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Progress in the synthesis of complex chains of plant and microbial , 2009: 181-198 ISBN: 978-81-7895-424-0 Editor: Nikolay E. Nifantiev

Structures of bacterial 7 polysaccharides

Yuriy A. Knirel N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences Leninsky Prospekt 47, Moscow 119991, Russia

Abstract This chapter is devoted to the composition and structure of various bacterial surface glycopolymers: of Gram-negative , - wall anionic polysaccharides of Gram-positive bacteria, including teichoic and lipoteichoic acids, and mycobacterial lipoglycans. Extracellular polysaccharides, which build a protective capsule, participate in formation or are excreted as slime, are considered too. The occurrence of both and non-carbohydrate groups as components of the is surveyed. Various structural types of the polysaccharides are discussed, including homopolysaccharides and heteropolysaccharides built up of or oligosaccharide-phosphate repeating units. Attention is paid to the mode of the attachment of various polysaccharides to the cell surface.

Correspondence/Reprint request: Dr. Yuriy A. Knirel, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, Moscow 119991, Russia. E-mail: [email protected] 182 Yuriy A. Knirel 1. Introduction Glycopolymers are components of the cell envelope of various bacteria. In Gram- negative bacteria, the cell envelope consists of the inner (cytoplasmic) and outer membrane and a rigid (murein) layer in between. The outer leaflet of the outer membrane consists mainly of (LPS, endotoxin). Gram-positive bacteria lack the outer membrane and have a much thicker peptidoglycan layer. Their cell- wall polysaccharides are linked to peptidoglycan or anchored into the cytoplasmic membrane. Although mycobycteria stain slightly Gram-positive, they posses Gram- negative rather than Gram-positive cell envelope features, i.e. a thin peptidoglycan layer and a lipid bilayer outer membrane. Important lipoglycans protrude through the . Bacteria of yet another group, , have a differently composed rigid layer called pseudomurein. Bacterial cells may be surrounded by a polysaccharide capsule or a S-layer. The latter consists of proteins or glycoproteins that are self-aggregated to crystal-like planar structures by electrostatic or hydrophobic interactions to yield a porous envelope. As microorganisms spend a significant amount of energy on their synthesis, the polysaccharides should play an important role in bacterial life. Indeed, being located on the cell surface, they define the immunospecificity and are implicated in protection and of microorganisms. The knowledge of structural details of bacterial surface polysaccharides helps a better understanding of the mechanisms of pathogenesis of infectious diseases and development of diagnostic agents and efficient . Recently, an impressive progress in elucidation of bacterial polysaccharide structures has been achieved, mainly due to elaboration of novel modern methods of structural analysis, first of all high-resolution NMR spectroscopy and mass spectrometry. In the last decades, the composition and structure of bacterial polysaccharides have been repeatedly surveyed [1-9], and the annually updated Bacterial Carbohydrate Structure Database is available via Internet at http://www.glyco.ac.ru/bcsdb. In the present Chapter, structural features of the major classes of bacterial polysaccharides, including LPSs of Gram-negative bacteria, teichoic and teichuronoic acids of Gram-positive bacteria, lipoglycans of mycobacteria, capsular and other extracellular polysaccharides as well as S-layer glycoproteins are discussed.

2. Lipopolysaccharides of Gram-negative bacteria The LPS is the major constituent of the outer membrane of the cell envelope of Gram- negative bacteria. A complete LPS (S-form) has three domains, which differ in their chemical nature, genetics, biosynthesis and function. A polysaccharide portion of the LPS called O- specific polysaccharide (OPS, O-chain, O-antigen) is either a homopolymer or, more often, a regular heteropolymer built up of oligosaccharide (from di- to octa-saccaharide) repeating units (O-units). Based on the fine structure of the OPS, serologically distinct strains of bacterial species are classified into O-serotypes or O-serovars. The OPS is linked to a large oligosaccharide called core, which, in turn, is linked to the lipid moiety of the LPS, lipid A. The latter serves as an anchor that links by hydrophobic interactions the outer leaflet of the outer membrane composed mainly of the LPS to the inner phospholipid layer. Lipid A of endotoxic active LPSs is responsible for the biological activities of the LPS. Some possess a truncated LPS that is either devoid of the O-chain (R-form) or has a single O-unit linked to the core (SR-form). Structures of bacterial polysaccharides 183 2.1. O-specific polysaccharides O-antigens of some bacteria are homopolysaccharides. They often consist of common monosaccharides (e.g. various , and are known) but homopolymers of less common and derivatives, such as N-acyl derivatives of 4-amino-4,6-dideoxy-D- and -L-mannose in Vibrio cholera, occur as well [8]. Legionella pneumophila serogroup 1 strains possess a homopolymer of α-(1→4)- interlinked residues of 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D- galacto-non-2-ulosonic (legionaminic) acid, either 8-O-acetylated or not, whereas in other L. pneumophila strains a similar homopolysaccharide of the corresponding D-glycero-D-talo isomer has been identified [10]. Heteropolysaccharides are more widespread and more diverse in composition than homopolysaccharides. They may include usual sugars, like , , 6-deoxyhexoses, N-acetylhexosamines and hexuronic acids, as well as less common 6- deoxyamino and 6-deoxydiamino sugars, aminouronic and diaminouronic acids as well as higher monosaccharides, including , 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids as well as branched monosaccharides [3]. N-Acetyl and O-acetyl groups are common and other non-sugar substituents often occur too, such as O-methyl groups, N-linked hydroxy and amino acids, carboxyl-linked amino components, including amino acids, lactic acid ethers, pyruvic acid acetals, alditol phosphates and ethanolamine phosphate [3] (examples 1-8 are shown in Figure 1).

Figure 1. Examples of unusual monosaccharides and derivatives, components of bacterial polysaccharides. 1, amide of D-glucuronic acid with N-[(R)-1-carboxyethyl]-L-lysine; 2, 4- amino-4,6-dideoxy-D- N-substituted with N-[(R)-3-hydroxybutanoyl]-L-alanyl; 3, (R)-acetal of pyruvic acid with D-; 4, ether of (R)-lactic acid with L-; 5, N-acetyl-D-glucosamine phosphorylated with phosphocholine; 6, non-2-ulosonic acid 5-N-acetyl-7-N-formylpseudaminic acid; 7, branched octose yersiniose A; 8, higher carbocyclic monosaccharide caryose. 184 Yuriy A. Knirel

Often the OPSs of serologically distinct strains have quite different structures as, e.g., in Shigella dysenteriae [11], but in some bacteria they are related to various extents. For instance, the OPSs of O1-O13 all have a 6-deoxy-D- hexosamine (N-acetyl-D-quinovosamine, N-acetyl-D-fucosamine or di-N-acylbacillosamine) as the first monosaccharide of the O-unit, whose transfer to a lipid carrier initiates the O- antigen biosynthesis. They are also enriched in amino and diamino hexuronic acids and diamino non-2-ulosonic acids [12], and some of them are closely related in structure. For instance, the OPSs of various subgroups in P. aeruginosa serogroup O6 have the same O-unit and differ only in the mode of connection of the O-units to each other [by α- (1→2)-, α-(1→3)- or β-(1→3)-linkage]:

Other differences between P. aeruginosa OPSs are due to a replacement of one sugar isomer with another (e.g. D-QuiNAc with D-FucNAc in serogroup O4) or one N- acyl group with another [e.g. acetyl with (S)-3-hydroxybutanoyl at N-4 of QuiNAc4N in serogroup O3]. Some of the OPSs differ also in O-acetylation and uronic acid amidation, which are usually non-stoichiometric. Other non-stoichiometric modifications that mask the regularity of polymers are glycosylation (most often glucosylation), methylation and phosphorylation. The non-reducing terminus of the OPSs (mainly of homopolysaccharides) may be occupied by an O-methylated monosaccharide, which is usually one of the O-unit components. Examples are 3-O-methyl-D-rhamnose and 3-O-methyl-L-rhamnose (D- and L-acofriose) in the corresponding rhamnans or 3-O-methyl-D-mannose in D-mannans [6]. In V. cholerae O1, 2-O-methylation of the terminal D-Rha4N residue results in seroconversion from Inaba to Ogawa [13]. In some other cases, the OPSs are terminated with a monosaccharide that is different from the O-unit components. For instance, N-acyl derivatives of 2,3,4-triamino-2,3,4- trideoxy-D-galacturonamide occupies the non-reducing end of the OPS of Bordetella bronchiseptica and Bordetella parapertussis, which is a homopolymer of 2,3-diacetamido-2,3-dideoxy-D-galacturonamide [14]. The OPSs of pneumoniae O4 and O12 are terminated with a residue of Kdo, which is no component of the O-units [15]. In the OPS of the gastric bacterium Helicobacter pylori, which is built up of occasionally fucosylated N-acetyl-β-lactosamine repeating units, the terminal non- reducing O-unit often carries one or two α-L- residues giving rise to Lewis x and Lewis y antigen determinants, respectively (Figure 2) [16]. It is suggested that such molecular mimicry has been acquired in the course of long co-evolution of the bacterium with humans. Similar or even identical OPSs are sometimes found in taxonomically distant bacteria, even in those belonging to different families. For instance, bacteria Francisella tularensis, Vibrio anguillarum, Sh. dysenteriae type 7 and P. aeruginosa O6 possess a Structures of bacterial polysaccharides 185

Figure 2. Molecular mimicry by bacterial LPS of host glycoconjugates structures: Lewis x and Lewis y antigens by the O-antigen of Helicobacter pylori and GM1, GD1a and GQ1b gangliosides by the outer core of Campylobacter jejuni. common fragment of linear O-units, which is either α-D-GalpNA-(1→4)-α-D-GalpNA-(1→3)-D-QuipN or β-D-QuipN-(1→4)-α-D-GalpNA- (1→4)-α-D-GalpNA or both [4]. Examples of identical OPSs are a D-mannan shared by O9, K. pneumoniae O3 and Hafnia alvei PCM 1223 [17, 18] and a hompolymer of α-(1→2)-interlinked 4,6-dideoxy-4-formamido-D-mannopyranose residues shared by abortus and Yersinia enterocolitica O9 [19]. These phenomenon may result in false-positive diagnosis of infectious deseases. In Enterobacteriaceae, a specific OPS may be replaced or may coexist with another polysaccharide called the enterobacterial common antigen (ECA), a heteropolysaccharide having a trisaccharide repeating unit (9), which is known in several forms, including the core- lipid A-linked form [20]. Similarly, strains of P. aeruginosa from various O-serogroups produce a D-rhamnan (10) as a part of an LPS coexisting with the LPS having a serogroup- specific O-chain [21].

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10 186 Yuriy A. Knirel

O-specific polysaccharides may be synthesized by a combined pathway with other glycoconjugates. For instance, in P. aeruginosa O7, a single trisaccharide O-unit is either polymerized to give the O7a,b,c-polysaccharide or O-linked to a serine residue of pilin, a glycoprotein of the somatic pili [22]. O-specific polysaccharides consisting of blocks of O-units having different structures are known. In K. pneumoniae O1, two homopolysaccharide domains called I and II are connected to afford a copolymer 11 [15]. The OPS of K. pneumoniae O2a,c (12) includes a galactan I domain associated with a heteropolysaccharide domain.

11

12

2.2. Core oligosaccharide In the LPS core, a lipid A-proximal domain is usually structurally conserved within the genus or even family. For instance, in all representatives of Enterobacteriaceae, the inner core is the same pentasaccharide consisting of two Kdo and three L-glycero-D- manno- (13) residues (Figure 3). The α-Kdop-(2→4)-α-Kdop is common for the LPS of most bacteria studied, whereas others contain α-Kdop 4- phosphate (14) [23]. In a few LPSs, Kdo is replaced with its 3-hydroxylated derivative, D-glycero-D-talo-oct-2-ulosonic acid (Ko) (15). The Kdo linkage that connects the core and lipid A is acid-labile which provides an excellent opportunity to cleave the carbohydrate portion of the LPS from the lipid moiety. Common components of the inner core region are phosphate or diphosphate, which may link ethanolamine to the core. Uronic acid residues may be present in addition to or instead of phosphate groups, e.g. in the LPS core of Proteus and Klebsiella. Therefore, the core is generally a (highly) negatively charged domain in the LPS. The inner core region may be decorated with other single monosaccharides or bear an outer core oligosaccharide (in Enterobacteriaceae, Pseudomonadaceae and some other bacteria). The core structure in the R-form LPS and S- or SR-form LPS may be different. E.g., in P. aeruginosa, two core glycoforms exist, which differ in the site of attachment of a L- rhamnose residue (Figure 4) [24]. As an alternative to the O-unit in the SR-LPS, an oligosaccharide extension that is different from the O-unit may be linked to the core. In Proteus LPS, these have been found to be almost as diverse in structure as the OPSs [25].

Figure 3. Components of the inner region of the LPS core. 13, L-glycero-D-manno-heptose; 14, 3- deoxy-D-manno-oct-2-ulosonic acid (Kdo) 4-phosphate; 15, D-glycero-D-talo-oct-2-ulosonic acid (Ko). Structures of bacterial polysaccharides 187

Figure 4. Full structure of the R-, SR- and S-form lipopolysaccharides of Pseudomonas aeruginosa O10a,10b (from ref. [24]). The core occurs in two glycoforms that differ in the position of a rhamnose residue; only one of the glycoforms is substituted with the O-antigen. Dotted lines indicate non-stoichiometric substitution.

As some OPSs, the core region may be terminated with a domain whose structure resembles those of host glycoconjugates. A consequence of such molecular mimicry may be post-infectious autoimmune diseases, as e.g. acute neuromuscular paralysis that is often associated with Campylobacter jejuni enteritis. The reason of the disease is the expression of ganglioside glycans on the short-chain C. jejuni LPS (Figure 2) and, as a result, induction of 188 Yuriy A. Knirel anti-GM1 and anti-GD1a (Gullian-Barré syndrome) or anti-GQ1b (Miller-Fisher syndrome) autoantibodies [26]. An additional region called primer occurs between the homopolysaccharide O-chain and the core. It belongs to the OPS biosynthesis pathway and is necessary for the initation of the O-antigen synthesis. The most common primer is β-D-GlcpNAc, which is present, e.g., in all K. pneumoniae O-serotypes [15] and in common polysaccharide antigen of P. aeruginosa [27]. In some bacteria, in addition to the primer, a more complex non-repetitive oligosaccharide domain (adaptor) occurs between the OPS and the core.

2.3. Lipid A Typical lipid A comprises a disaccharide bisphosphate backbone, which carries up to seven O-acyl groups. In most bacteria, the constituent monosaccharides are GlcN but one or both GlcN residues may be replaced with 2,3-diamino-2,3-dideoxy-D-glucose [28]. Four of the acyl groups (usually 3-hydroxyacyl groups) are linked directly to the monosaccharides and called primary, whereas the others called secondary are attached to the hydroxy groups of the primary fatty acids. The arrangement of six fatty acids may be symmetrical, e.g. as in lipid A of P. aeruginosa (Figure 4), or asymmetrical with the GlcN residue proximal to the core bearing four acyl groups, as in all Enterobacteriaceae. The biological activity of lipid A and the LPS as the whole, including profound immunostimulatory and inflammatory capacity, is defined by the number and the nature of acyl groups and depends also on the presence of phosphate groups. The latter may carry cationic substituents, such as ethanolamine or 4- amino-4-deoxy-L-arabinopyranose, which confer the resistance of bacteria to cationic antimicrobial peptides. In some bacteria the lipid A structure shows a significant deviation from the structure discussed above. For instance, in Rhizobia sp. Sin-1, lipid A is not phosphorylated but includes 2-amino-2-deoxy-D-gluconic acid (or its 2,3-dehydro derivative) in place of α-GlcN-1-P and β-GlcN bears a single very long secondary acyl group, 27-hydroxyoctacosanoic acid [29].

3. Cell-wall polysaccharides of Gram-positive bacteria 3.1. Teichoic and lipoteichoic acids Common cell-wall components of Gram-positive bacteria are teichoic and lipoteichoic acids, which represent polyol phosphate-containing polymers often including also glycosyl moieties. Teichoic acids are covalently linked by phosphodiester bridges to muramic acid residues in peptidoglycan. Lipoteichoic acids are linked to a glycolipid that anchors them in the cytoplasmic membrane. Teichoic acids are necessary for the viability of bacilli being involved in ion exchange, permeability of the cell wall to nutrients and antibiotics and control of the activity of autolytic enzymes and adhesins. As other surface polysaccharides of bacteria, they define the immunospecificity and binding of specific bacteriophages. Being widely distributed in Gram-positive bacteria and greatly diverse in structure, teichoic acids are applicable to the systematics of these microorganisms. Teichoic acids differ in composition of polyols and sugars, positions of their phosphorylation and glycosylation, the presence of other (mainly O-acyl) groups and basal structure. There are two major structural types of teichoic acids. Type I teichoic acids are polymers of a polyol phosphate, in which the polyol is most often glycerol or ribitol; in addition, erythritol, arabinitol and mannitol have been Structures of bacterial polysaccharides 189 found. These polymers are not true polysaccharides. Some of them lack any carbohydrate at all, whereas in the others the polyol is glycosylated. Poly(glycerol phosphates) are most abundant in Gram-positive bacteria. They are often glycosylated with glucose, galactose or N-acetylamino sugars, which are linked as mono- or oligo-saccharide side chains at position 2 or 1 of glycerol. Lateral phosphate groups [30], O-acetyl groups and O-linked amino acids occur as well. The most common amino acid is D-alanine, whose residues being protonated provide counterions for control of the net anionic charge of teichoic acids [31]. Most common type I ribitol teichoic acids are 1,5-poly(ribitol phosphates). Glucose, rhamnose, 3-O-methylrhamnose and N-acetylamino sugars have been found as ribitol- linked lateral glycosyl substituents. These polymers may contain also ribitol phosphate or glycerol phosphate side chains as well as O-linked L-lysyl, D-alanyl and succinyl groups or a pyruvic acid acetal [32]. Side-chain groups often occur in non-stoichiometric quantities and may differ in different monomer units. A 3,5-poly(ribitol phosphate) has been found only in Nocardiopsis and is unsubstituted [33]. Typical type I teichoic acids of subtilis, substituted 1,3-poly(glycerol phosphate) and 1,5-poly(ribitol phosphate), are linked to peptidoglycan at position 6 of N-acetylmuramic acid via a highly conserved domain (adaptor) consisting of a β-D- ManpNAc-(1→4)-α-D-GlcpNAc-P disaccharide-phosphate and one to three glycerol phosphate monomers (Figure 5) [34]. of DSM 20233 possesses a long 1,3- poly(glycerol phosphate) chain linked to a diglucosyl bisacyl glycerol (Figure 6) [35]. Streptococcus sp. produces a relatively short 1,3-poly(glycerol phosphate) lipoteichoic acid having an average chain length of 10 glycerophosphate residues and anchored to a minor membrane glycolipid β-D-Galf-(1→3)-acyl2Gro [36]. Poly(mannitol phosphates) are present in Brevibacterium species. Four types of monomer units have been identified in 1,6-poly(mannitol phosphate) of Brevibacterium iodinum, including unsubstituted units and those bearing 2-linked β-D-Glc or 4,5-linked pyruvic acid (S)-acetal or both [37]. The latter substituent endows the with additional negative charges.

Figure 5. 1,3-Glycerol teichoic acid of Bacillus subtilis 168 and 1,5-ribitol teichoic acid of Bacillus 1 2 3 subtilis W23. R = H or D-Glc, R = H or D-Ala, R = H, D-Ala or D-Glc, n = 40, m = 1-3. 190 Yuriy A. Knirel

1 Figure 6. Lipoteichoic acid of Staphylococcus aureus DSM 20233. R = H, D-Ala or α-D-GlcpNAc; R 2 and R = acyl groups C14-C19 partially methylated at positions 9, 14 or 17, n = 45-50.

Known type I teichoic acids with other polyols in the main chain are 1,4-poly(erythritol phosphate), which has been found, e.g., in Glycomyces tenuis [38]. It consists of ca. 23 units, five units being substituted with β-D-GlcpNAc. An oligosylated poly(arabinitol phosphate) with unknown position of the phosphodiester linkage has been reported in Agromyces cerinus [39]. Type II teichoic acids comprise both glycosyl or oligosyl moeity and polyol (glycerol or ribitol) in the main chain. In the known type II glycerol teichoic acids, D- glycerol-3-phosphate (D-Gro-3-P) residue is always present and is usually glycosylated at position 1. The polymers with sugars phosphorylated at position 6 are the most widespread but substitution at position 3 or 4 occurs too. All known poly(glycosylribitol phosphates) include 1-O-glycosylated D-ribitol-5-phosphate (D-Rbo-5-P). Type II teichoic acids often possess mono- or oligosaccharides side chains, which are usually linked to the sugar moiety of the main chain. Composition of their carbohydrate moiety is diverse and they often include unusual monosaccharides, such as 2-acetamido-4-amino-2,4-dideoxy-D-galactose (D-FucNAc4N), and unusual non-sugar components, such as choline. The common antigen of Streptoсcoccus pneumoniae (C-polysaccharide) belongs to type II teichoic acids. It contains Rbo-5-P and one or two phosphocholine (Cho-P) groups [40]:

A variant of the C-polysaccharide with both choline residues replaced with ethanolamine residues has been found in Streptococcus mitis SK598 [41]. In pneumococcal lipoteichoic acid (F-antigen), the C-polysaccharide is β-(1→3)-linked to a α-D-Glc-(1→3)-acyl2Gro lipid anchor [42]. In addition to the two major types, teichoic acid with mixed structures are known. They have a main chain that consists of alternating glycerol phosphate and either glycosylphosphate (found only in Staphylococcus) or glycosylglycerol phosphate units (occur exclusively in Nocardiopsis dassonvillei) [9]. The latter consists of →1)-Gro-3-P and β-D-GalpNAc-(1→2)-Gro-3-P units, the sugar residue being 3-O-phosphorylated in ssp. dassonvillei or 4-O-phosphorylated and 3-O-succinylated in ssp. albirubida. Structures of bacterial polysaccharides 191

3.2. Teichuronic acids and other anionic polysaccharides Gram-positive bacteria that lack teichoic acids generally have functionally similar phosphate-free anionic polysaccharides that, as teichoic acids, are linked to peptidoglycan. Such polymers may also replace teichoic acids when bacteria are cultivated in phosphate- limited media. Most known anionic polysaccharides contain uronic acid residues and are called teichuronic acids. They are highly diverse in composition and have a repeating unit from disaccharide to pentasaccharide and higher. A rather simple teichuronic acid having a →4)-β-D-ManpNAcA-(1→6)-α-D-Glcp-(1→ disaccharide repeating unit occurs in Micrococcus luteus. This polysaccharide is attached at position 6 of MurNAc of peptidoglycan via a GlcNAc 1-phosphate residue located at the reducing end of the teichuronic acid [43]. An unusual anionic polysaccharide having a homopolymer backbone of α-(2→4)- interlinked 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid (Kdn) and lateral 8-O-(β-glucopyranosyl) groups has been found in phytopathogenic streptomycete strains [44]. Oligomers of Kdn bearing β-D-galactose or its 3-O-methyl derivative at position 9 have been identified in Streptomyces sp. MB-8 [45]. By analogy with teichuronic acids, these anionic cell-wall polymers may be called teichulosonic acids. Various anionic cell-wall polysaccharides often coexist in one bacterial strain. For instance, three anionic polymers occur simultaneously in plant pathogenic Streptomyces sp.: a teichuronic acid with a disaccharide repeating unit →6)-α-D-Glcp-(1→4)-β-D- ManpNAc3NAcA-(1→, a β-glucosylated Kdn polymer and a β-glucosylated 1,5-poly(ribitol phosphate) [44]. Neutral polysaccharides are uncommon in Gram-positive cell-wall, a galactomannan of Streptomyces sp. VKM Ac-2125 being among few exceptions [46].

4. Cell-wall lipoglycans of mycobacteria Mycobacterial cell-wall glycopolymers differ significantly from polysaccharides of other Gram-positive bacteria as they comprise different structural motifs and contain abundant lipid portions. Two major lipoglycans are the mycolyl complex and lipoarabinomannan. The primary structure of the arabinogalactan is quite well defined in tuberculosis (Figure 7) [47]. Its main chain represents a linear galactan consisting of about 30 alternating 5- and 6-linked D-Galf residues. Arabinan chains are attached to the galactan core at position 5 of some 6-substituted D-Galf residues. They are composed mainly of 5-linked D- Araf residues with some D-Araf branching at position 3. The non-reducing end of the arabinan chains has a branched domain of six D-Araf residues. Position 5 of about two-thirds of the terminal and penultimate arabinofuranosyl groups in this domain are occupied by long-chain α-alkyl-β-hydroxyacyl (mycolyl) groups in clusters of four. The arabinogalactan is linked via a →4)-α-L-Rhap-(1→3)-α-D-GlcpNAc-(1→P domain to postion 6 of muramic acid residues of peptidoglycan. Similar motifs are characteristic of other mycobacteria studied. have been found also in the cell-wall of related genera of actinomycetes, Rhodococcus and Nocardia [48]. Lipoarabinomannan of mycobacteria is likewise complex. Although not yet fully elucidated, its basic structure can be presented as shown in Figure 8[49]. The main chain 192 Yuriy A. Knirel

Figure 7. Structural model of the mycolyl arabinogalactan complex of Mycobacterium tuberculosis. MurAc, N-acetylmuramic acid; Myc, mycolyl.

Figure 8. Structural model of the lipoarabinomannan of Mycobacterium tuberculosis. Ins, myo- inositol; man stands for α-D-Manp, α-D-Manp-(1→2)-α-D-Manp or α-D-Manp-(1→2)-α-D-Manp- (1→2)-α-D-Manp; acyl stands for a long-chain fatty acid, e.g. palmitic or tuberculostearic acid. Structures of bacterial polysaccharides 193 is an α-(1→6)-linked D-mannan consisting of 20-26 D-Manp residues and having branched regions, in which each sugar residuebears a single α-D-Manp residue at position 2. An α-(1→5)-linked arabinofuranose chain is attached close to the non-reducing end of the mannan core (presumably at position 2 of the penultimate D-Manp residue). In part further branched α-(1→5)-linked D-Araf oligomers are connected as branches at position 3 of some D-Araf residues. The arabinan domain includes totally 50-70 D-Araf residues. The non-reducing end of the chains may be capped with α-D-Manp or α- (1→2)-linked mannobiose or mannotriose. The mannan core is linked to mannosyl- phosphatidyl-myo-inositol (MPI) anchor, whose lipid moiety includes in most cases one palmitoyl and one tuberculostearoyl group. In some mycobacteria, there are succinate and other ester-linked substituents, which provide the lipoarabinomannan with negative charge.

5. Extracellular polysaccharides Cells of some bacteria, both Gram-negative and Gram-positive, are surrounded by a polysaccharide capsule (K-antigen) or a slime. The extracellular polysaccharides has usually anionic character due to the presence of acidic monosaccharides (uronic and aldulosonic acids) or non-sugar acids (lactic acid ethers, pyruvic acid acetals, amino acids, dicarboxylic acids, esters of phosphoric acid). The bacterial capsule plays a protective role. As cell-wall polysaccharides, most capsular polysaccharides (CPSs) are heteropolysaccharides built up of oligosaccharide repeats. In such bacteria as E. coli, K. pneumoniae and S. pneumoniae they are diverse in structure. The CPSs of E. coli K1, K92 and Neisseria meningitides type B are homopolymers of N-acetylneuraminic acid [1]. That of Sinorhizobium fredii HH103 is a polymer of 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutanoylmino)-L-glycero-L- manno-non-2-ulosonic acid, in which monomers are linked via the 3-hydroxybutanoyl group [50]. The capsule of Typhi (so called Vi-antigen) is a 1,4-linked partially 3-O-acetylated homopolymer of N-acetyl-D-galactosaminuronic acid [1]. Phosphorylated CPSs are rather common. Many of them contain an alditol, e.g. poly(β-D-ribofuranosyl-(1→1)-D-ribitol 5-phosphate) from type B, and thus resemble teichoic acids; others have an oligosaccharide-phosphate repeating unit [51]. Some CPSs have zwitterionic character; for instance, the CPS repeating unit of 638R includes 4-amino-4,6-dideoxy-D-galactose and ether-linked (S)-3-hydroxybutyric acid (Figure 9) [52]. Such CPSs exhibit peculiar biological activities, particularly, initiate immunomodulatory T cell response [53].

Figure 9. Structure of the zwitterionic capsular polysaccharide from Bacteroides fragilis 638R. Hep stands for L-glycero-D-manno-heptose. 194 Yuriy A. Knirel

The K-antigens of E. coli represented by ca. 80 structural types are classified to four groups based on the genetic locus location, the ability to be expressed at low temperatures and the type of lipid anchor [54]. In groups 1 and 4, the CPS at least partially is linked to the core-lipid A region of the lipopolysaccharide. In group 2 and presumably in group 3, part of the CPS molecules are linked to phosphatidic acid, which functions as an anchor, whereas the rest is unsubstituted. The CPSs of K. pneumoniae are similar to those of E. coli group 1 K-antigens, and CPSs of N. meningitides and H. influenzae resemble K-antigens of groups 2 and 3 [49]. In Gram-negative bacteria, CPS may have the same structure as the OPS of the same bacterium or, sometimes, another bacterium. Some CPSs resemble glycoconjugates of the host and, as a result, are non-immunogenic or little immunogenic. In addition to (2→8)-linked polysialic acid of E. coli K1 and N. meningitides type B, such polysaccharides are produced, e.g., by E. coli K5 (N-acetylheparosan) [1], [55] and Pateurella multocida [56] (). Examples of extracellular slime-like polysaccharides are alginic acid found in and some Pseudomonas species [12] and colanic acid (M-antigen) produced by many enteric bacteria [1]. Alginates are highly acidic partially O- acetylated linear polysaccharides built up of D-mannuronic and L-guluronic acids in various ratios. Colanic acid is a branched polymer, and its repeating unit includes six monosaccharides as well as O-acetyl groups and acetal-linked pyruvic acid (Pyr) [1,57]:

In E. coli K-12, colanic acid repeats are ligated to a significant proportion to the LPS core-lipid A moiety [57]. In a number of bacteria, extracellular polysaccharides are components of a biofilm, a structural community of microorganisms encapsulated within a self-developed matrix of polymeric substances and adherent to a living or inert surface. Recently, an important role of biofilm in many infectious processes nas been recognized. For instance, extracellular (1→6)-linked poly(N-acetyl-β-glucosamine), often called polysaccharide adhesin, mediates intercellular adherence and adherence to a solid surface of Staphylococcus species and some other bacteria. A more detailed study of this polysaccharide produced by Staphylococcus epidermidis RP62A revealed a structural heterogeneity due to partial N-deacetylation and partial O-succinylation of GlcN residues giving rise to both positively and negatively charged groups [58]. Structures of bacterial polysaccharides 195 6. S-layers The glycan moieties of some S-layer glycoproteins studied resemble those of LPSs of Gram-negative bacteria as they both contain monosaccharides that are uncommon for other natural , such as D-rhamnose, D-glycero-D-manno-heptose, 3-amino- 3,6-dideoxy-D-glucose and 3-amino-3,6-dideoxy-D-galactose, 2,3-diamino-2,3-dideoxy- D-mannuronic acid etc. [49, 59]. Moreover, S-layer glycoproteins include structurally diverse repetitive long polysaccharide chains analogous to the OPSs and core-like oligosaccharides, as can be exemplified by the glycan of Thermoanaerobacter thermohydrosulfuricus L111-69 [60]:

Another peculiar feature of Th. thermohydrosulfuricus is the termination of the polysaccharide chain with a methylated monosaccharide L-acofriose (compare homopolysaccharide O-antigens), whose addition is believed to stop the chain elongation during biosynthesis. In most bacterial S-layers, glycans are O-glycosidically linked to threonine but in the Th. thermohydrosulfuricus glycoprotein, a novel type of carbohydrate-polypetide O-glycosidic linkage, that between galactose and tyrosine, has been discovered. In the S-layer of Paenibacillus alvei CCM 2051, a different polysaccharide is linked to the protein via a core oligosaccharide that is similar to the core of Th. thermohydrosulfuricus L111-69. This finding shows that certain structural motifs in S- layers may remain conserved even amongst phylogenetically distinct bacteria [59]. Some microorganisms may express differently composed S-layer glycans on the cell surface. S-layer glycoproteins are characteristic also for Archaea but, as opposite to bacteria, they have predominantly short-chain glycans that are N-linked to asparagine. Halobacteria are an exception as they produce long-chain polysaccharides, which are strongly negatively charged due to extensive sulfation [49]. S-layers are non-covalently bound to the outer membrane in Gram-negative bacteria or to peptidoglycan in Gram-positive bacteria. Although one speculate about the biological role of S-layers, their particular function(s) remains largely unknown.

7. Summary Lipopolysaccharide is the main component of the outer membrane of Gram-negative bacteria. It consists of an OPS chain (O-antigen), lipid A and an intervening core oligosaccharide. The OPS defines the serological O-specificity of the bacterium. Some O-antigens are homopolysaccharides but heteropolysaccharides are more widespread and are extraordinarily diverse in composition and structure. The O-antigens consisting of two repetitive homo- or hetero-polysaccharide domains are known. Lipid A is a conserved domain of the LPS, which anchors the LPS molecule into the membrane and is responsible for biological (endotoxic) activities of the LPS. Some bacteria produce a 196 Yuriy A. Knirel truncated LPS that is devoid of any O-antigen or have a single O-antigen repeat (O-unit) linked to the core-lipid A moiety. Teichoic and lipoteichoic acids are characteristic polysaccharides of Gram-positive bacteria that are attached to peptidoglycan or a cell-wall glycolipid, respectively. They are involved in ion exchange and permeability of the cell wall to nutrients and antibiotics. Teichoic acids include a polyol phosphate and often also a glycosyl moiety either in the main chain or as a lateral group. Teichuronic acids or other anionic polysaccharides may replace teichoic acids in some Gram-positive bacteria or under certain environmental conditions. The cell-wall of mycobacteria is distinguished by the occurrence of two major lipoglycans, the mycolyl arabinogalactan complex and lipoarabinomannan, which show little structural diversity. Their carbohydrate moieties are characterised by various non- repetitive motifs. Extracellular glycopolymers are found in both Gram-negative and Gram-positive bacteria. Some exopolysaccharides are bound to the cell surface and build a protective capsule (K-antigen), whereas others are released to the environment as a slime or enter into biofilm, which plays an important role in bacterial virulence. Some bacteria produce extracellular polysaccharides of only one type, whereas others are characterized by various structures. The CPSs having a zwitterionic character are able to stimulate T cell- dependent immune response that can confer protection against bacterial pathogens. Another glycopolymers exterior to the cell wall are S-layer glycoproteins, whose glycan chains are much more diverse in structure than those of eukaryotic glycoproteins. A common theme in the field is the occurrence of the same or closely related polysaccharides in different bacteria, which in some particular cases may complicate serodiagnostics of infectious diseases. Some bacterial glycans contain a domain that resembles host structures, a feature that may help the bacterium to escape adaptive immune response and may lead to post-infectious autoimmune diseases.

Acknowledgements I am grateful to Prof. Dr. Otto Holst (Research Center Borstel, Borstel, Germany) for critical reading of the manuscript.

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