**5. Gram-negative lipopolysaccharide (LPS): Structure and function**

LPS is essential in the structure and function of the external membrane of gram-negative bacterial cell walls. LPS intervenes in the transportation of hydrophobic molecules to the interior of bacterial cells and are an essential factor in host-microorganism interactions.

LPS is an amphipathic glycoconjugate that constitutes 10% to 15% of the total molecules in the external membrane and represents 75% of the total of bacterial surface [1]. There are three different LPS domains: a) Lipid A, which is the domain that is anchored to the membrane and the hydrophobic and endotoxic portions of the structure; b) The core oligosaccharide, which is the domain that connects lipid A to antigen O and is divided into the inner core and the outer core. The inner core is joined to lipid A and consists of unusual monosaccharides, including 2-keto-3-deoxy-octanoate (Kdo) and L-glycero-D-mannoheptose.

The outer core is joined to the O antigen and is made of common sugars such as hexoses and hexosamines [2]; and c) The O polysaccharide, which is the hydrophilic and immunodominant domain of LPS and is an oligosaccharide of repeated units that is projected from the core toward the exterior of the bacterial surface.

The O antigen has a polysaccharide chain that varies in length with up to 40 repeated units of dideoxyhexoses. At least 20 different sugar molecules may compose the O antigen, including molecules that are rarely found in nature, such as abequose, colitose, paratose and tyvelose. These components are strain-specific. The O antigen displays a large degree of inter-species and intra-species variation, which is related to the nature, order and union of the different sugars (**Figure 1**) [3].

**Figure 1.** Schematic representation of lipopolysaccharide structure. Smooth-type (left), Semi-Rough type with only one O-chain subunit (center) and rough-type (right).

The O antigen is the immunodominant part of LPS and therefore is the easiest target for the humoral response of the host. For this reason, the O antigen is the basis for the serological classification of gram-negative bacteria. The O antigen is recognised by the innate immune response and participates in complement activation and in the inhibition of the formulation of the complex that attacks the membrane [6,7].

## **6. Biosynthesis of LPS**

LPS is the primary component in the surface of gram-negative bacteria. The synthesis of LPS structures, which consist of lipid A, the core and antigen O, begins in the cytoplasm, where these structures are assembled. The structures are translocated to compartments such as the periplasm until the final destination is reached, which is the surface of the external membrane. The synthesis process has been widely studied in *E. coli* and *Salmonella.* The biosynthesis and exportation pathways of LPS are common among the majority of gramnegative bacteria, as is explained below (**Figure 2**). However, unique characteristics may exist in certain bacteria with respect to the types of enzymes and particular pathways.

The formation of lipid A is carried out in the internal face of the cytoplasmic membrane, and nine enzymes participate: LpxA, LpxC, LpxD, LpxH, LpxB, LpxK, KtdA, LpxL and LpxM. The biosynthesis of LPS begins with the formation of uridine diphosphate-diacyl- *D*glucosamine (UDP-diacyl-GlcN) from uridine diphosphate *N*-acetylglucosamine (UDP-GlcNAc). This reaction is catalysed by the enzymes LpxA, LpxC and LpxD and results in two 3-OH fatty acid chains in the 2 and 3 position of UDP-glucosamine (UDP-GlcNAc) to form UDP-2,3-diacyl- glucosamine (UDP-diacyl-GlcN). Subsequently, this molecule is hydrolysed by LpxH to form lipid X, the enzyme LpxB condenses the lipid X and its precursor UDP-diacyl-GlcN to form disaccharide-1-P and the enzyme LpxK phosphorylates this molecule at the 4 position of the disaccharide-1-P molecule to form the lipid IVA. Subsequently, KdtA incorporates the waste from 3-deoxy-D-manno-octulosonic acid (Kdo) in the 6' position of lipid IVA using the nucleotide cytidine monophosphate-3-deoxy-Dmanno-octulosonic acid (CMP-Kdo) as a donor to produce Kdo2 – lipid IVA, which is exposed to more reactions catalysed by LpxL and LpxM to form Kdo2 – lipid A. The enzyme LpxL adds a second lauryl, and LpxM adds a residue of myristoyl to the distal glucosamine unit.

Notably, the acyltransferases – LpxA, LpxD, LpxL and LpxM – selectively catalyse the different substrates and employ different acyl donors. For the first steps of the synthesis pathway of lipid A, the enzymes LpxA, LpxB and LpxD are required, with 3R-hydroxyacyl - Acyl Carrier Protein (3R-hydroxyacyl-ACP) serving as a donor. This compound is dehydrated by FabZ to form trans-2-acyl-ACP, which is also used as a donor of fatty acids in the biosynthesis of phospholipids. The synthesis of other LPSs in bacteria, such as *Neisseria meningitidis*, also involve trans-2-acyl-ACP[8]. Importantly, the structure of lipid A is the most conserved compared to the structure of the core oligosaccharides and antigen O [9,10].

## **6.1. The core oligosaccharides**

74 The Complex World of Polysaccharides

the different sugars (**Figure 1**) [3].

The outer core is joined to the O antigen and is made of common sugars such as hexoses and hexosamines [2]; and c) The O polysaccharide, which is the hydrophilic and immunodominant domain of LPS and is an oligosaccharide of repeated units that is

The O antigen has a polysaccharide chain that varies in length with up to 40 repeated units of dideoxyhexoses. At least 20 different sugar molecules may compose the O antigen, including molecules that are rarely found in nature, such as abequose, colitose, paratose and tyvelose. These components are strain-specific. The O antigen displays a large degree of inter-species and intra-species variation, which is related to the nature, order and union of

**Figure 1.** Schematic representation of lipopolysaccharide structure. Smooth-type (left), Semi-Rough

The O antigen is the immunodominant part of LPS and therefore is the easiest target for the humoral response of the host. For this reason, the O antigen is the basis for the serological classification of gram-negative bacteria. The O antigen is recognised by the innate immune response and participates in complement activation and in the inhibition of the formulation

LPS is the primary component in the surface of gram-negative bacteria. The synthesis of LPS structures, which consist of lipid A, the core and antigen O, begins in the cytoplasm, where

type with only one O-chain subunit (center) and rough-type (right).

of the complex that attacks the membrane [6,7].

**6. Biosynthesis of LPS** 

projected from the core toward the exterior of the bacterial surface.

The assembly of lipid A from the core oligosaccharides (Kdo2 – lipid A) is the next step in the synthesis of LPS. This step is performed on the cytoplasmic surface of the internal membrane by glycosyltransferases, which are associated with the membrane and with nucleotide sugars as donors.

The core oligosaccharides normally contains 10 to 15 monosaccharides and may be divided into two structural regions, which are the inner core and the outer core, which are ultimately connected to lipid A and antigen O, respectively, in the final structure of LPS. The inner core contains residues of Kdo and Hep (L-glycero-D-manno-heptose). Kdo is the most conserved component in the nuclear region of the LPS. In contrast, the outer core is more variable,

depend on the strain. However, the vertebral column of the oligosaccharide is typically composed of six units, and upon joining with other units, the column forms structures. The sugars commonly found in the core oligosaccharides are D-glucose, D-galactose, Kdo and Hep [9,10].

## **6.2. The O antigen**

The majority of O antigens are heteropolymers, although a portion of O antigens may be composed of a single monosaccharide. The synthesis of the O antigen is performed in the same location as the core oligosaccharides, and this synthesis also uses nucleotide sugars as donors. In the majority of bacteria, a cluster of genes known as *rfb* codes the enzymes necessary for 1) the biosynthesis of the nucleotide sugars of antigen O, 2) the transfer of the sugars to form the polysaccharide chain (glycosyltransferases) and 3) the assembly and transfer of antigen O toward the periplasm. The synthesis routes of the nucleotide sugars are grouped according to the nucleotide that bonds to the sugar, which may be CDP, UDP, dTDP or GDP. Antigen O may be a homopolymer or a heteropolymers, and the sugars may be formed linearly or in a ramification. Glycosyltransferases may be grouped according to their function, and they carry undecaprenyl phosphate, which is also used for the synthesis of capsular polysaccharides and peptidoglycans.

The following hypotheses have been proposed regarding the assembly and transfer process of antigen O: a) a pathway dependent on Wzy, which is the prototype system; b) a pathway dependent on ABC transporters, which are typically used by linear polysaccharide structures; c) a pathway dependent on synthase, which involves glycosyltransferases capable of synthesising within a single polypeptide and is an uncommon pathway and finally d) seroconversion reactions, in which the addition of acetyl residues or glucose residues modifies antigen O. Within the prototype pathway dependent on Wzy, in bacteria such as *Salmonella enterica* and *E. coli*, a multi-step process occurs. When the lipid A-core and the O antigen are synthesised, they are transported to the periplasm. The protein MsbA, which displays homology with MDR (multi-drug resistant) eukaryote proteins, transports the lipid A-core, and Wzx transports the O antigen, which was previously polymerised by the proteins Wzy and Wzz. With the help of WaaL, the structures of the lipid A-core and the O antigen are assembled, finally producing the LPS [10].

### **6.3. LPS and its transportation to the external membrane**

When an LPS is formed, it must pass through the periplasmic space to reach the external membrane [9,10], and this process is facilitated by protein LptA (periplasmic), LptB (cytosolic), LptC, LptF, LptG (internal membrane) and LptD and LptE (external membrane). Several of these proteins act in complexes. For example, in the case of the transporter ABC, LptBFG and LptA and LptC translocate the LPS to the internal side of the external membrane such that the proteins LptD and LptE place it on the surface of the membrane. It has been observed an absence of LptA or LptB or both causes the accumulation of LPS in the periplasm [11–17].

In the majority of bacteria, the genes that code for the enzymes involved in the biosynthesis of the O antigen are found in clusters. However, in the case of *Helicobacter pylori*, which is a pathogenic bacteria of the human stomach, these enzymes are found distributed throughout its chromosome, which probably contributes to the fact that the assembly pathway of its LPS has not been completely characterised. However, several enzymes that participate in the synthesis of *H. pylori* LPS has been identified and characterised, including several glycosyltransferases [18,19]. The glycosyltransferase WecA and the ligase WaaL also participate in the biosynthesis of *H. pylori* LPS. However, translocases are typically not involved in the translocation of the O antigen to the periplasm for its assembly with the lipid A-core structure [20]. Only the participation of a translocase named Wzk, which directs N-glycosylation in other bacteria, has been observed, and this fact suggests that the translocase Wzk of *H. pylori* could indicate an evolutionary connection between the biosynthesis pathways of LPS and glycoproteins [18]. Recently, it has been observed that there is an analogy and homology between the biosynthesis of LPS and the biosynthesis of glycoproteins in other bacteria, such as *S. enterica, P. aeruginosa* PAO1, *Neisseria* spp.*, Paenibacillus alvei, Campylobacter jejuni and E. coli O8.* This homology could have enormous biotechnological potential. However, further studies are required to confirm this fact [21].

76 The Complex World of Polysaccharides

Hep [9,10].

**6.2. The O antigen** 

periplasm [11–17].

of capsular polysaccharides and peptidoglycans.

O antigen are assembled, finally producing the LPS [10].

**6.3. LPS and its transportation to the external membrane** 

depend on the strain. However, the vertebral column of the oligosaccharide is typically composed of six units, and upon joining with other units, the column forms structures. The sugars commonly found in the core oligosaccharides are D-glucose, D-galactose, Kdo and

The majority of O antigens are heteropolymers, although a portion of O antigens may be composed of a single monosaccharide. The synthesis of the O antigen is performed in the same location as the core oligosaccharides, and this synthesis also uses nucleotide sugars as donors. In the majority of bacteria, a cluster of genes known as *rfb* codes the enzymes necessary for 1) the biosynthesis of the nucleotide sugars of antigen O, 2) the transfer of the sugars to form the polysaccharide chain (glycosyltransferases) and 3) the assembly and transfer of antigen O toward the periplasm. The synthesis routes of the nucleotide sugars are grouped according to the nucleotide that bonds to the sugar, which may be CDP, UDP, dTDP or GDP. Antigen O may be a homopolymer or a heteropolymers, and the sugars may be formed linearly or in a ramification. Glycosyltransferases may be grouped according to their function, and they carry undecaprenyl phosphate, which is also used for the synthesis

The following hypotheses have been proposed regarding the assembly and transfer process of antigen O: a) a pathway dependent on Wzy, which is the prototype system; b) a pathway dependent on ABC transporters, which are typically used by linear polysaccharide structures; c) a pathway dependent on synthase, which involves glycosyltransferases capable of synthesising within a single polypeptide and is an uncommon pathway and finally d) seroconversion reactions, in which the addition of acetyl residues or glucose residues modifies antigen O. Within the prototype pathway dependent on Wzy, in bacteria such as *Salmonella enterica* and *E. coli*, a multi-step process occurs. When the lipid A-core and the O antigen are synthesised, they are transported to the periplasm. The protein MsbA, which displays homology with MDR (multi-drug resistant) eukaryote proteins, transports the lipid A-core, and Wzx transports the O antigen, which was previously polymerised by the proteins Wzy and Wzz. With the help of WaaL, the structures of the lipid A-core and the

When an LPS is formed, it must pass through the periplasmic space to reach the external membrane [9,10], and this process is facilitated by protein LptA (periplasmic), LptB (cytosolic), LptC, LptF, LptG (internal membrane) and LptD and LptE (external membrane). Several of these proteins act in complexes. For example, in the case of the transporter ABC, LptBFG and LptA and LptC translocate the LPS to the internal side of the external membrane such that the proteins LptD and LptE place it on the surface of the membrane. It has been observed an absence of LptA or LptB or both causes the accumulation of LPS in the

**Figure 2.** The biosynthetic pathway and transport of lipopolysaccharide. **A.** A representation of the biosynthetic pathway of the structures of LPS in *E. coli*. **B.** The assembly and transport of the LPS; the antigen O is assembled to the structure core-lipid A in the periplasm and is later transported toward the outer membrane. The names of the enzymes involved in these processes are: 1. LpxA, LpxC, LpxD; 2. LpxH; 3. LpxB; 4. LpxK, KdtA, LpxL, LpxM; 5. Glycosyltransferases; 6 and 10. WaaL; 7. Wzx; 8. Wzy, Wzz; 9. MsbA; 11. Lpt B C F G; 12. LptA; 13. Lpt D E. The lipid A (Yellow circles), the core (Red ovals) and antigen O (Blue ovals)
