**1. Introduction**

126 The Complex World of Polysaccharides

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Polysaccharide capsules are structures found on the cell surface of a broad range of bacterial species. The polysaccharide capsule often constitutes the outermost layer of the cell, and therefore is often involved in mediating direct interactions between the bacteria and its environment. It is due to these interactions that polysaccharide capsules have been implicated as important virulence factors for many bacterial pathogens.

Bacterial extracellular polysaccharides (EPS) may be classified as either capsular polysaccharides (CPS), where the polysaccharide is intimately associated with the cell surface, or as slime polysaccharides, where the polysaccharide is loosely associated with the cell [1]. Differentiation between these forms is difficult since CPS may be released from the cell, giving the appearance of a slime polysaccharide [1]. In turn, distinguishing between CPS and other cell surface polysaccharides, such as O-antigenic moieties of lipopolysaccharide (LPS), may also be difficult, since CPS may be found associated with LPS [1].

Capsular polysaccharides are highly hydrated molecules that are over 95% water [2]. They are often linked to the cell surface of the bacterium via covalent attachments to either phospholipid or lipid-A molecules, although some CPS may be associated with the cell in the absence of a membrane anchor [1, 3]. Capsular polysaccharides can be either homo- or heteropolymers composed of repeating monosaccharides joined by glycosidic linkages [4]. The multiple hydroxyl groups present within each monosaccharide may be involved in the formation of the glycosidic bond, therefore, any two monosaccharides may be joined in a number of configurations, which leads to large structural diversity among CPS types. In the case of human pathogens, a large number of different capsule serotypes have been identified, and certain CPS or K-antigens have been associated with specific infections [4]. For example, the *Escherichia coli* K1 antigen, a homopolymer of α2,8-linked Nacetylneuraminic acid (NeuNAc), is the major cause of neonatal meningitis [5]. While

© 2012 Reckseidler-Zenteno, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

bacterial species may demonstrate great structural diversity in synthesizing capsules, chemically identical capsular polysaccharides may also be synthesized by different bacterial species. The *Neisseria meningitidis* group B capsular polysaccharide is identical to the K1 polymer of *E. coli*, and the *E. coli* K18, K22, and K100 antigens have the same constituents and structure as the *Haemophilus influenza* serotype b capsule [6, 7]. The conservation of CPS types between bacterial species raises interesting questions regarding the evolution of capsules and the transmission and acquisition of capsule biosynthesis genes [4].

The genetic loci necessary for the production of bacterial capsules are primarily clustered at a single chromosomal locus, which allows for the coordinate regulation of a large number of genes that may be involved in both the biosynthesis and export of capsular polysaccharides [4, 1]. In most bacterial species, the capsule gene clusters demonstrate conserved genetic organization. The capsules of *E. coli* have been classified into four groups based on genetic organization and biosynthetic criteria and capsules of other bacteria may resemble these prototypes. Group 1 capsules include the *E. coli* K30 capsule and the capsules of *Klebsiella* sp. and *Erwinia* sp. [8]. Group 2 capsules include the *E. coli* K1 and K5 capsules, as well as the capsules produced by *Neisseria* sp. and *Haemophilus* sp. Group 3 capsules include the *E. coli* K10 capsule. Group 4 capsules include the capsules of *E. coli* K40 and 0111. Both group 2 and group 3 capsule gene clusters are organized into 3 regions. Regions 1 and 3 are involved in the export and modification of the capsular polysaccharides and are conserved between members of the group, while region 2 contains the genes responsible for the biosynthesis of the capsule and is usually serotype specific [4, 8]. Generally these regions are organized into one transcriptional unit and the regions within the capsule locus are divergently transcribed [4, 8]. In addition, some genes within a region may be translationally coupled, such as the *kpsU* and *kpsC* genes in group 2 *E. coli* capsules, and the *kpsM* and *kpsT* genes in group 3 *E. coli* capsules, which allows for balanced expression of two proteins [4].

The A+T composition of capsule gene clusters is often significantly higher than the rest of the chromosome, suggesting a common ancestry of capsule genes in gram-negative bacteria [9]. It is likely that these A+T rich regions have been horizontally transferred between bacterial species. In addition, the A+T ratio of region 2 DNA of group 2 *E. coli* capsule gene clusters compared to regions 1 and 3 confirms that capsule diversity has been achieved in part through the acquisition of different region 2 sequences [4].

The production of a polysaccharide capsule is widespread in pathogenic bacteria. A number of functions have been assigned to bacterial capsules including: prevention of dessication, adherence, resistance to non-specific host immunity, resistance to specific host immunity, and mediating the diffusion of molecules through to the cell surface [4, 1].

Capsules may form a hydrated gel around the surface of the bacterial cell, which may protect the bacteria from the harmful effects of dessication [10]. This may increase the survival of encapsulated bacteria outside of the host, promoting the transmission of pathogenic bacteria from one host to another [4]. Mucoid isolates of *E. coli*, *Acinetobacter calcoaceticus*, and *Erwinia stewartii* are more resistant to drying than isogenic nonmucoid strains [11]. Studies with *E. coli* have shown that the expression of genes encoding for the colanic acid capsule is increased by dessication [11]. In addition, alginate production by *Pseudomonas aeruginosa* is triggered by high osmolarity, which may be a consequence of dessication [12].

128 The Complex World of Polysaccharides

bacterial species may demonstrate great structural diversity in synthesizing capsules, chemically identical capsular polysaccharides may also be synthesized by different bacterial species. The *Neisseria meningitidis* group B capsular polysaccharide is identical to the K1 polymer of *E. coli*, and the *E. coli* K18, K22, and K100 antigens have the same constituents and structure as the *Haemophilus influenza* serotype b capsule [6, 7]. The conservation of CPS types between bacterial species raises interesting questions regarding the evolution of

The genetic loci necessary for the production of bacterial capsules are primarily clustered at a single chromosomal locus, which allows for the coordinate regulation of a large number of genes that may be involved in both the biosynthesis and export of capsular polysaccharides [4, 1]. In most bacterial species, the capsule gene clusters demonstrate conserved genetic organization. The capsules of *E. coli* have been classified into four groups based on genetic organization and biosynthetic criteria and capsules of other bacteria may resemble these prototypes. Group 1 capsules include the *E. coli* K30 capsule and the capsules of *Klebsiella* sp. and *Erwinia* sp. [8]. Group 2 capsules include the *E. coli* K1 and K5 capsules, as well as the capsules produced by *Neisseria* sp. and *Haemophilus* sp. Group 3 capsules include the *E. coli* K10 capsule. Group 4 capsules include the capsules of *E. coli* K40 and 0111. Both group 2 and group 3 capsule gene clusters are organized into 3 regions. Regions 1 and 3 are involved in the export and modification of the capsular polysaccharides and are conserved between members of the group, while region 2 contains the genes responsible for the biosynthesis of the capsule and is usually serotype specific [4, 8]. Generally these regions are organized into one transcriptional unit and the regions within the capsule locus are divergently transcribed [4, 8]. In addition, some genes within a region may be translationally coupled, such as the *kpsU* and *kpsC* genes in group 2 *E. coli* capsules, and the *kpsM* and *kpsT* genes in group 3 *E.* 

The A+T composition of capsule gene clusters is often significantly higher than the rest of the chromosome, suggesting a common ancestry of capsule genes in gram-negative bacteria [9]. It is likely that these A+T rich regions have been horizontally transferred between bacterial species. In addition, the A+T ratio of region 2 DNA of group 2 *E. coli* capsule gene clusters compared to regions 1 and 3 confirms that capsule diversity has been achieved in

The production of a polysaccharide capsule is widespread in pathogenic bacteria. A number of functions have been assigned to bacterial capsules including: prevention of dessication, adherence, resistance to non-specific host immunity, resistance to specific host immunity,

Capsules may form a hydrated gel around the surface of the bacterial cell, which may protect the bacteria from the harmful effects of dessication [10]. This may increase the survival of encapsulated bacteria outside of the host, promoting the transmission of pathogenic bacteria from one host to another [4]. Mucoid isolates of *E. coli*, *Acinetobacter calcoaceticus*, and *Erwinia stewartii* are more resistant to drying than isogenic nonmucoid strains [11]. Studies with *E. coli* have shown that the expression of genes encoding for the

capsules and the transmission and acquisition of capsule biosynthesis genes [4].

*coli* capsules, which allows for balanced expression of two proteins [4].

part through the acquisition of different region 2 sequences [4].

and mediating the diffusion of molecules through to the cell surface [4, 1].

Capsular polysaccharides may promote adherence of bacteria to both surfaces and other bacterial cells, which may facilitate colonization of a particular niche and may lead to the formation of biofilms [13]. Cell-surface polysaccharides have been shown to mediate the attachment of bacterial cells to one another, leading to biofilm formation and persistence of the organisms during colonization [1, 14].

Capsular polysaccharides are one of the components responsible for resistance to the nonspecific immunity of the host. The presence of a capsule is thought to confer resistance to non-specific host defense mechanisms such as complement and complement-mediated opsonophagocytosis [4]. Bacterial capsules may resist complement-mediated killing by providing a permeability barrier to complement components, which masks the underlying cell surface structures that activate complement [15]. The capsule may also act in concert with O-antigens to confer resistance to complement-mediated killing [16]. As a result, a combination of cell surface structures is responsible for conferring resistance to killing by the complement cascade [4]. Finally, capsules are responsible for resistance to complementmediated opsonophagocytosis. This resistance may be due to steric effects, which results in the capsule acting as a barrier between the C3b deposited on the bacterial surface and the C3b receptors present on phagocytes [4]. Alternatively, the resistance to opsonophagocytosis may be due to the net negative charge of the polysaccharide capsule [17].

Capsules may also confer resistance to the specific immune response of the host. Although most capsular polysaccharides can elicit an immune response, some capsules are poorly immunogenic [4]. Examples of such capsules include those containing NeuNAc, such as the *E. coli* K1 capsule or the capsule of *Neisseria meningitidis* serogroup B, and the *E. coli* K5 capsule, which is similar to desulfoheparin [18, 19]. Because these capsules are structurally similar to polysaccharides encountered on host tissue, these capsules are poorly immunogenic, and elicit a poor antibody response in the host [20].

*Burkholderia pseudomallei*, the causative agent of melioidosis, is a gram-negative, facultatively anaerobic, motile bacillus that is commonly found in the soil and stagnant waters in a number of regions around the world, particularly in areas that fall between 20o north and 20o south of the equator [21, 22]. Infection by *B. pseudomallei* is often due to either direct inoculation into wounds and skin abrasions or to inhalation of contaminated material [22, 23, 24,]. This would explain the prevalence of the disease among rice farmers as well as helicopter pilots in the Vietnam War who developed melioidosis due to inhalation of contaminated dust [24]. Melioidosis may present as an acute pneumonia or an acute septicemia, which is the most severe form of the disease. The disease may also manifest as a chronic infection involving long-lasting suppurative abscesses in numerous sites in the body. Infection with *B. pseudomallei* may even result in a subclinical infection and remain undetected for a number of years. Both the chronic and subclinical forms generally remain undiagnosed until activated by a traumatic event or a decrease in immunocompetence [25]. *B. pseudomallei* is

inherently resistant to a number of antibiotics, and even with aggressive antibiotic therapy, the mortality rate remains high, and the incidence of relapse is common [26, 27].

At the time our studies were initiated some cell-associated antigens had been identified and characterized in *B. pseudomallei*. Cell-associated antigens include exopolysaccharide (EPS) and lipopolysaccharide (LPS) [28, 29, 30]. The EPS produced by *B. pseudomallei* was determined to be an unbranched polymer of repeating tetrasaccharide units with the structure -3)-2-*O*-acetyl-β-D-Gal*p*-(1-4)-α-D-Gal*p*-(1-3)-β-D-Gal*p*-(1-5)-β-D-KDO*p*-(2- [31, 32]. The role of EPS in virulence was not known, but sera from patients with melioidosis had been shown to contain antibodies against EPS [30]. Two other EPS structures were also identified; a branched 1,4-linked glucan polymer ((CP-1a) and a triple-branched heptasaccharide repeating unit composed of rhamnose, mannose, galactose, glucose, and glucuronic acid (CP-2) [33]. The genes involved in the synthesis of these capsules, and the role of these capsules in virulence had not been identified. The LPS of *B. pseudomallei* was structurally characterized and reported to contain two types of O-polysaccharide moieties termed type I O-PS and type II O-PS [34, 35]. Type II O-PS was found to be an unbranched heteropolymer with repeating D-glucose and L-talose residues with the structure -3)-β-Dglucopyranose-(1-3)-6-deoxy-α-L-talopyranose-(1-. Type II O-PS had been shown to be involved in serum resistance [36]. Type II O-PS mutants also demonstrated reduced virulence in three animal models of *B. pseudomallei* infection [36]. Type I O-PS was determined to be an unbranched homopolymer with the structure -3)-2-*O*-acetyl-6-deoxy-β-D-manno-heptopyranose-(1-, however, the role for this polysaccharide in infection had not been defined, nor the genes responsible for its biosynthesis been identified.

*B. thailandensis* is a nonpathogenic soil organism originally isolated in Thailand [37]. Based on biochemical, immunological, and genetic data, *B. pseudomallei* and *B. thailandensis* are closely related species. However, these two organisms differ in a number of ways and have been classified into two different species [38]. The rRNA sequence of *B. thailandensis* differs from that of *B. pseudomallei* by 15 nucleotides, and there are significant differences in genomic macrorestriction patterns between these organisms [39]. The biochemical profiles of these two species differ in that *B. thailandensis* can utilize L-arabinose whereas *B. pseudomallei*  does not [38, 40]. The most distinct difference between these two species, however, is their relative virulence. The 50% lethal dose (LD50) for *B. pseudomallei* in the Syrian hamster model of acute melioidosis is <10 organisms, whereas the LD50 for *B. thailandensis* is approximately 106 organisms [38]. It has also been shown that the two species can be differentiated based on their propensity to cause disease in humans. Environmental strains isolated in Thailand that are able to assimilate L-arabinose are not associated with human infection, whereas clinical isolates are not able to utilize L-arabinose [41].
