Inhibiting Lipopolysaccharide Biogenesis: The More You Know the Further You Go

Abstract

Gram-negative bacteria are intrinsically resistant to many antibiotics because they are surrounded by an outer membrane that creates a robust permeability barrier. The outer membrane has an unusual asymmetric structure with a periplasmic leaflet composed of phospholipids and an outer leaflet composed of lipopolysaccharides. Because lipid biosynthesis is completed in the inner membrane of these didermic bacteria, these components must be transported across the cell envelope and properly assembled to expand the outer membrane during growth and division. Lipopolysaccharide molecules are transported over a multi-protein transenvelope bridge that is powered by ATP hydrolysis in the cytoplasm. This review discusses how this bridge is assembled and functions and how lipopolysaccharide transport is regulated to ensure balanced growth of all envelope layers. A combination of approaches and new experimental tools have significantly advanced our understanding of this molecular machine and contributed to the development of new antimicrobials that interfere with transport.

Keywords: ABC transporter; intermembrane bridges; intermembrane transport; lipid transport; lipopolysaccharide; outer-membrane biogenesis.

Figure 1

Protein and lipid transport pathways in outer membrane biogenesis. The Lol system shuttles lipoproteins (green) from the inner membrane to the outer membrane. Unfolded outer-membrane proteins (pink) are translocated into the periplasm by the Sec machinery, then brought to the Bam complex (yellow) by soluble chaperones. Bam then folds and inserts β-barrel proteins into the outer membrane. AsmA-like proteins (turquoise) are proposed to facilitate transport of phospholipids between the inner and outer membranes. Cytoplasmic LPS is flipped to the periplasmic leaflet of the inner membrane (not shown) and then delivered to the outer membrane by the Lpt proteins (blue). Abbreviations: Bam, β-barrel assembly machine; IM, inner membrane; Lol, localization of lipoproteins; LPS, lipopolysaccharide; Lpt, LPS transport system; OM, outer membrane; OMP, outer-membrane protein.

Figure 2

Compounds that target various steps in LPS biogenesis superimposed on a current model for LPS transport to the cell surface. Proposed targets or processes are shown as dashed lines. In the cytoplasm, compounds that target LpxC (gold) inhibit the first committed step of LPS biosynthesis. MsbA (dark blue) inhibitors disrupt the essential step of flipping LPS to the periplasmic leaflet of the inner membrane. The natural product antibiotic colistin is proposed to target the LPS pathway at the inner membrane. Murepavadin inhibits LPS transport in Pseudomonas spp. and has been proposed to target LptD, though its specific mechanism of action remains to be elucidated. The natural product thanatin has been proposed to interfere with Lpt bridge formation and/or stability, though, like murepavadin, the specific details of its mechanism are not yet known. Abbreviations: LPS, lipopolysaccharide; Lpt, LPS transport system.

Figure 3

The movement of the LptC transmembrane helix is a targetable, on-pathway LPS transport intermediate. (a) ATP-binding-defect mutations in LptB reduce LPS transport in vivo. The deletion of the LptC transmembrane helix (LptC^Δ™) restores LPS transport in these complexes. (b) A class of MCPs (e.g., zosurabalpin) inhibit LPS transport in vivo in Acinetobacter baumannii. (c) Only MCPs with bulky substituents (purple) inhibit the transport of LPS by A. baumannii LptB₂FGC to LptA in vitro. When the LptC transmembrane helix is removed, all MCPs inhibit LPS transport by forming a stable complex with LPS and LptFG. Abbreviations: LPS, lipopolysaccharide; Lpt, LPS transport system; MCP, macrocyclic peptide.

Figure 4

Regulatory mechanisms to maintain LPS homeostasis. When LPS levels are low in the OM, mislocalized phospholipids are degraded by PldA (green), releasing products that eventually lead to the stabilization of LpxC (gold), leading to increased LPS biosynthesis. When LPS biosynthesis outpaces LPS transport by Lpt (blue), LPS built up in the IM binds to YejM (salmon), which displaces LapB (teal), leading to FtsH (purple)-mediated degradation of LpxC. Abbreviations: IM, inner membrane; LPS, lipopolysaccharide; Lpt, LPS transport system; OM, outer membrane.