**8. Variability of the LPS**

In bacterial pathogens, the most variable structures are those that are expressed on the cell surface. Intra-species genetic individuality has inheritable traits that may be observed in a sample of individuals. The genetic variability is derived from the combination of a certain number of genes, which each exists as a family of alleles that differ in structure and function. An example is the family located in the biosynthetic locus of the O antigen, and an example of this phenomenon at the population level is the hypervariability in the structure of this biomolecule [39]. Among the components of the surface of the gram-negative bacteria, LPS is the primary constituents of the external membrane, which is a heterogeneous surface that is significantly involved in the process of the microorganism adapting to its environment.

LPS primarily consists of conserved segments, such as lipid A and the core, and secondarily consists of a hypervariable segment, which is the O antigen. The conserved domains of the LPS are shared regions among bacterial species, which intervene in the development and in the survival of the bacteria. The O antigen may display modifications such alterations in the length of the oligosaccharide chain and changes in the surface composition and in the chemical configuration, due to the addition of glycosyl or fucosyl groups or even nonhydrocarbonated substitutes, such as acetyl or methyl groups, which could affect the cellular structure [40].

### **8.1. Intra-species variability in the composition of the O antigen**

82 The Complex World of Polysaccharides

**8. Variability of the LPS** 

*rfb* [1,18,19*.*]

Wzy exerts its action over the long-chain O antigen.

moving the O unit bonded to UndP through the internal membrane before the polymerase

The majority of the O antigen operons are constitutively expressed and are preceded by a sequence of 39 bp, known as JUMPStart **(Figure 5B)**.This sequence includes two elements known as *ops*, which are involved in the recruiting of elongation factors during the transcription. The JUMPStart region is controlled by RfaH, which acts as a positive regulator for the genes involved in the outer core of LPS and also increases the expression of the locus

**Figure 5. A.** The location of the genes involved in the synthesis of the O antigen in several species of *Enterobacteriaceae*. **B.** The JUMPStart sequence of *S. flexneri* strain IB1833**.** The *ops* sequence is indicated in bold for eight base pairs. A residue of thymine that is highly conserved in the far 3' is indicated by

In bacterial pathogens, the most variable structures are those that are expressed on the cell surface. Intra-species genetic individuality has inheritable traits that may be observed in a sample of individuals. The genetic variability is derived from the combination of a certain number of genes, which each exists as a family of alleles that differ in structure and function. An example is the family located in the biosynthetic locus of the O antigen, and an example of this phenomenon at the population level is the hypervariability in the structure of this biomolecule [39]. Among the components of the surface of the gram-negative bacteria, LPS is the primary constituents of the external membrane, which is a heterogeneous surface that is significantly involved in the process of the microorganism adapting to its environment.

cursive text, and repeated sequences are underlined. Accession number: U118168.1.

The heterogeneity in the expression of LPS may provide a medium to discriminate among the bacterial species. This heterogeneity is responsible for the well-known ladder profile that can be detected in silver-stained SDS-PAGE gels (**Figure 6).** This method is used to determine the number and repeated units of oligosaccharides that constitute the O antigen, which has been useful in epidemiological studies [41]. The smooth strains contain the entire LPS, whereas the semi-rough strains have a subunit of the O antigen, and the rough strains lose the subunits of the O antigen.

**Figure 6. LPS profiles in SDS-PAGE.** The smooth forms of LPS display a ladder profile that is strainspecific. **A)** *C. freundii* E9750; **B)** *S. senftenberg* 74210; **C)** *E. coli* O157:H7; **D)** *S. marcescens* biotype TC.

In **Table 1**, examples are presented of various sequences from different genera and species, which mark the variability of intra-species and inter-species LPS that impedes the immune response control of bacterial infections, among other issues.


D-Col, D-colitose; L-Fuc, L-fucose; D-Gal, D-galactose; D-Gal2NAc, 2-*N*-acetylgalactosamine; D-Glc, D-glucose; D-Glc2NAc, D-2-*N*-acetylglucosamine; D-Per, D-perosamine; D-Per4NAc, D-4-*N*-acetylperosamine.

**Table 1.** Examples of different structures of the bacterial O antigen.

An increase in temperature during microbial growth causes changes in the concentration of carbohydrates in LPS, which modifies their composition [42]. In a study on *in vitro* passages of the *C. freundii* E9750 strain cultivated under different temperatures, variability was observed in the ladder profiles of the O antigen, in the concentration of carbohydrates and in the agglutination reactions with the anti-O serum of *C. freundii* E9750 and *Salmonella senftenberg* 74210 [43]. According to the ladder profiles of the isolates of *C. freundii* E9750, six profiles were distinguished: A (control strain), B, C, D, E (immunoreactive isolates) and F (isolates that had lost the immunoreactive chain). These profiles were generated according to their similarity. Each LPS profile displayed a typical ladder profiles (**Figure 7).**

The different profiles from the isolates obtained from the sub-cultures of *C. freundii* E9750 were out of phase with respect to the control. This result may be explained by variation in the number of oligosaccharide units present in the O antigen. However, when the length of the chain is increased, the differences are more marked, and the bands of the ladder profiles are out of phase, as Lawson et al., report with strains of *S. enterica* serovar Typhimurium [41]. There are other reports in the literature on the variability of the length of the O antigen chain as a response to change in temperature, as in strains of *P. aeruginosa* and enteric bacteria [44,45].

Several isolates of *C. freundii* E9750 displayed cross-reactivity in agglutination tests with the anti-O serum of *S. senftenberg* 74210, which suggests changes in the conformation of the epitopes that may be associated with the addition of glucosyl groups or residues derived from N-acetyl [46].

84 The Complex World of Polysaccharides

bacteria [44,45].

D-Col, D-colitose; L-Fuc, L-fucose; D-Gal, D-galactose; D-Gal2NAc, 2-*N*-acetylgalactosamine; D-Glc, D-glucose; D-

An increase in temperature during microbial growth causes changes in the concentration of carbohydrates in LPS, which modifies their composition [42]. In a study on *in vitro* passages of the *C. freundii* E9750 strain cultivated under different temperatures, variability was observed in the ladder profiles of the O antigen, in the concentration of carbohydrates and in the agglutination reactions with the anti-O serum of *C. freundii* E9750 and *Salmonella senftenberg* 74210 [43]. According to the ladder profiles of the isolates of *C. freundii* E9750, six profiles were distinguished: A (control strain), B, C, D, E (immunoreactive isolates) and F (isolates that had lost the immunoreactive chain). These profiles were generated according

The different profiles from the isolates obtained from the sub-cultures of *C. freundii* E9750 were out of phase with respect to the control. This result may be explained by variation in the number of oligosaccharide units present in the O antigen. However, when the length of the chain is increased, the differences are more marked, and the bands of the ladder profiles are out of phase, as Lawson et al., report with strains of *S. enterica* serovar Typhimurium [41]. There are other reports in the literature on the variability of the length of the O antigen chain as a response to change in temperature, as in strains of *P. aeruginosa* and enteric

Several isolates of *C. freundii* E9750 displayed cross-reactivity in agglutination tests with the anti-O serum of *S. senftenberg* 74210, which suggests changes in the conformation of the

Glc2NAc, D-2-*N*-acetylglucosamine; D-Per, D-perosamine; D-Per4NAc, D-4-*N*-acetylperosamine.

to their similarity. Each LPS profile displayed a typical ladder profiles (**Figure 7).**

**Table 1.** Examples of different structures of the bacterial O antigen.

**Figure 7. LPS profiles in SDS-PAGE stained with silver.** Profiles of colonies of *C. freundii* E9750. A) isolates at a temperature of 42°C; B) isolates at a temperature of 37°C. M= weight markers.

The isolates of *C. freundii* E9750 that display variability suggest heterogeneity in the composition or conformation of the epitopes of the O antigen. The cross-reactivity of the isolates of *C. freundii* and *S. senftenberg* 74210 is associated with the specificity of the epitopes of the O polysaccharide that these species share. The addition at O antigen of glucosyl groups or derived N-acetyl residue could be involved in these cross-reactions.
