Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Progress in the synthesis of complex carbohydrate chains of plant and microbial polysaccharides, 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: lipopolysaccharides of Gram-negative bacteria, cell- wall anionic polysaccharides of Gram-positive bacteria, including teichoic and lipoteichoic acids, and mycobacterial lipoglycans. Extracellular polysaccharides, which build a protective capsule, participate in biofilm formation or are excreted as slime, are considered too. The occurrence of both monosaccharides and non-carbohydrate groups as components of the polysaccharide is surveyed. Various structural types of the polysaccharides are discussed, including homopolysaccharides and heteropolysaccharides built up of oligosaccharide 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 peptidoglycan (murein) layer in between. The outer leaflet of the outer membrane consists mainly of lipopolysaccharide (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 cell wall. Bacteria of yet another group, Archaea, 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 virulence 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 vaccines. 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 pathogenic bacteria 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 glucans, galactans and mannans are known) but homopolymers of less common sugars and sugar derivatives, such as N-acyl derivatives of 4-amino-4,6-dideoxy-D-mannose 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 hexoses, pentoses, 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 heptoses, 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 monosaccharide 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-glucose N-substituted with N-[(R)-3-hydroxybutanoyl]-L-alanyl; 3, (R)-acetal of pyruvic acid with D-galactose; 4, ether of (R)-lactic acid with L-rhamnose; 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 Pseudomonas aeruginosa 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 Klebsiella 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-fucose 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.