Synthesis, secretion and assembly of extracellular complex carbohydrates in Gram-negative bacteria

Complex carbohydrates are essential biopolymers ubiquitously expressed in all kingdoms of life. On cell surfaces, they usually perform architectural functions by fortifying the cell boundary, aiding in osmo-regulation, defining an extracellular matrix, and mediating cell-to-cell communications, among many other roles. We understand fairly well how polypeptides are transported across or integrated into biological membranes. Similar mechanisms for polysaccharides, which range from acidic to water-insoluble hydrophobic polymers, remain mostly unexplored, despite playing critical roles in many physiological and pathological processes. My research seeks to fill this gap. We integrate structural biology approaches with biochemistry, glycobiology, biophysics, bioinformatics, and molecular biology to delineate how high molecular weight complex carbohydrates are synthesized and deposited on the cell surface. Leveraging microbial, viral, plant and vertebrate model systems, we provide atomistic descriptions of mechanistically distinct secretion systems. Understanding these processes on a molecular level aids novel drug and biomaterial developments. This proposal combines two research directions on microbial extracellular polysaccharides synthesized and secreted by fundamentally different mechanisms. First, cellulose, a linear glucose polymer, is an important biofilm component of many enterobacteria, including E. coli. Biofilms pose a particular threat to human health, causing ~80% of nosocomial infections. Cellulose is synthesized by a ‘synthase-dependent’ pathway in which a membrane-embedded enzyme synthesizes and secretes the polymer. Most enterobacteria modify cellulose with phosphoethanolamine during secretion to stabilize it on the cell surface. We provided the first insights into the molecular organization of the supramolecular cellulose synthase complex. Our future research direction will address how cellulose biosynthesis is controlled, how cellulose is modified in the periplasm, and how it is transported across the periplasm and the outer membrane. Second, Gram-negative bacteria are protected by an outer membrane containing lipopolysaccharides (LPS) in the extracellular leaflet. LPS molecules contain variable O antigen polysaccharides that significantly extend the bacteria’s outermost protective coat and provide survival benefits to many human pathogens. Prior to attachment to the conserved LPS core, O antigens are completely assembled on a lipid linker inside the cell and transported to the periplasm by an ATP- fueled ABC transporter, called WzmWzt. We currently lack any mechanistic insights into how ABC transporters translocate biopolymers, such as polysaccharides, teichoic acids and polypeptides, which are all potent virulence factors. Our X-ray and cryo electron microscopy structures of WzmWzt in O antigen-free states provided the first insights into the transporter’s function. We now seek to determine the mechanism by which O antigens are secreted. This will be accomplished biochemically by reconstituting O antigen translocation in vitro, and structurally by determining snapshots of WzmWzt during substrate translocation.