Structure of the essential inner membrane lipopolysaccharide-PbgA complex

The following content is an introduction to the 2020 paper published in Nature, “Structure of the essential inner membrane lipopolysaccharide-PbgA complex” by Clairfeuille et al. The paper discusses the role of PbgA in the LPS biosynthesis regulation and has key implications for novel antibiotic research in the future.

For survival, bacteria have a cell envelope surrounding the cytoplasm that gives the cell its shape, selectively allows the passage of molecules into and out of the cytoplasm, and protects the cell (1). The bacteria fall into two groups, depending on their cell envelope: Gram-negative bacteria and Gram-positive bacteria. E. coli is an example of a Gram-negative bacteria (1).

E. coli’s cell envelope, shown in Figure 1, consists of an inner membrane (IM), a peptidoglycan cell wall, and an outer membrane (OM) which is unique to Gram-negative bacteria (1). The IM is a phospholipid (PL) bilayer (2). Proteins responsible for energy production, lipid biosynthesis, protein secretion, and transport are located in the IM due to a lack of intracellular organelles (1,2). The peptidoglycan cell wall, found in the periplasmic space between the IM and OM, is made up of repeating units of the disaccharide N-acetyl glucosamine-N-acetyl muramic acid (NAG-NAM), cross-linked by pentapeptide side chains (1,3). This rigid cell wall is responsible for the maintenance of E. coli’s characteristic rod shape (1). The peptidoglycan layer is connected to the OM through a lipoprotein, murein/Braun’s lipoprotein (Lpp) (1). The OM is an asymmetric lipid bilayer that is essential for E.coli’s survival because it acts as the first line of defence against external threats (1,2). It prevents the entry or exit of large, hydrophobic molecules and works together with the peptidoglycan cell wall to provide mechanical strength to the bacterial cell, protecting it from osmotic lysis (4). The OM is made of PLs in the inner leaflet and lipopolysaccharide (LPS) glycolipid molecules in the outer leaflet (5). The OM also consists of OM proteins (Omps), exopolysaccharides (EPS), flagella and type I fimbria (6). EPS, flagella, and fimbria are nonessential structures; therefore, they are not present in all E. coli strains (6). There are three important Omps: OmpC, OmpF, and OmpA (6). OmpC and OmpF regulate the entry of small molecule solutes into the cytoplasm while OmpA maintains E. coli’s cell surface integrity (6).

Figure 1: The cell envelope of E. coli. The cell envelope is made of the inner membrane, peptidoglycan cell wall, and outer membrane. The inner membrane consists of a phospholipid (PL) bilayer. The peptidoglycan cell wall can be found in the periplasmic space between the IM and OM. It is made up of the NAG-NAM disaccharide, cross-linked by pentapeptide side chains. The peptidoglycan cell wall is connected to the OM via murein or Braun’s lipoprotein, Lpp (coloured dark blue). The OM consists of PLs in its inner leaflet, LPS molecules in its outer leaflet, and outer membrane proteins, Omps (coloured green). The outer membrane may also contain nonessential structures such as exopolysaccharides (EPS), flagella and type I fimbria.

Figure 2: An image of the structure of LPS. The structure has 3 primary regions, the O-Antigen, Core Oligosaccharide, and Lipid A.

LPS are glycolipids comprised of three primary regions. The first is the lipid A region, which is typically made up of a bis-phosphorylated glucosamine disaccharide that carries fatty acids in ester and amide linkages. This region is connected to the second core oligosaccharide region via 2-keto-3 deoxy-octulosonic acid (Kdo). The third region is the O-chain, consisting of repeating oligosaccharide units and differs from one bacterium to another.

LPS provides a permeability barrier that prevents the entry of harmful molecules. High density of saturated fatty acids cause broadly and strong interaction with the acyl chain, which provides hydrophobic character that inhibits the movement of hydrophilic molecules through the outer membrane, and it synthesizes a low fluidity of the membrane bilayer. Moreover, the core oligosaccharide and O-antigen part of LPS provide hydrophilic character (5). In addition, divalent cations between LPS molecules stabilize the high negativity of the membrane, which is caused by the presence of the phosphate group. Polyionic interaction within the outer membrane promotes LPS packing, and constructs LPS as a permeability barrier (7).

LPS also contributes to impacting the virulence of the bacteria cell. LPS (endotoxin) is more stable by comparison with bacterial exotoxins, and is a primary molecule found on the surface of bacterial pathogens. LPS is recognized by the host immune system as a pathogen-associated molecular pattern (PAMP), and the immune response could be so strong as to be toxic to the host cell. O-antigen plays an important role in the infection process by enabling attachment, colonization of the host, and the avoidance of host defense mechanisms. Some bacteria evade the host response by masking their LPS with various O-antigen(7). Although bacterial evasion could not prevent antibody production, the length of O-antigen chain prevents the complement at bacterial cell surface (5).

The LPS biosynthesis pathway is crucial for the structural makeup of gram-negative bacteria’s outer membrane (8). Reflected in the structure of LPS, its synthesis is dependent on the formation of its three regions: Lipid A, the core oligosaccharide, and the O-antigen. LPS synthesis occurs within the cytoplasm along the inner membrane surface.

The LPS biosynthesis starts with the conserved pathway of lipid A synthesis as displayed in Figure 3 (9). This pathway begins with a UDP-N-acetylglucosamine (UDP-GlcNAc) molecule (8). Lipid A synthesis involves the addition of hydrophobic fatty acid chains to UDP-GlcNAc catalyzed by LpxA, LpxC and LpxD, forming UDP-diacyl-GlcN. Although the biosynthesis begins with the acylation of UDP-GlcNAc, catalyzed by LpxA, this reaction is thermodynamically unfavourable (10). Thus, the first committed step of lipid A biosynthesis is the deacetylation reaction catalyzed by LpxC. LpxC is a crucial enzyme as it catalyzes the non-reversible step in lipid A synthesis which is the deacetylation of UDP-3-O-(acyl)-GlcNAc. LpxC also has a unique sequence compared to other deacetylases and plays a regulatory role in lipid A biosynthesis, which makes it an attractive target for antibiotic development (8). Following LpxD action, UDP-diacyl-GlcN undergoes a series of reactions catalyzed by three other enzymes, LpxH, LpxB and LpxK, to compose lipid IVA. Two Kdo molecules are added to Lipid IVA, catalyzed by the bifunctional KdtA. Kdo2-LipidIVA is further modified with acyltransferases, LpxL and LpxM, to form Kdo2-Lipid A. Kdo2-Lipid A is the active form that is used for the addition of core oligosaccharides and overall assembly of LPS.

Figure 3: Image from William and Raetz displaying Kdo2-Lipid A synthesis, the first part of LPS synthesis. Kdo2-Lipid A synthesis involves the first committed step in LPS synthesis, catalyzed by LpxC, which is the deacetylation of UDP-3-O-(acyl)-GlcNAc (red box) (11).

The core oligosaccharides are added to lipid A on the cytoplasmic surface of the inner membrane through membrane-bound glycosyltransferases and nucleotide sugar donors (8). The core oligosaccharides have two components: the inner core and the outer core. The inner core is the conserved region of the core oligosaccharides and includes Kdo molecules as well as the L-glycero-D-manno-heptose molecule (Hep) (9). The formation and attachment of Hep are mediated by enzymes synthesized by the gmhD operon. On the other hand, the outer core region is less conserved. The outer core oligosaccharides are synthesized by gene products of the waaQ operons.

The O-antigen polymers are added to the outer core oligosaccharides through glycosyltransferases and nucleotide sugar donors (8). The rfb gene cluster enzyme derivatives contribute to O-antigen diversity through the creation of enzymes for varying sugar-nucleotide precursors. The rfb operon also synthesizes glycosyltransferases, polymerases, and proteins needed for O-antigen transport through the inner membrane.

LPS transport begins with the movement of LPS from the inner membrane to the outer membrane and involves MsbA translocation (12). MsbA flippase catalyzes the flipping of the Lipid A core moiety across the inner membrane. Following complete synthesis, the movement of the mature LPS molecule to the cell surface is assisted by the LPT molecular machine. Broadly, the transport of LPS from the inner membrane to the outer membrane can be divided into three key steps: LPS detachment from the inner membrane, LPS transport across the periplasm, and LPS insertion and assembly in the outer membrane at the cell surface (12).

LPS assembly begins on the internal surface of the E.coli membrane. The rate of LPS assembly is controlled by LpxC. Prior to the completion of LPS biosynthesis, the lipid undergoes further modifications when it is flipped to the external surface of the inner membrane. Following synthesis completion, LPS is transported to the outer membrane’s external surface via a protein bridge that connects both the inner and outer membranes (2).

Feedback inhibition is a key cellular control mechanism where the activity of a key enzyme within a pathway is inhibited by that same enzyme's end product(s). This control mechanism is essential in controlling and regulating LPS biosynthesis. It is currently unknown but it has been suspected that LPS or a precursor of LPS is the feedback signal responsible for LPS regulation (2).

Figure 4: Regulatory pathway of LPS biogenesis.

There are 3 scenarios involving LPS biosynthesis: typical LPS biosynthesis, LPS excess, and LPS deficiency.

1. Beginning with normal LPS synthesis that takes place within the cell cytoplasm, the enzyme LpxC controls the biosynthesis of LPS while utilizing precursors located in the cytoplasm . Following biosynthesis, the immature LPS is flipped onto the external surface of the inner membrane and is then transported to the outer membrane. The FtsH enzyme, guided by interactions with LapB, degrades LpxC which disrupts LPS biosynthesis (2). However, Clairefeuille and colleagues show that a protein, PbgA, inhibits LapB-FtsH activity to promote LPS biosynthesis. LPS synthesis and degradation of LpxC.

2. When LPS is being synthesized in excessive amounts, it will accumulate on the external surface of the inner membrane and bind to PbgA (2). Thereby, PbgA will lessen its control on the LapB-FtsH complex activity, allowing for the degradation of LpxC to restore normal LPS levels. When LPS is being synthesized in excess, it will begin to accumulate on the external surface of the inner membrane and bind to PbgA. Bound to LPS, the protein will relax its inhibitory control on FtsH-LapB to promote LpxC degradation and therefore, restores normal LPS levels.

3. There is a truncation mutation of PbgA that leads to the depletion of LPS. This is most likely because the mutant fails to strongly inhibit the LapB-FtsH interaction that degrades LpxC and thereby, promotes LpxC degradation (2). PLs then fill in the gaps that are left by the LPS in the outer membrane, enabling greasy antibiotics and detergents to penetrate local PL bilayers, and large soluble compounds to leak through transient boundary defects where LPS and the PL phases meet (2).

Figure 5: Pymol structure of PbgA. Crystal structure of PbgA, an essential inner transmembrane protein in E. coli that is used for regulating LPS synthesis and outer membrane homeostasis. The C-terminal periplasmic domain is depicted in green. The N-terminal domain is a five-transmembrane domain depicted in red, yellow, orange, purple, and cyan.

PbgA, also known as YejM, is an essential protein in E. coli that is required for regulating LPS synthesis and maintaining membrane homeostasis (13).

As shown in Figure 5, PbgA is an inner membrane protein with a five-transmembrane-domain N terminus (residues 1-190) that is essential for growth and a nonessential C-terminal periplasmic domain (residues 191-586). Nonsense mutations that cause truncations in the periplasmic domain in PbgA cause phenotypes consistent with defects in outer membrane assembly, including reduced LPS/PL ratio, vancomycin sensitivity, temperature sensitivity, and leakage of periplasmic proteins (3).

PbgA is structurally related to LtaS, an enzyme found in many gram-positive bacteria that synthesizes lipoteichoic acids. PbgA, like LtaS, contains a hydrophobic binding pocket in its periplasmic domain that is required for protein activity. However, the crystal structure of the PbgA domain indicates that it lacks residues required for LtaS catalytic activity, so it is unlikely to have a homologous enzymatic function (3).

In S. Typhimurium, cells that use a two-component regulatory system, PhoPQ, require PbgA to coordinate this process. The PhoPQ system induces changes to the outer membrane to protect cells from infection by sensing changes in the environment. One of the changes that occurs is an increase in the content of the PL, cardiolipin (CL). PbgA was found to bind to CL in vitro and since the deletion of PbgA showed no increase in CL content, it led to the assumption that it acts as a transporter that brings CL from the inner membrane to the outer membrane (13).

However, other studies have shown that PbgA may not act as a cardiolipin transporter as it lacks an outer membrane partner while other complexes that transport substrates from the inner membrane to the outer membrane have inner membrane, periplasmic, and outer membrane components. Additionally, if PbgA were essential for cardiolipin synthesis, then it would be expected that cardiolipin deficiency would lead to toxic levels of PbgA, but this does not occur. Finally, because PbgA is known to have an impact on outer membrane permeability, but truncations of PbgA periplasmic domain show outer membrane permeability defects, indicating that both the inner membrane and periplasmic domain of PbgA are involved in the same function. Therefore, it is unlikely that PbgA is involved in cardiolipin transport (13).

A recent study suggests that PbgA serves a larger but undefined role in envelope assembly, such as preventing excessive turnover of LpxC and promoting LpxC accumulation by shielding it from the FtsH-LapB proteolytic system. They also found that the N-terminal transmembrane of PbgA alone interacts with the LapB component of the FtsH-LapB proteolytic system to promote LpxC accumulation (3).

Polymyxins are an important class of antibiotics used in the treatment of systemic infections caused by multidrug-resistant gram-negative bacteria such as pseudomonas aeruginosa (14). Currently, these antibiotics are used as a last line of treatment against such infections (14). The main drugs in clinical use within this antibiotic class are Polymyxin B and Polymyxin E (also called colistin), which target infections of the urinary tract, meninges, and bloodstream (14).

Polymyxins target the lipid A core of LPS (15). These antibiotics destabilize the PLs and LPS present in the outer membrane of gram-negative bacteria (14). Since polymyxins are positively charged, they electrostatically interact with the phosphate groups on both of the negatively charged phosphorylated sugars that make up lipid A (14). This causes the divalent cations (such as calcium and magnesium) from the phosphate groups within the membrane lipids to become displaced, creating increased permeability (14). This leads to the outer membrane becoming disrupted, allowing small molecules and other intracellular contents to leak out of the cell and cause bacterial cell death (14).

In addition, polymyxins can neutralize the endotoxin effect of pathogens (14). Since the endotoxic part of gram-negative bacteria corresponds to the lipid A core, polymyxins can bind to the LPS that was released as a result of cellular death (14). This results in the neutralization of the endotoxin, preventing its effects in circulation (14).

Figure 6: Colistin electrostatically interacts with the lipid A core of LPS, creating a disruption of the membrane and allowing small molecules to leak out of the cell.

Figure 7: The enzyme EptA modifies LPS and reduces the negative charge of lipid A; therefore, prevents colistin from binding and makes bacteria resistant to the antibiotic.

Polymyxins are an extremely important and clinically relevant class of drugs since they are the last line of defence against gram-negative bacteria that are resistant to all other antibiotics (15). Unfortunately, there is an emergence of bacteria that are also resistant to polymyxins (15). One bacterial resistance mechanism that has been discovered is due to the expression of EptA, a protein part of the same family as PbgA (16). This protein has been shown to remodel the surface of the bacteria, making it resistant to colistin (16). EptA does this by modifying the phosphate groups on both of the phosphorylated sugars on lipid A; thereby, reducing its overall negative charge (16). This makes the bacteria resistant to polymyxins because they can no longer have the previously mentioned electrostatic interactions and thus cannot bind to the modified lipid A core (16). With polymyxin resistance becoming a bigger problem, more research on PbgA will be beneficial for future antibiotic discovery.

Antibiotic resistance is a growing concern today and for our future. Gram-negative bacteria have been particularly associated with antibiotic resistance due to the presence of LPS in their cell wall, acting as a protective barrier against drugs. The ability of these bacteria to rapidly evolve and develop resistance to antibiotics has made it an ongoing challenge for scientists to find new ways to combat these infections. Understanding the pathways in which LPS is synthesized and regulated demonstrates importance pertaining to antibiotic drug targeting. Clairfeullie et al. contribute to this research through characterizing PbgA and demonstrating how manipulation of this protein could possibly be utilized in the lowering of LPS levels and subsequently virulence by E. coli.

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