Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis

Abstract

Gram-negative bacteria decorate their outermost surface structure, lipopolysaccharide, with elaborate chemical moieties, which effectively disguises them from immune surveillance and protects them from the onslaught of host defences. Many of these changes occur on the lipid A moiety of lipopolysaccharide, a component that is crucial for host recognition of Gram-negative infection. In this Review, we describe the regulatory mechanisms controlling lipid A modification and discuss the impact of modifications on pathogenesis, bacterial physiology and bacterial interactions with the host immune system.

Figure 1 |. The cell envelope of Gram-negative bacteria.

a | A cryoelectron tomography image of an Escherichia coli cell, showing the characteristic inner membrane (IM) and outer membrane (OM) (scale bar of 200 nm). b | Schematic of the Gram-negative cell envelope, showing the typical inner and outer bilayers that are separated by the periplasm, which contains peptidoglycan (PG). The outer leaflet of the outer membrane contains lipopolysaccharide (LPS), which is anchored to the membrane by the LPS lipid A domain. The inner leaflet of the outer membrane and also the entire inner membrane are composed of phospholipids only, and both bilayers can contain a range of different types of membrane protein. c | The lipid A and inner core (Kdo (3-deoxy-d-manno-octulosonic acid)) portion of LPS are shown. Unmodified lipid A consists of a β-1′,6-linked disaccharide of glucosamine that is both phosphorylated and fatty acylated. This basic structure can be extensively modified after synthesis. d | The LPS modifications that occur in Salmonella spp. The enzymes responsible are controlled by either the PmrAB two-component system (red) or the PhoPQ two-component system (blue), or have no known two-component regulatory system (green). The various possible modifications include the addition of 4-amino-4-deoxy-l-arabinose (aminoarabinose) moieties (by ArnT) and phosphoethanolamine moieties (by EptA and EptB), as well as phosphorylation (by LpxT), deacylation (by PagL and LpxR; resulting in loss of acyl chains, as indicated by dashed lines), acylation (by PagP) and hydroxylation (by LpxO). Transcription of the gene encoding LpxO is modestly induced by PhoPQ, but LpxO remains active in conditions in which PhoPQ is inactive, suggesting that the enzyme acts independently of this two-component system.

Figure 2 |. Transcriptional and post-translational regulation of lipid A modification enzymes.

a | Transcriptional control of lipid A modification enzymes includes gene regulation by two-component systems such as PhoPQ, leading to acylation and deacylation of lipid A by upregulating transcription of the genes encoding the enzymes PagP and PagL, respectively. The two-component system PmrAB leads to the addition of 4-amino-4-deoxy-l-arabinose (aminoarabinose; l-Ara4N) and phosphoethanolamine (pEtN) to lipid A by upregulating transcription of the genes encoding the innermembrane enzymes ArnT and EptA, respectively, which modify lipid A as it is transported to the outer membrane^,. Expression of EptB (another phosphoethanolamine transferase) is repressed by the small RNA (sRNA) MgrR, which is induced by PhoPQ. Translation of the phoP mRNA is repressed by the sRNA MicA, which leads to the loss of regulation by the PhoPQ system. The sRNA MicF increases degradation of the lpxR mRNA, which encodes a lipid A deacylase. b | Post-translational control of lipid A modification systems includes inhibition of the kinase LpxT (which phosphorylates lipid A during transport to the outer membrane) by the small peptide PmrR, which is upregulated by the PmrAB system in response to high levels of Fe³⁺ (REF. 33). Post-translational regulation is also mediated by substrate availability. Membrane perturbation can lead to the displacement of phospholipids from the inner leaflet to the outer leaflet of the outer membrane, placing these donor substrates in close proximity to the acyltransferase PagP, and thus enhancing enzyme activity. PagP cleaves the phospholipid substrate, restoring the composition of the outer membrane and increasing the integrity of the permeability barrier by further acylating lipid A. LpxR deacylates lipid A, but this activity is inhibited by the aminoarabinose lipid A modification.

Figure 3 |. Toll-like receptor 4–MD2 signalling.

This simplified Toll-like receptor 4 (TLR4)–MD2 signalling schematic illustrates the two responses—the myeloid differentiation primary response protein 88 (MYD88)-dependent and TIR domain-containing adaptor inducing IFNβ (TRIF) (or MYD88-independent) pathways—that can be differentially stimulated on binding of lipid A to the TLR4–MD2 complex. This binding occurs through the association of lipid A with lipid A-binding protein (LBP) and CD14, and leads to the production of cytokines and clearance of the pathogen. The MYD88-dependent pathway leads to the production of pro-inflammatory cytokines, whereas the less inflammatory TRIF pathway occurs after endocytosis of the TLR4–MD2 receptor and stimulates the expression of interferon (IFN)-inducible genes that are important for adjuvanticity but are less inflammatory than those cytokines induced by the MYDD88-dependent pathway.

Figure 4 |. Lipid A modification strategies that promote survival in the host.

Helicobacter pylori, Yersinia pestis and Vibrio cholerae have evolved different mechanisms of lipid A modification to aid colonization of their respective niches inside the host. Differences in structure between the pairs of lipid A molecules are highlighted in red. a | The human stomach is the sole niche of H. pylori, and the bacterium has adapted to this environment by constitutively modifying lipid A to a form that resists cationic antimicrobial peptides (CAMPs) and evades the lipid A receptor, Toll-like receptor 4 (TLR4). Surface expressed H. pylori lipid A consists of a tetra-acylated form that lacks the 4′-phosphate group and is substituted at the C1 position with a phosphoethanolamine. Lipid A modification mutants present a hexa-acylated, bis-phosphorylated species that is a TLR4 agonist. b |When residing in the flea vector, Y. pestis produces an endotoxic hexa-acylated lipid A that is a strong immunostimulant in humans. Following transmission to the human host, the bacterium senses a shift in temperature (from 21–27 °C to 37 °C) and synthesizes tetra-acylated lipid A, which escapes detection by TLR4, and immune stimulation is thereby curtailed. c | V. cholerae normally inhabits freshwater, estuarine and oceanic environments and often colonizes marine organisms such as copepods, which are important for cholera transmission. Copepod colonization provides an environment that probably induces lipid A modifications which are crucial for marine and host survival, a prospect that is currently under investigation. The V. cholerae O1 El Tor biotype modifies lipid A with a glycine or diglycine residue, providing resistance to CAMPs, whereas the classical V. cholerae O1 biotype remains susceptible.