Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model

Abstract

Gram-negative bacteria have a double-membrane cellular envelope that enables them to colonize harsh environments and prevents the entry of many clinically available antibiotics. A main component of most outer membranes is lipopolysaccharide (LPS), a glycolipid containing several fatty acyl chains and up to hundreds of sugars that is synthesized in the cytoplasm. In the past two decades, the proteins that are responsible for transporting LPS across the cellular envelope and assembling it at the cell surface in Escherichia coli have been identified, but it remains unclear how they function. In this Review, we discuss recent advances in this area and present a model that explains how energy from the cytoplasm is used to power LPS transport across the cellular envelope to the cell surface.

Fig. 1. LPS transport pathway in E. coli

LPS is synthesized on the cytoplasmic side of the inner membrane (IM) and flipped to the periplasmic side by an ABC transporter, MsbA. LPS is then transported to the cell surface via the Lpt pathway. This pathway consists of seven essential proteins, LptA-G. LPS is extracted from the IM in an ATP-dependent manner by the ABC transporter LptB₂FG and transferred to LptC, which forms a complex with LptB₂FG. LptC consists of a single membrane spanning domain and a large periplasmic domain, which forms a periplasmic bridge with soluble protein LptA and the N-terminal region of LptD. LPS transverses the aqueous periplasmic space through this protein bridge and reaches the cell surface with the help of the C-terminal domain of LptD, which forms a β-barrel structure plugged by the outer membrane (OM) lipoprotein LptE. LPS is composed of lipid A, the inner and outer core oligosaccharides, and the O antigen, which is highly variable and absent in E. coli K-12. EtN, ethanolamine; Gal, D-galactose; Glc, D-glucose; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; P, phosphate.

Fig. 2. The periplasmic protein bridge

The figure illustrates a model of the periplasmic protein bridge comprised of LptC, LptA and LptD. The C-terminal periplasmic region of LptC (yellow; PDB ID: 3MY2), LptA (pink; PDB ID: 2R19) and N-terminal region of LptD (orange; PDB ID: 4Q35) are stacked to illustrate the Lpt bridge. Two LptA molecules in the trigonal crystal form (PDB ID: 2R1A) were replaced by C-LptC and N-LptD. The number of LptA molecules in the bridge is unknown.

Fig. 3. The outer membrane translocon

The figure illustrates a model of the two protein plug-and-barrel in the OM comprised of LptD (orange; PDB ID:4Q35) and LptE (cyan; PDB ID:4Q35).

Fig. 4. Regulation of the formation of the Lpt bridge

The biogenesis of the functional LPS LptDE translocon requires disulfide bond rearrangements at the OM. LptD and LptE are targeted to the OM via the Bam and Lol pathways, respectively. LptD has four cysteines, two in the N-terminal periplasmic region (Cys31 and Cys173) and two in the β-barrel domain (Cys724 and Cys725). LptD with a disulfide bond between Cys31 and Cys173 forms a non-functional complex with LptE, followed by several disulfide bond rearrangements to produce a functional translocon with native disulfide bonds (Cys31-Cys724 and Cys173-Cys725). Functional translocon formation permits N-LptD to interact with LptA, resulting in a functional LPS transporter including the IM complex, LptB₂FGC. It is unknown how the interaction between LptA and LptC is regulated.

Fig. 5. The PEZ model

LPS transport from the IM to LptC, and from LptC to LptA, requires energy derived from ATP hydrolysis. LPS binding sites both in LptC and LptA are constantly occupied by molecules of LPS. The observation that multiple rounds of ATP hydrolysis are required to transport LPS to the cell surface, and that LPS binding sites in LptC and LptA are always filled, suggests that ATP is needed to push a continuous stream of LPS through the Lpt bridge. Therefore, the PEZ model suggests that LPS transport occurs by analogy to a PEZ candy dispenser, in which PEZ candies filling the dispenser are pushed by a spring at the bottom of the dispenser. In this model, LPS molecules in the outer leaflet of the IM are pushed towards LptC via the action of LptB₂FG, in a process that depends on ATP hydrolysis in the cytoplasm, which is mediated by the ATPase LptB in the complex. LPS is then pushed from LptC to LptA and across the Lpt periplasmic bridge towards the LptDE translocon, in a process that also involves ATP hydrolysis mediated by LptB₂FGC. LPS is then proposed to cross the translocon with the lipid portion of LPS being directly inserted into the outer leaflet of the OM without entering the lumen of the LptD barrel, while the sugar portion of LPS goes through the barrel.