Mechanistic studies of lipopolysaccharide transport in Gram-negative bacteria

Abstract

Gram-negative bacterial infections are a significant threat to human health due to increasing resistance to all classes of clinically used antibiotics. Gram-negative bacteria are extremely challenging to kill because of their unique cell envelope, which consists of an inner phospholipid membrane, an outer membrane, and a layer of peptidoglycan in the aqueous space (periplasm) between the two membranes. The outer membrane is an unusual asymmetric bilayer containing lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. LPS is a large, highly charged glycolipid, and lateral interactions between LPS molecules create a dense electrolyte mesh that prevents entry of hydrophobic molecules, including most antibiotics. To maintain this protective barrier, cells must quickly and accurately transport millions of LPS molecules from their synthesis site at the cytoplasmic face of the inner membrane, across the aqueous periplasm, and through the outer membrane to the cell surface. The pathway that is responsible for this transport, the lipopolysaccharide transport (Lpt) machinery, consists of seven essential proteins that are proposed to form a transenvelope bridge to transport LPS. This thesis establishes biochemical systems that demonstrate the formation of a bridge across which LPS molecules move from the inner membrane to the outer membrane. We also address how the Lpt machinery recognizes the LPS substrate and achieves efficient transport. In chapter two, we describe a reconstitution of LPS transport from purified components that has enabled the first direct visualization of the Lpt protein bridge. Chapter three describes the development of a quantitative fluorescence-based assay to monitor membrane-to-membrane LPS transport and allow characterization of factors that influence the rate of LPS transport. In chapter four, we identify a cluster of residues required for LPS substrate recognition by the inner membrane Lpt complex and identify a role for LptC in coupling ATP hydrolysis and LPS transport. In chapter five, we characterize a suppressor mutation that allows survival in the absence of LptC and provides insight into its functional role in the Lpt pathway. Finally, in chapter six, we describe progress toward establishing a purification of the transenvelope Lpt protein bridge, with the goal of allowing future structural characterization. Together, these results show how the Lpt machinery accomplishes rapid and efficient LPS transport to the cell surface. Minor disruptions to LPS transport increase outer membrane permeability and cause antibiotic susceptibility, and major disruptions cause cell death, making the LPS transport machinery an attractive target for antibiotic development. Detailed structural and mechanistic knowledge of the transport machinery will greatly facilitate the development of novel antibiotics that disrupt LPS transport.