Mechanism and Inhibitors of Lipopolysaccharide Transport

Abstract

Gram-negative bacterial infections pose a significant health threat due to their increasing resistance to all classes of currently used antibiotics. Gram-negative bacteria are especially challenging to kill because of their unique cell envelope. This envelope is composed of two membranes that are separated by an aqueous compartment known as the periplasm. The inner membrane is a phospholipid bilayer, and the outer membrane is an unusual asymmetric bilayer containing lipopolysaccharide on the outer leaflet and phospholipids on the inner leaflet. Lipopolysaccharide is a large, highly charged, glycolipid and lateral interactions between lipopolysaccharide molecules creates an electrolyte mesh that forms a permeability barrier that prevents the entry of drugs that are normally able to kill other types of bacteria. Lipopolysaccharide synthesis occurs in the cytoplasm and on the cytoplasmic face of the inner membrane. It is then flipped across the inner membrane by the ATP-powered flippase MsbA. Transport across both the periplasm and the outer membrane is mediated by the seven-protein lipopolysaccharide transport machine (LptB2FGCADE), which forms a bridge that connects the inner and the outer membranes. Transport by LptB2FGCADE is powered by ATP-hydrolysis by the inner-membrane components LptB2FGC. In this thesis, we present progress towards understanding how the lipopolysaccharide transporter works, how it coordinates lipopolysaccharide transport to phospholipid biogenesis and progress towards antibiotics that target lipopolysaccharide transport. In chapter two, we describe one of the first two reported MsbA inhibitors. We find that this compound decouples MsbA’s ATPase activity from its lipopolysaccharide transport activity. In chapter three, we report the development of new tools to observe intermediate lipopolysaccharide transport states in vivo and in vitro. These results are guided by a crystal structure of LptB2FGC. We use these newly developed tools to understand how lipopolysaccharide transport is coordinated to ATP hydrolysis, and how LptB2FGC maintains efficient LPS transport. In chapter four, we use the tools developed in chapter three to investigate the mechanism of action of three compounds that interfere with LptB2FGC in vivo. We show that polymyxin B, which is currently the clinical drug of last resort against multidrug resistant Gram-negative infections, interacts specifically with LptB2FGC in vivo and in vitro. This presents a new avenue for making improved polymyxin analogues. We find that the effect of novobiocin on Lpt depends on ATP concentration, which sheds light into how novobiocin interacts with LptB2FGC. We also show that inhibition of fatty acid biosynthesis causes an inhibition of lipopolysaccharide transport. We speculate that this may form the basis of how the cell coordinates phospholipid biogenesis with lipopolysaccharide biogenesis. In chapter five, we report that a novel macrocyclic antibiotic functions (MCP-1) by inhibiting LptB2FGC activity. We report a 3.1 Å resolution co-complex of lipopolysaccharide, LptB2FG and MCP-1. We show that MCP-1 has a novel non-competitive binding mode. Our structural and biochemical results provide insight both into how MCP-1 functions and how LptB2FGC functions. In chapter six, we report the cryo-EM structure of full membrane-to-membrane LptB2FGCADE protein bridge, with each component at 3.5 Å resolution or better. We find that the transmembrane helix of LptC serves to regulate LptB2FGC activity by sensing proper bridge assembly. Future work can leverage the chemical, biochemical and structural tools described in this thesis to gain a better understanding of how lipopolysaccharide is transported, how the cell coordinates lipopolysaccharide transport, and how to develop drugs that target lipopolysaccharide transport.