**1. Introduction**

*B. fragilis* is an obligate anaerobic bacterium which colonizes the intestinal tract of the human gut, and essentially all other mammals. It is an integral component of the normal gastrointestinal flora [1, 2]. It is classified as a Gram-negative, nonspore forming and anaerobic bacilli. This mammalian symbiont and opportunistic pathogen depends on its capsular layer for virulence as well as for symbiosis in the mammalian gut [3, 4]. Eight capsule polysaccharides can be expressed on its surface, depending on the environmental niche of the organism, designated as CPSA through CPSH [5–10]. Capsular polysaccharide A is one of the eight polysaccharides found on the surface of *B. fragilis,* and is the most abundant*.* CPSA plays a role in abscess formation when the bacterium localizes outside of its normal niche in the gastrointestinal tract or during surgical procedures [11]. However, this view has been challenged when it was found that treating the animal with the CPSA and then introducing the abscess-inducing bacteria resulted in the immune system of the animal protecting itself against the production of abscesses. Furthermore, few studies have also claimed that the abscess formation by *B. fragilis* actually prevents infection in the wound by other pathogenic bacteria [12, 13].

CPSA is a unique polymer. It has both negatively and positively charged motifs present on each repeating monomer, making it a zwitterionic molecule [7, 14] (**Figure 1**). The presence of this zwitterionic character has been attributed to the novel immunologic activity displayed by CPSA. The zwitterionic character has been shown to modulate the mammalian immune system by interacting with the adaptive immune system [15]. Elimination of either charge group in CPSA results in a lack of *in vivo* activation of the T-cells [16, 17].

CPSA modulates the immune system by its stimulation of a T-cell dependent form of immunity that provides protection against the formation of the intraabdominal abscesses. At the molecular level, CPSA interacts with the MHCII pathway similar to traditional protein antigens [18]. The first step is endocytosis of CPSA by the antigen-presenting cells like dendritic cells. Once in the endosome, CPSA is depolymerized based on the chemical reaction, deaminative cleavage [19]. This cleaving is mediated by nitric oxide, that has been generated by the upregulation of inducible nitric oxide synthase (iNOS). The 130 kDa CPSA is processed

#### **Figure 1.**

*Tetrameric repeat unit of the CPSA found on* B. fragilis*. It consists of an acetamido-4-amino-6 deoxygalactopyranose (AADGal), 4,6-pyruvate galactose (4,6-pyr-gal), N-acetylgalactosamine (GalNAc), and a galactofuranose (Galf) sugar.*

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**Figure 2.**

*present on CD4+ T-cell.*

model of colonic irritation [24].

**2. CPSA gene locus**

*Biosynthesis of the Immunomodulatory Molecule Capsular Polysaccharide…*

to 15 kDa units. After being processed, the endosomes fuse with lysosomes and exocytic vesicles to form MIIC vesicle carrying HLA-DR and the accessory molecule HLA-DM. HLA-DM catalyzes the binding of MHCII to CPSA fragments, which is then presented to the CD4+ T cell receptor (**Figure 2**). This leads to the proliferation of the CD4+ T cell population, that produces IL-10, which is responsible for providing protection against the formation of intra-abdominal abscesses [15, 20]. CPSA can restore the immune system from a variety of autoimmune disorders, making it a promising candidate for a therapeutic drug. Colonization of nude mice with wild type *B. fragilis*, that produces the zwitterionic capsular polysaccharide A, protected animals from antibiotic induced experimental autoimmune encephalomyelitis (EAE), while animals infected with mutant *B. fragilis* deficient in the production of the polysaccharide were not protected [12, 21]. In germ free animal models of Inflammatory Bowel Disease (IBD), it was found that CPSA alone without the bacterial carrier was enough to stimulate normal immune system function and prevent intestinal inflammatory disease [22, 23]. CPSA has been given therapeutically to decrease pro-inflammatory cytokine production in an experimental

*Depolymerization of CPSA in antigen presenting cell. 1. Internalization of CPSA in an endosome. 2. iNOS upregulation produces NO, which cleaves 130 kDa CPSA to ~15 kDa units. 3. Endosome fuses with the lysosome. 4. Endo-lysosome fuses with exocytic vesicle to form MIIC vesicle which has HLA-DR, HLA-DM and processed polysaccharide. In here, processed polysaccharide is loaded on HLA-DR with the help of HLA-DM. 5, 6. The loaded HLA-DM is presented on the surface of the antigen presenting cell to be recognized by alpha beta TCR* 

CPSA is a polymer of a tetramer repeated approximately 160 times. Its size is estimated to be 110 kDa [25]. The CPSA tetrameric repeat unit consists of an

*DOI: http://dx.doi.org/10.5772/intechopen.96937*

*Biosynthesis of the Immunomodulatory Molecule Capsular Polysaccharide… DOI: http://dx.doi.org/10.5772/intechopen.96937*

#### **Figure 2.**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

infection in the wound by other pathogenic bacteria [12, 13].

*in vivo* activation of the T-cells [16, 17].

found on the surface of *B. fragilis,* and is the most abundant*.* CPSA plays a role in abscess formation when the bacterium localizes outside of its normal niche in the gastrointestinal tract or during surgical procedures [11]. However, this view has been challenged when it was found that treating the animal with the CPSA and then introducing the abscess-inducing bacteria resulted in the immune system of the animal protecting itself against the production of abscesses. Furthermore, few studies have also claimed that the abscess formation by *B. fragilis* actually prevents

CPSA is a unique polymer. It has both negatively and positively charged motifs

CPSA modulates the immune system by its stimulation of a T-cell dependent form of immunity that provides protection against the formation of the intraabdominal abscesses. At the molecular level, CPSA interacts with the MHCII pathway similar to traditional protein antigens [18]. The first step is endocytosis of CPSA by the antigen-presenting cells like dendritic cells. Once in the endosome, CPSA is depolymerized based on the chemical reaction, deaminative cleavage [19]. This cleaving is mediated by nitric oxide, that has been generated by the upregulation of inducible nitric oxide synthase (iNOS). The 130 kDa CPSA is processed

*Tetrameric repeat unit of the CPSA found on* B. fragilis*. It consists of an acetamido-4-amino-6-*

*deoxygalactopyranose (AADGal), 4,6-pyruvate galactose (4,6-pyr-gal), N-acetylgalactosamine (GalNAc),* 

present on each repeating monomer, making it a zwitterionic molecule [7, 14] (**Figure 1**). The presence of this zwitterionic character has been attributed to the novel immunologic activity displayed by CPSA. The zwitterionic character has been shown to modulate the mammalian immune system by interacting with the adaptive immune system [15]. Elimination of either charge group in CPSA results in a lack of

**106**

**Figure 1.**

*and a galactofuranose (Galf) sugar.*

*Depolymerization of CPSA in antigen presenting cell. 1. Internalization of CPSA in an endosome. 2. iNOS upregulation produces NO, which cleaves 130 kDa CPSA to ~15 kDa units. 3. Endosome fuses with the lysosome. 4. Endo-lysosome fuses with exocytic vesicle to form MIIC vesicle which has HLA-DR, HLA-DM and processed polysaccharide. In here, processed polysaccharide is loaded on HLA-DR with the help of HLA-DM. 5, 6. The loaded HLA-DM is presented on the surface of the antigen presenting cell to be recognized by alpha beta TCR present on CD4+ T-cell.*

to 15 kDa units. After being processed, the endosomes fuse with lysosomes and exocytic vesicles to form MIIC vesicle carrying HLA-DR and the accessory molecule HLA-DM. HLA-DM catalyzes the binding of MHCII to CPSA fragments, which is then presented to the CD4+ T cell receptor (**Figure 2**). This leads to the proliferation of the CD4+ T cell population, that produces IL-10, which is responsible for providing protection against the formation of intra-abdominal abscesses [15, 20].

CPSA can restore the immune system from a variety of autoimmune disorders, making it a promising candidate for a therapeutic drug. Colonization of nude mice with wild type *B. fragilis*, that produces the zwitterionic capsular polysaccharide A, protected animals from antibiotic induced experimental autoimmune encephalomyelitis (EAE), while animals infected with mutant *B. fragilis* deficient in the production of the polysaccharide were not protected [12, 21]. In germ free animal models of Inflammatory Bowel Disease (IBD), it was found that CPSA alone without the bacterial carrier was enough to stimulate normal immune system function and prevent intestinal inflammatory disease [22, 23]. CPSA has been given therapeutically to decrease pro-inflammatory cytokine production in an experimental model of colonic irritation [24].

#### **2. CPSA gene locus**

CPSA is a polymer of a tetramer repeated approximately 160 times. Its size is estimated to be 110 kDa [25]. The CPSA tetrameric repeat unit consists of an acetamido-4-amino-6-deoxygalactopyranose (AADGal), 4,6-pyruvate galactose (4,6-pyr-Gal), N-acetylgalactosamine (GalNAc), and a galactofuranose (Galf) sugar (**Figure 3**) [26]. The structure of CPSA has previously been well investigated using total correlated spectroscopy and NOESY NMR [27]. Three-dimensional structure of a highly related PSA2 molecule shows a right-handed helix with two repeating units per turn, and a pitch of 20 Å. The zwitterionic motif is formed with alternating anionic carboxylate lying in repeated grooves and the cationic-free amines exposed on the outer surface of the carbohydrate [12, 28].

Although the chemical composition of CPSA is known, yet the biochemical pathway involved in its production is poorly documented [29, 30]. The location of the proposed CPSA locus was knocked out, making a mutant *B. fragilis* which did not express CPSA on its surface, thereby confirming the location of the biosynthetic locus (**Figure 4**). Within the CPSA locus, there are eleven genes, of which nine express proteins similar to other proteins involved in various other polysaccharide biosynthesis (**Table 1**).

### **2.1 Initiating the CPSA biosynthesis**

The function of the nine genes have been elucidated and a pathway has been constructed (**Figure 5**). The identity of the genes present in the CPSA gene locus

#### **Figure 3.**

*Tetrameric repeat of CPSA.*


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membrane.

**Figure 5.**

*Pathway of CPSA biosynthesis.*

**Table 1.**

*Biosynthesis of the Immunomodulatory Molecule Capsular Polysaccharide…*

**ORF Size (aa) Size (kDa) Role Accession no.** *wzx* 482 56 flippase AAK68914.1 *wcfM* 364 43 galactopyranose mutase AAK68915.1 *wcfN* 291 34 glycosyltransferase AAK68916.1 *wzy* 434 43 polymerase AAK68917.1 *wcfO* 357 40 pyruvyltransferase AAK68918.1 *wcfP* 378 44 glycosyltransferase AAK68919.1 *wcfQ* 268 32 glycosyltransferase AAK68920.1 *wcfR* 407 45 aminotransferase AAK68921.1 *wcfS* 195 23 glycosyltransferase AAK68922.1

suggests that the most likely route for assembling the complex bacterial polysaccharide is a Wzy-dependent pathway in which the repeat unit oligosaccharides are assembled one sugar at a time on the cytosolic face of the bacterial inner membrane [31]. Assembly of the oligosaccharide takes place on a C55 isoprenoid bactoprenol [32]. It is a hydrophobic anchor which holds the growing polymer in the cell

The enzymes responsible for the synthesis of the first sugar, AADGal, in the tetrameric repeat, and the enzyme that catalyzes the transfer of this sugar to the bactoprenol anchor have been well characterized [33]. AADGal is synthesized by the sequential action of a dehydratase and an aminotransferase, which is then transferred to the bactoprenyl anchor by a hexose phosphate initiating transferase.

*DOI: http://dx.doi.org/10.5772/intechopen.96937*

*Functions of the gene products in the CPSA biosynthesis operon.*

#### **Figure 4.**

*CPSA locus in the* B. fragilis *genome.*

*Biosynthesis of the Immunomodulatory Molecule Capsular Polysaccharide… DOI: http://dx.doi.org/10.5772/intechopen.96937*


#### **Table 1.**

*Bioactive Compounds - Biosynthesis, Characterization and Applications*

amines exposed on the outer surface of the carbohydrate [12, 28].

biosynthesis (**Table 1**).

**2.1 Initiating the CPSA biosynthesis**

acetamido-4-amino-6-deoxygalactopyranose (AADGal), 4,6-pyruvate galactose (4,6-pyr-Gal), N-acetylgalactosamine (GalNAc), and a galactofuranose (Galf) sugar (**Figure 3**) [26]. The structure of CPSA has previously been well investigated using total correlated spectroscopy and NOESY NMR [27]. Three-dimensional structure of a highly related PSA2 molecule shows a right-handed helix with two repeating units per turn, and a pitch of 20 Å. The zwitterionic motif is formed with alternating anionic carboxylate lying in repeated grooves and the cationic-free

Although the chemical composition of CPSA is known, yet the biochemical pathway involved in its production is poorly documented [29, 30]. The location of the proposed CPSA locus was knocked out, making a mutant *B. fragilis* which did not express CPSA on its surface, thereby confirming the location of the biosynthetic locus (**Figure 4**). Within the CPSA locus, there are eleven genes, of which nine express proteins similar to other proteins involved in various other polysaccharide

The function of the nine genes have been elucidated and a pathway has been constructed (**Figure 5**). The identity of the genes present in the CPSA gene locus

**108**

**Figure 4.**

**Figure 3.**

*CPSA locus in the* B. fragilis *genome.*

*Tetrameric repeat of CPSA.*

*Functions of the gene products in the CPSA biosynthesis operon.*

suggests that the most likely route for assembling the complex bacterial polysaccharide is a Wzy-dependent pathway in which the repeat unit oligosaccharides are assembled one sugar at a time on the cytosolic face of the bacterial inner membrane [31]. Assembly of the oligosaccharide takes place on a C55 isoprenoid bactoprenol [32]. It is a hydrophobic anchor which holds the growing polymer in the cell membrane.

The enzymes responsible for the synthesis of the first sugar, AADGal, in the tetrameric repeat, and the enzyme that catalyzes the transfer of this sugar to the bactoprenol anchor have been well characterized [33]. AADGal is synthesized by the sequential action of a dehydratase and an aminotransferase, which is then transferred to the bactoprenyl anchor by a hexose phosphate initiating transferase. Within the CPSA biosynthesis locus, there is a predicted aminotransferase gene, *wcfR*, and a hexose phosphate initiating transferase, *wcfS*, but no predicted dehydratase was found. However, a gene encoding a potential dehydratase, *ungD2*, has been identified elsewhere in the *B. fragilis* genome. When this gene was knocked out by Coyne *et al*, they found out that, synthesis of the seven out of the eight capsular polysaccharides was stopped. Initial studies with UngD2 and WcfR did not show any promise in the synthesis of AADGal. Hence a previously well characterized dehydratase, PglF [34], from *Campylobacter jejuni* was used to provide the substrate needed for WcfR function. The coupling of these enzymes together led to the production of AADGal (**Figure 6**) [35, 36]. This also points to the notion that depending on homology alone for functional assignment of genes, is not always right, and wet lab results are needed to confirm the function of the gene product.

The synthesized UDP-AADGal was further used as a potential substrate for WcfS, identified as the initiating hexose phosphate transferase. Studies done by Mostafavi et al. demonstrated that WcfS was indeed the initiating hexose phosphate transferase, which lead to the formation of the bactoprenyl linked monosaccharide (**Figure 7**) [33].

As mentioned previously, assembly of the polysaccharides in bacterial cells is done on a C55 bactoprenyl anchor. It is produced by the condensation of farnesyl diphosphate (FPP) to eight units of isopentenyl diphosphate (IPP), done by the enzyme undecaprenyl diphosphate synthase (UPPS). A major drawback of using this compound in in vitro assays is that, it does not have easily distinguishable chromophores associated to it, hence very few rapid assays are available to detect and quantify the activity of enzymes associated with polysaccharide synthesis. To circumvent this problem, the Troutman lab developed fluorescent analogues of the native bactoprenyl, which are easily traceable [25, 37]. Assays done using these analogues take a short time to reveal valuable information about the enzymes when compared to traditional assays, which follow the more tedious route of using radioactive labeled substrates. Mostafavi et al. used a p-nitroaniline bactoprenyl phosphate analogue to find out the function of WcfS (**Figure 7**) [33].

**Figure 6.** *Biosynthesis of AADGal.*

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**Figure 8.**

*General reaction scheme for a glycosyltransferase (GTs).*

*Biosynthesis of the Immunomodulatory Molecule Capsular Polysaccharide…*

Cell surface polysaccharides are nothing but complex carbohydrates. They play important roles in a number of biological processes such as cell growth, cell to cell interactions, immune response, and inflammation. The polysaccharides are synthesized by a class of enzymes known as glycosyltransferases [38]. Glycosyltransferases are an enzyme superfamily responsible for the attachment of carbohydrate moieties to a wide array of acceptors that include nucleic acids, polysaccharides, proteins, lipids, and carbohydrates. The majority of glycosyltransferases are sugar nucleotide-dependent enzymes, and utilize nucleoside diphosphate sugars (NDP-sugars) as donors for the glycosidic bond formation. In other cases, the sugar donors can also be lipid phosphates and unsubstituted

The glycosyltransferases have been classified by sequence homology into 96 families in the Carbohydrate Active enZyme database (CAZy), each of which catalyze the reaction as shown in **Figure 8** [40]. Chain elongation of the oligosaccharide units in complex carbohydrates is achieved by the addition of monosaccharide units through the action of different glycosyltransferases in a specific sequence. The CAZy database provides a highly powerful predictive tool, as the structural fold and mechanism of action are invariant in most of the families [22]. Therefore, where the structure and mechanism of a glycosyltransferase member for a given family has been reported, some assumptions about other members of the family can be made. Substrate specificity, however, is more difficult to predict, and requires experimen-

Determining both the sugar donor and acceptor for a glycosyltransferase of unknown function can be challenging, and it is one of the reasons there are significantly fewer well characterized isoprenoid linked sugar glycosyltransferases when compared to the glycosyltransferases responsible for synthesizing disaccharides or the oligosaccharides [40]. The less reports on isoprenoid linked sugar transferases can be attributed to the fact that, a high throughput method has not yet been developed which will enable for faster characterization. Another challenge in characterizing the glycosyltransferases is the availability of rare sugars, as most of the bacterial polysaccharides contain rare sugars. Rare sugars, such as rhamnose or fucose, may provide the bacterial polysaccharides with additional biological properties compared to those composed of more common sugar monomers [23, 41]. Rare sugars are monosaccharides that are not commonly found in nature, in comparison to D-glucose, D-galactose, D-fructose, D-xylose, D-ribose, and L-arabinose which are more abundant [23]. Moreover, the traditional methods like radioisoptopic

**2.2 Glycosyltransferases involved in CPSA biosynthesis**

tal characterization of individual glycosyltransferases.

*DOI: http://dx.doi.org/10.5772/intechopen.96937*

phosphate [39].

**Figure 7.** *Biosynthesis of bactoprenyl linked monosaccharide.*
