**9. The importance of the variability of the O antigen of LPS**

The modifications that are present in the O antigen and that cause its variability play an important role in infections by gram-negative bacteria, given that the modifications may influence adherence, colonisation and the ability to evade the host's defence mechanisms.

## **9.1. The role of the variation of LPS in the immune response.**

LPS activates not only the innate immune response but also the adaptive response. The first contact that LPS has with the immune system is with lipid A, which is recognised by the receptors involved in the innate immune response, while the structure of the O antigen participates in the adaptive response (synthesis of antibodies). LPS is a potent stimulator of the cells of the immune system, given that it induces the production of pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β) and acute phase proteins.

Although the variability is greater in the O antigen within LPS, there is also variability within lipid A. This variability is due to the length of the chains and the saturation of its acyl groups. Because these groups are strong immunostimulants, both the changes in the number of their chains and the presence of phosphorylations within the structure of the lipid A may influence its biological activity [61].

90 The Complex World of Polysaccharides

**Figure 10.** Model of the phase variation of the regulatory region of the *gtr* operon. Illustration of the interaction of DNA proteins in the regulatory region of the *grt* P22 operon, which consists of methylation and demethylation in the GATC sequence in the activated and deactivated phases. Adapted from [60].

Understanding the variation of the LPS structure is important because the composition and the length of the O antigen chain may be an indicator of the virulence, and this characteristic

The modifications that are present in the O antigen and that cause its variability play an important role in infections by gram-negative bacteria, given that the modifications may influence adherence, colonisation and the ability to evade the host's defence mechanisms.

LPS activates not only the innate immune response but also the adaptive response. The first contact that LPS has with the immune system is with lipid A, which is recognised by the receptors involved in the innate immune response, while the structure of the O antigen participates in the adaptive response (synthesis of antibodies). LPS is a potent stimulator of the cells of the immune system, given that it induces the production of pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-

**9. The importance of the variability of the O antigen of LPS** 

**9.1. The role of the variation of LPS in the immune response.** 

often differs within a single bacterial strain [7].

1β) and acute phase proteins.

Lipid A is the structure of LPS that is recognised by the TLR4 receptors, which are part of the so-called Toll Like Receptors (TLRs), which are expressed by cells of the innate immune system and are stimulated by pathogen-associated molecular patterns (PAMPs). The stimulation of the LPS in certain cells of the monocyte-macrophage lineage, lymphoid cells and even cells that are not part of the immune system, such as epithelial cells, endothelial cells and vascular cells, occurs with the participation of other molecules, such as LPS binding protein (LBP), CD14 and MD-2. The transduction signals of TLR4 are divided into MyD88-dependent and MyD88-independent (also called TRI-dependent) groups. These signals may be regulated at various levels. For example, the RP105 and SIGIRR (Single immunoglobulin IL-IR related molecule) molecules inhibit the start of the signalling cascade [62].

Recognition through TLR4 is crucial for the control of infection, but changes in the signalling pathways may cause sepsis or evasion of the pathogen. The importance of signalling via TLR4-MD2 in response to gram-negative pathogens make this pathway an alternative to search for therapeutic targets not only for infectious diseases but also for other diseases with inflammatory aetiology, such as cancer, atherosclerosis, asthma and autoimmune conditions.

Antagonists of TLR4-MD2 have been identified, and several of these are based on the lipid A structures and other inhibitor molecules [63–65]. The intention is to use this type of antagonist therapy to treat septic shock. Additionally, several TLR4 antagonists primarily those that activate the TRAF (TNF receptor- associated factor) or TRIM (Tripartite motif) pathways have been proposed as adjuvants.

However, certain pathogens have the ability to modify the structure of lipid A and its detection by the host. For example, some isolates of *P. aeruginosa* are capable of modifying the structure of lipid A into a penta-acylated moiety, which does not activate the TLR4 and allows the immune response to be evaded. Other isolates of *P. aeruginosa* colonise respiratory pathways of patients with CF during its adaptation, producing hexa-acylated structures that are highly pro-inflammatory [61].

The large variability that the O antigen displays allows for the existence of various clones within a single species, which offers a selective advantage in the niche occupied by this clone and is precisely the interaction between the O antigen and the immune system that permits this advantage.

Many pathogens have the capability of varying the antigens that are attached to their surface and therefore can vary their antigenic composition. This variation is typically

mediated by the regulation of the expression of genes. By varying their antigenicity, the pathogens have a greater ability to evade the immune response of the host, and this variability makes it more difficult to design vaccines for these pathogens [66].

The O antigen is considered to be highly immunogenic and induces the production of antibodies that may activate the complement pathway, either through the classic pathway or an alternate pathway, which leads to cellular death or phagocytosis. Certain modifications in the oligosaccharide chain of the O antigen may alter the interaction of the complement pathway. Several O antigens of pathogens are similar to host molecules and facilitate invasion through mimicking in the host; for example, O antigens of the LPS of *H. influenzae*  and of *N. gonorrhoeae* mimic epitopes of glycosphingolipids [67].

The mimicking property may also serve to evade the immune system, as is the case of *H. pylori.* The chains of the O antigen that contain the surface of the LPS of *H. pylori* express Lewis antigens, mainly Lex and Ley, although some isolates may contain other antigens, such as Lea, Leb, Lec, Sialyl-Lex and H-1, in addition to type A and B blood groups [68].

The expression of Lewis antigens and their fucosylation have biological effects in the pathogenesis of this bacterium. The O antigen of *H. pylori* exhibits molecular mimicking with the Lewis antigens of the host within the gastric epithelium. The expression of the Lewis antigens is subject to phase variation, given that the regulation of the glycosyltransferase genes is regulated by SSM, which in the O antigen structure promotes variations among the strains. The antigenic mimicking is essentially involved in the evasion of the immune system and gastric adaptation. Several studies show that mimicking also plays a role in the colonisation and adhesion of Lex of the bacteria with the galectin-3 of a gastric receptor.

Moreover, *H. pylori* is capable of evading the binding effect of surfactant protein D, which is expressed in the gastric mucosa and is a component of the innate immune response [69]. This microorganism impedes the bonding of the surfactant protein through the variation of its LPS. This phenomenon is associated with changes in the fucosylation of the O antigen chain. In addition, the expression of Lewis antigens affects both the inflammatory response and the polarisation of the T cells that are triggered after an infection. Because it is a chronic pathogen, several studies have shown that *H. pylori* may induce anti-Lewis auto-reactive antibodies, which enable the gastric mucosa to be recognised and contribute to the development of gastric atrophy [70].

### **9.2. Changes in the LPS related to resistance**

The hydrophobic antibiotics that reach the interior of the cells due to the permeability of the external membrane are aminoglycosides, macrolides, rifamycins, novobiocin, fusidic acid and cationic peptides. The tetracycline and the quinolones use pathways that are mediated by lipids and porins. The central region of the LPS is important because it provides a barrier against hydrophobic antibiotics and other components; isolates that express a long LPS have intrinsic resistance to these factors [71].

The polymyxins, which include polymyxin B and colistin (polymyxin E), belong to a group of natural antimicrobials that are found in eukaryotic cells; this group is known as the cationic antimicrobial peptides. The polymyxins are active against gram-negative pathogens, such as *P. aeruginosa, Acinetobacter baumanii, Klebsiella* spp., *E. coli* and other *Enterobacteriaceae* [71–73].

The LPS has a negative charge and provides integrity and stability to the external membrane of the bacteria. Polymyxin has a positive charge, displacing the Mg+2 or Ca+2 and bonding to LPS, which as a consequence, destabilises and destroys the internal and external membrane [74].

Gram-negative bacteria may develop resistance to colistin and polymyxin B. The most important mechanisms involve modifications in the external membrane through changes of the LPS. The modification of the LPS occurs with the addition of 4-amino-4-deoxy-Larabinose (Lara4N) to a phosphate group in lipid A. This addition causes an decreases in the negative charge of the lipid A, which decreases the affinity of the positively charged polymyxins [ 68–70].

The biosynthesis of LAra4N is mediated by the regulatory systems PmrA/PmrB and PhoP/PhoQ [26]. One of the primary roles of the activation of PmrAB is the modification of the LPS. These modifications include additions of Ara4N and pEtN to the lipid A and of pEtN to the core of LPS. The modifications mask phosphate groups with positive charges, thereby affecting the electrostatic interaction with certain cationic compounds. The biosynthesis of LAra4N depends on the genes of the operon of resistance to polymyxin, which is known as *arn*. This operon includes the genes *pmrHFIJKLM* [71–73].
