surrounded by an outer membrane (OM) composed of lipopolysaccharide (LPS) that creates a formidable permeability barrier preventing the uptake of many drugs. The spread of acquired antibiotic resistance mechanisms among Gram-negative bacteria combined with the intrinsic resistance conferred by the OM has severely limited treatment options for infections with these organisms. Therefore, the development of new antibiotics effective against Gram-negative bacteria is an urgent medical need. To enable the discovery of these treatments, our CARBIRU team will uncover new vulnerabilities and targets required for OM assembly using E. coli as a model organism. Our approach will be three-pronged. Aim 1 will focus on the essential Bam machine that assembles beta-barrel proteins in the OM. We have purified and characterized different states of the six-member complex, including an intermediate state engaged with a substrate. To determine which states of the machine are the most effective to target with drugs that disrupt the OM permeability barrier, we will use our nanobody screening platform to identify nanobodies that selectively bind purified complexes locked in different conformations. The ability of the nanobodies to engage with surface exposed Bam epitopes on cells and promote cell killing or permeability will then be assessed. Structures of the nanobody-bound complexes will also be determined using cryo-EM methods that successfully elucidated the structure of the stalled Bam machine. The results will provide insights into the mechanism of Bam function and define susceptible domains within the machine. The second aim will investigate the role of the essential membrane protein YejM in regulating LPS synthesis. LpxC is the committed step in the pathway, and it has long been known to be subjected to proteolysis by the FtsH protease. However, it has remained unclear how LpxC proteolysis is regulated to coordinate LPS synthesis with OM assembly. We will test the hypothesis that YejM is responsible for this coordination and elucidate the regulatory mechanism. The results will teach us how the important drug target LpxC is regulated in enterobacterial pathogens and identify new ways to disrupt LPS synthesis for antibiotic development. Finally, we will investigate the mechanism by which undecaprenyl-phosphate (Und-P) is recycled. Und-P is the lipid carrier used for the synthesis of most cell surface polysaccharides, including peptidoglycan and the O-antigen of LPS. Our genetic and bioinformatic analyses identified two conserved protein families as candidates for the long-sought flippases that recycle Und-P. We will investigate their role in Und-P transport and how their inactivation affects OM assembly and OM modifications that promote antibiotic resistance. Because Und-P recycling is central to cell envelope assembly, the results will define an attractive new class of targets for antibiotics or antibiotic potentiators. Overall, our results with these conserved systems will be highly relevant to the development of novel treatments for infections with a broad-spectrum of Gram-negative pathogens.