Introduction: '''

Gram-Negative Bacteria:'''

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 cell¹ (Silhavy, Kahne, & Walker, 2010). 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 bacteria¹ (Silhavy, Kahne, & Walker, 2010).

E. coli''’s cell envelope, shown in figure X, consists of an inner membrane (IM), a peptidoglycan cell wall, and an outer membrane (OM) which is unique to Gram-negative bacteria¹ (Silhavy, Kahne, & Walker, 2010). The IM is a phospholipid (PL) bilayer² (Bishop, 2020). 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 (Bishop, 2020; Silhavy, Kahne, & Walker, 2010). 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 (Fivenson, & Bernhardt, 2020; Silhavy, Kahne, & Walker, 2010). This rigid cell wall is responsible for the maintenance of E. coli''’s characteristic rod shape¹ (Silhavy, Kahne, & Walker, 2010). The peptidoglycan layer is connected to the OM through a lipoprotein, murein/Braun’s lipoprotein (Lpp)¹ (Silhavy, Kahne, & Walker, 2010). 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 (Bishop, 2020; Silhavy, Kahne, & Walker, 2010). 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 lysis⁴ (Guest et al., 2020). The OM is made of PLs in the inner leaflet and lipopolysaccharide (LPS) glycolipid molecules in the outer leaflet⁵ (Bertani & Ruiz, 2018). The OM also consists of OM proteins (Omps), exopolysaccharides (EPS), flagella and type I fimbria⁶ (Wang, Ma, & Wang, 2021). EPS, flagella, and fimbria are nonessential structures; therefore, they are not present in all E. coli'' strains⁶ (Wang, Ma, & Wang, 2021). There are three important Omps: OmpC, OmpF, and OmpA⁶ (Wang, Ma, & Wang, 2021). OmpC and OmpF regulate the entry of small molecule solutes into the cytoplasm while OmpA maintains E. coli''’s cell surface integrity⁶ (Wang, Ma, & Wang, 2021).

Figure X: 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 non essential structures such as exopolysaccharides (EPS), flagella and type I fimbria.

LPS Structure'''

https://www.ocl-journal.org/articles/ocl/full_html/2020/01/ocl200025s/ocl200025s.html

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 Function

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, and it synthesizes a low fluidity of the membrane bilayer. 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.

LPS also contributes to impacting the virulence of the bacteria cell. LPS is more stable by comparing with bacterial exotoxins, and is the primary

https://www.ncbi.nlm.nih.gov/books/NBK554414/#:~:text=The%20primary%20function%20of%20LPS,inhabitation%20in%20the%20gastrointestinal%20tract. ⁷ (Farhana)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091223/⁵

X.0 LPS synthesis'''

The LPS biosynthesis pathway is crucial for the structural makeup of gram-negative bacteria’s outer membrane⁸ (Wang & Quinn, 2010). 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.

X.1 Lipid A synthesis'''

The LPS biosynthesis starts with the conserved pathway of lipid A synthesis as displayed in Figure X '''⁹ (Whitfield & Trent, 2014). This pathway begins with a UDP-N-acetylglucosamine (UDP-GlcNAc) molecule⁸ (Wang & Quinn, 2010). 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 unfavourable¹⁰ (Barb & Zhou, 2008). 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 development⁸ (Wang & Quinn, 2010). Following LpxD action, UDP-diacyl-GlcN undergoes a series of reactions catalyzed by three other enzymes, LpxH, LpxB and LpxK, to compose lipid IV_A. Two Kdo molecules are added to Lipid IV_A, catalyzed by the bifunctional KdtA. Kdo₂-LipidIV_A 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.

X.2 Core Oligosaccharides synthesis/addition'''

The core oligosaccharides are added to lipid A on the cytoplasmic surface of the inner membrane through membrane-bound glycosyltransferases and nucleotide sugar donors⁸ (Wang & Quinn, 2010). 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 (Whitfield & Trent, 2014). 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.

X.3 O-antigen addition'''

The O-antigen polymers are added to the outer core oligosaccharides through glycosyltransferases and nucleotide sugar donors⁸ (Wang & Quinn, 2010). 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.

Figure X''': 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 (Williams & Raetz, 2007).

LPS transport '''

LPS transport begins with the movement of LPS from the inner membrane to the outer membrane and involves MsbA translocation (Sperandeo et al., 2017). MsbAflippase 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 (Sperandeo et al., 2017).

Regulation '''

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 membranes² (Bishop, 2020).

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 regulation² (Bishop, 2020).

There are 3 scenarios involving LPS biosynthesis that will be discussed: typical LPS biosynthesis, LPS excess, and LPS deficiency.

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 biosynthesis² (Bishop, 2020). However, Clairefeuille and colleagues show that a protein, PbgA, inhibits LapB-FtsH activity to promote LPS biosynthesis (2020).

Figure _. LPS synthesis and degradation of LpxC. The enzyme LpxC controls the biosynthesis of LPS, utilizing precursors located within the E.coli''''cell cytoplasm. After being flipped to the external surface of the inner membrane through an ABC transporter, the mature LPS is transported to the outer membrane using LPT machinery. The enzyme FtsH, aided by interactions with the protein LapB, degrades LpxC.

Figure _. Inhibition of FtsH-LapB activity. PbgA is a protein that inhibits the actions of FtsH-LapB to promote LPS biosynthesis.

Next, when LPS is being synthesized in excessive amounts, it will accumulate on the external surface of the inner membrane and bind to PbgA² (Bishop, 2020). Thereby, PbgA will lessen its control on the LapB-FtsH complex activity, allowing for the degradation of LpxC to restore normal LPS levels.

Figure _. LPS excess. 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.

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 degradation² (Bishop, 2020). 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 meet² (Bishop, 2020).

Figure_. PbgA truncation mutation leads to LPS depletion. A depletion of LPS occurs when there is a PbgA truncation mutation, most likely due to the mutant failing to inhibit FtsH-LapB strongly enough. Therefore, PLs will attempt to fill in the gaps left by the depletion of LPS in the outer membrane. This enables greasy antibiotics and detergents to penetrate as well as large soluble compounds to leak through.

https://journals.asm.org/doi/10.1128/ecosalplus.ESP-0001-2018

X: PbgA'''

X.1 PbgA Structure '''

PbgA, also known as YejM, is an essential protein in E. coli'' that is required for regulating LPS synthesis and maintaining membrane homeostasis¹² (Simpson et al., 2020).

As shown in Figure X, 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 proteins³ (Fivenson & Bernhardt, 2020).

Figure X: 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.

X.2 PbgA protein similarity '''

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 function³ (Fivenson & Bernhardt, 2020).

X.3 Initial Proposed Function of PbgA'''

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 membrane¹² (Simpson et al., 2020).

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 transport¹² (Simpson et al., 2020).

X.4 Novel Discovery of PbgA Function'''

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 accumulation³ (Fivenson & Bernhardt, 2020).

Polymyxin Antibiotics and Antibiotic Resistance:'''

X.1. Polymyxin Antibiotics'''

Polymyxins are an important class of antibiotics used in the treatment of systemic infections caused by multidrug-resistant gram-negative bacteria such as pseudomonas aeruginosa¹³ (Shatri & Tadi, 2022). Currently, these antibiotics are used as a last line of treatment against such infections¹³ (Shatri & Tadi, 2022). 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 bloodstream¹³ (Shatri & Tadi, 2022).

X.2. Mechanism of Action of Colistin'''

Polymyxins target the lipid A core of LPS¹⁴ (Clairfeuille et al., 2020). These antibiotics destabilize the PLs and LPS present in the outer membrane of gram-negative bacteria¹³ (Shatri & Tadi, 2022). Since polymyxins are positively charged, they electrostatically interact with the phosphate groups on both of the negatively charged phosphorylated sugars that make up lipid A¹³ (Shatri & Tadi, 2022). This causes the divalent cations (such as calcium and magnesium) from the phosphate groups within the membrane lipids to become displaced, creating increased permeability¹³ (Shatri & Tadi, 2022). 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 death¹³ (Shatri & Tadi, 2022).

In addition, polymyxins can neutralize the endotoxin effect of pathogens¹³ (Shatri & Tadi, 2022). 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 death¹³ (Shatri & Tadi, 2022). This results in the neutralization of the endotoxin, preventing its effects in circulation¹³ (Shatri & Tadi, 2022).

Figure X:''' 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.

X.3. Antibiotic Resistance'''

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 antibiotics¹⁴ (Clairfeuille et al., 2020). Unfortunately, there is an emergence of bacteria that are also resistant to polymyxins¹⁴ (Clairfeuille et al., 2020). One bacterial resistance mechanism that has been discovered is due to the expression of EptA, a protein part of the same family as PbgA¹⁵ (Xu et al., 2018). This protein has been shown to remodel the surface of the bacteria, making it resistant to colistin¹⁵ (Xu et al., 2018). EptA does this by modifying the phosphate groups on both of the phosphorylated sugars on lipid A; thereby, reducing its overall negative charge¹⁵ (Xu et al., 2018). 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 core¹⁵ (Xu et al., 2018). With polymyxin resistance becoming a bigger problem, more research on PbgA will be beneficial for future antibiotic discovery.

Figure X: '''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.

'''

Conclusion:'''

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''.