Antibiotic-resistant pathogens pose a significant threat to human health. The NIH identified several Gram- negative species of particular concern due to increasing antibiotic resistances. The cell envelope of Gram- negative bacteria consists of two membranes and lipopolysaccharides (LPS) form an important component of the extracellular leaflet of the outer membrane. LPS are important cell wall components that control diffusion acrosstheoutermembrane,stabilizethecellenvelope,andassistinescapinghostimmunedefenses,among otherfunctions.ImpairedLPSbiosynthesiscorrelateswithincreasedsusceptibilitytoantimicrobialtreatments. LPS contain a conserved core, consisting of lipid-A attached to an oligosaccharide backbone, and a hypervariable region, called the O antigen. O antigens are primarily linear complex carbohydrates that reduce the efficacy of complement-mediated cell lysis and phagocytosis as part of the innate immune response. O antigens are synthesized via two fundamentally different mechanisms. One pathway relies on assembling the polymers from short oligosaccharides in the periplasm, the other involves moving fully-assembled O antigens fromthecytosolictotheperiplasmicsideoftheinnermembranewiththehelpofanABCtransporter.Notonly is ABC transporter-mediated secretion of O antigens an important process for microbial pathogenicity, the transport of a substrate several times the size of the ABC transporter itself is fascinating from a molecular level. Taking advantage of an already determined O antigen-translocating ABC transporter structure, we propose a structural biology approach to unravel the mechanism of O antigen translocation and to identify uniquefeaturesoftheOantigenthatregulatetransporteractivity. ABC transporters use ATP binding and hydrolysis to cycle between conformations that mediate substrate translocation.OurOantigenABCtransporterstructurerepresentsanucleotide-freeconformation,inwhichthe transporter forms a continuous channel across the membrane that could accommodate a translocating O antigen. We speculate that conformational changes associated with nucleotide binding induce O antigen translocation by about 1-2 sugar units per ATP hydrolyzed. To reveal the molecular mechanism of O antigen translocation, we seek to determine the structure of the ABC transporter in a nucleotide-bound closed conformation (Aim 1). Further, many bacterial species signal completion of O antigen biosynthesis by modifying the polymer?s growing end with specific groups, such as carbohydrate, phosphate, or methyl moieties. These ?capped O antigens? can only be exported by transporters containing a carbohydrate-binding domain (CBD) attached to their nucleotide-binding domain. This domain was removed from our O antigen transporter to facilitate crystallization. To reveal how the CBD binds its substrate and modulates transporter functions,weproposetodeterminethestructureandsubstratebindingpropertiesoftheisolatedCBD(Aim2A) andtodeterminethearchitectureoftheCBD-containingfull-lengthOantigentransporter(Aim2B).
Many multi-drug resistant pathogens are Gram-negative bacteria. These species are surrounded by two membranes, of which the outer membrane contains lipopolysaccharides (LPS) that contain a conserved core and a hypervariable region, calledtheOantigen.Oantigensarecomplexcarbohydratesthataresynthesizedinside the cell and have to be transported to the periplasmic space for attachment to the LPS core.Thecurrentproposalseekstoidentifythemechanismofthistransportprocess.
