Lipid Trafficking: How diverse architectures of lipid transport systems shuttle lipids to the bacterial outer membrane.

By: Michelle Rudden

Twitter: @RuddenMichelle


Highly conserved domains in MCE proteins are used as structural building block to synthesise diverse transport architectures capable of shuttling hydrophobic lipid molecules between the inner and outer membrane of bacteria.


One of the defining hallmarks of Gram negative bacteria is their cell wall, composed of two phospholipid (PL) bilayer membranes separated by the periplasmic space which contains a thin layer of peptidoglycan. The inner membrane (IM) faces the cytoplasm while the outer membrane (OM) is exposed to the environment. The physical properties of the OM is a crucial selective barrier often preventing entry of toxic compounds, antibiotics and host innate immune compounds such as cationic antimicrobial peptides (CAMPs). Any mutations or changes affecting the physical integrity of the OM can reduce pathogenicity and increase antibiotic susceptibly, as a result it is a therapeutic target for many bacterial diseases. The OM is an asymmetric bilayer composed of an inner leaflet of phospholipids and an outer leaflet rich in lipopolysaccharide (LPS) in which several non-specific porins and specific uptake channels are embedded. The LPS-bilayer structure of the OM is more rigid compared to normal phospholipid bilayers and is largely impermeable to hydrophobic molecules by slowing down passive diffusion and narrow pores limit by size the uptake of hydrophilic molecules. The transport system for trafficking LPS molecules from the cytoplasm has been described in detail with a model comprising seven essential proteins (with most of the structures resolved) that spans the periplasmic space to deliver the LPS molecule to the outer leaflet of the OM. However there is no detailed mechanism described for the transport of nascent phospholipids in the inner leaflet and exactly how the asymmetry is maintained between the leaflets of the OM still remains unclear.

In a paper published this month in Cell, Ekiert et al, (2017) propose a mechanism by which proteins belonging to the mammalian cell entry (MCE) superfamily form hexameric assemblies that can form diverse protein channels with hydrophobic pores capable of shuttling hydrophobic molecules between the IM and OM in E. coli.  MCE proteins are ubiquitous in double membraned bacteria including E. coli and some membrane bound organelles such as chloroplasts where they are suggested to be involved in transport of lipid molecules either from the OM to the IM or as exporters driving molecules out of the cell.

Lipid molecules are hydrophobic and are poorly soluble in water, the hydrophilic periplasmic space is the major barrier in lipid transport across the membranes. In the LPS transport model protein components assemble to form a periplasm spanning bridge with a hydrophobic core that protects the lipid tails of LPS facilitating the movement of LPS molecules toward the OM. This led Ekiert et al (2017) to propose a similar mechanism by which MCE proteins assemble to form a hydrophobic pore allowing the transport of phospholipids between inner and outer membranes.

In this paper X-Ray and Cryo-electron microscopy (Cryo-EM) structures are reported for three MCE proteins found in E. coli each producing a distinct architecture with conserved MCE hexameric ring modules. The crystal structure of the MCE protein MlaD in E. coli revealed a single homo-hexameric ring assembled with a hydrophobic core that facilitates the transport of phospholipids. MlaD forms a complex with three other proteins anchored in the IM (Fig 1). MlaD does not span the periplasmic space, it recruits periplasmic lipid binding protein MlaC that delivers the phospholipids to the outer membrane.

Cryo EM structures of two different MCE proteins non-homologous to MlaD revealed different architectures that span the periplasm implicating their role in moving hydrophobic substrates across the membrane. PqiB forms a needle and syringe like structure that forms a periplasmic bridge (Fig 1). PqiB contains three MCE domains (MCE1, MCE2, MCE3) each forming a hexameric ring stacked on top of each other to form the hollow barrel of the structure (Fig. 1). The needle like structure of PqiB is a six helix coiled coil, one helix contributed from the C-terminus of each polypeptide chain. The needle contains a hollow hydrophobic lumen that runs through the barrel thus allowing for the movement of lipid molecules across the periplasm.  The YebT MCE protein also forms a periplasmic bridge with a unique structure. YebT contains seven MCE domains that stack on top of each other as hexameric rings forming an elongated tube,  like both MlaD and PqiB, YebT is anchored in the IM by N-terminal trans membrane (TM) helices.  The elongated tube also contains a hydrophobic lumen which allow the transport of hydrophobic substrates. Both YebT and PqiB are greater than 200 Å in length which is approximately the width of the periplasm. Ekiert et al., (2017) propose that YebT and PqiB provide a continuous pathway for lipid translocation across membranes without the need for a soluble periplasmic binding protein in a manner similar to that of the current LPS model.

Figure 1. Model for MCE-Mediated Phospholipid Transport across the Periplasm in E. coli
Figure 1. Model for MCE-Mediated Phospholipid Transport across the Periplasm in E. coli. Left, model for the Mla lipid transport system. MlaC likely serves as a soluble lipid carrier protein to transport lipids between the OM MlaA-OmpC/OmpF complex and the IM MlaFEDB complex. Middle left, PqiB may interact with OM lipoprotein PqiC and IM protein PqiA to mediate periplasmic lipid transport. Middle right, YebT may associate with YebS in the IM as part of a larger transport complex. Right, model of LPS transport system based on other studies (Okuda et al., 2016), which has also been suggested to form a bridge for LPS transport across the periplasm using a structurally unrelated framework.

Ekiert and colleagues’ present a neat structural argument for role of MCE domains in generating diverse architectures that are capable of spanning the periplasm and may function to traffic lipid across membranes. However the authors do state that there is still “a great deal that we don’t understand about the components of MCE transporters and how they function together”. This leads to several exciting open questions like do these systems function together or alone and are all three needed in E. coli? What is the directionality of lipid transport? What are the substrates for these systems? How phospholipids are transported and inserted in the outer membrane could be essential to understand how assembly and asymmetry is maintained in the OM.



Source: Ekiert et al., (2017) Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 169, 273–285

Using a periplasmic binding protein as a biosensor for thiamine

By Sophie Rugg

Twitter: @sophiejrugg

Thiamine, also known as vitamin B1, is an essential micronutrient with an important role in metabolism for all life forms. Thiamine can’t be synthesised by animals, and so has to be obtained from their diet. Until recently, detecting thiamine was limited to either the use of expensive high-performance liquid chromatography (HPLC), or a slow assay involving microbial growth. This is because there is no antibody available that is specific for thiamine, making commonly used high throughput detection techniques such as enzyme-linked immunosorbent assay (ELISA) impossible.

Periplasmic binding proteins are components of the ABC transporters of Gram-negative bacteria, and bind their substrates with high affinity and specificity to enable them to be transported into the bacterial cell. These properties of high affinity binding and specificity make periplasmic binding proteins ideal for use as the recognition element in a biosensor. As bacterial ABC transporters are used for the import of nutrients, including a transporter for thiamine, many of these binding proteins have evolved to recognise small molecules which it may be difficult to raise an antibody against.

Edwards et al., (2016) developed a biosensor for thiamine based on the periplasmic binding protein for thiamine from Escherichia coli. This binding protein was incorporated into dye-encapsulating liposomes in order to amplify the signal. Immobilised to the surface of a streptavidin coated plate is biotin conjugated thiamine analogue. The thiamine analogue is connected to the biotin via a long polyethylene glycol (PEG)linker, so that the thiamine analogue doesn’t get in the way of the biotin binding the streptavidin coating. The immobilised thiamine analogue binds to the periplasmic binding protein with lower affinity than thiamine. After the thiamine containing sample has been added, any material not bound to the surface is removed. Any liposomes still stuck to the surface of the plate are lysed and the resulting dye concentration is inversely proportional to the thiamine concentration.

Overview of assay for thiamine detection taken from Edwards et al., (2016). Competitive assay with biotin conjugated thiamine analogue immobilized via streptavidin in microtiter plates and detected via periplasmic binding protein for thiamine conjugated to the lipid bilayer of dye encapsulating liposomes (left). After competition with sample thiamine, unbound materials are removed (middle) and liposomes remaining bound are lysed to release dye yielding a signal inversely proportional to thiamine concentration (right).

This work shows that periplasmic binding proteins can be used effectively in biosensors , particularly where there is no antibody available. With the wide range of periplasmic binding proteins evolved by bacteria to be able to transport nutrients into their cells, this technique is open to use across a wide range of applications provided that a suitable analogue of the substrate can be immobilised to the surface of a plate.

Source: Edwards et al., 2016. High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition. Analytical  Chemisty88 (16), pp 8248–8256. DOI: 10.1021/acs.analchem.6b02092