Chapter 13
Bacterial Extracellular Polysaccharides Kateryna Bazaka, Russell J. Crawford, Evgeny L. Nazarenko, and Elena P. Ivanova
Abstract Extracellular polysaccharides are as structurally and functionally diverse as the bacteria that synthesise them. They can be present in many forms, including cell-bound capsular polysaccharides, unbound “slime”, and as O-antigen component of lipopolysaccharide, with an equally wide range of biological functions. These include resistance to desiccation, protection against nonspecific and specific host immunity, and adherence. Unsurprisingly then, much effort has been made to catalogue the enormous structural complexity of the extracellular polysaccharides made possible by the wide assortment of available monosaccharide combinations, non-carbohydrate residues, and linkage types, and to elucidate their biosynthesis and export. In addition, the work is driven by the commercial potential of these microbial substances in food, pharmaceutics and biomedical industries. Most recently, bacteria-mediated environmental restoration and bioleaching have been attracting much attention owing to their potential to remediate environmental effluents produced by the mining and metallurgy industries. In spite of technological advances in chemistry, molecular biology and imaging techniques that allowed for considerable expansion of knowledge pertaining to the bacterial surface polysaccharides, current understanding of the mechanisms of synthesis and regulation of extracellular polysaccharides is yet to fully explain their structural intricacy and functional variability.
13.1 Introduction Bacteria secrete an intricate assortment of extracellular polymeric substances, including polysaccharides, proteins, and nucleic acids, that vary in molecular mass and structural properties (Jayaratne et al., 1993). Extracellular polysaccharides E.P. Ivanova (B) Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, VIC 3122, Australia e-mail:
[email protected]
D. Linke, A. Goldman (eds.), Bacterial Adhesion, Advances in Experimental Medicine and Biology 715, DOI 10.1007/978-94-007-0940-9_13, C Springer Science+Business Media B.V. 2011
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(EPSs) are produced by a broad range of bacterial species. They can be present in many forms, including that of capsular polysaccharides, which are also referred to as “cell-bound extracellular polymeric substances”. These are located on the outermost surface of a wide range of bacteria. The EPS can also be found in its unbound form, or “free EPS” (Dong et al., 2006; Roberts, 1996). In contrast to free EPS (otherwise known as slime), which maintains only limited association with the surface of the bacterial cells, capsular polysaccharides remain connected to cell surfaces by means of a covalent attachment to phospholipid or lipid A molecules present at the bacterial surface (Deng et al., 2000; Sørensen et al., 1990; Whitfield and Valvano, 1993). However, using the extent of attachment as a means to differentiate between capsular and free EPS can be complicated, since capsular polysaccharides may themselves be released into the growth medium (i.e. become “free”) as a consequence of the low stability of the phosphodiester linkage between the polysaccharide and the phospholipid membrane anchor. Moreover, certain free EPS molecules can also remain in close proximity to the cell surfaces in the absence of a detectable membrane anchoring mechanism (Roberts, 1996; Troy et al., 1971). These tightly attached capsular polysaccharides form a distinct structural layer (the capsule) which encloses the cell and serves as a protective layer. This layer acts as a shield, protecting the cell from major bacterial pathogens (Dong et al., 2006). The commonly described biological functions of bacterial EPS include resistance to desiccation (Roberson and Firestone, 1992), protection against nonspecific and specific host immunity, and adherence. Capsular polysaccharides are major determinants of virulence for many pathogenic bacteria as they may act to inhibit the complement-mediated bactericidal activity of human serum. They may also impede antibody opsonization and phagocytosis, promote colonisation of tissues and surfaces, and induce inflammation and aberrant complement activation that can be damaging to host tissues. In addition, the presence of the capsule may significantly increase the survival of a pathogen in the environment by acting as a permeability barrier that facilitates selective transportation of nutrients, whilst at the same time providing a protective barrier that excludes harmful substances such as antimicrobial agents. Most significantly, EPS play a major role in mediating the bacterial colonization of surfaces, biotic and abiotic, by enabling cell adhesion and co-aggregation via dipole interactions, covalent or ionic bonding, steric interactions, and hydrophobic association (Beveridge and Fyfe, 1985; Beveridge and Graham, 1991; Bruno and Yves, 2002; Decho, 1990; Flemming, 1995). Components of free EPS can be released onto surfaces that might otherwise be considered unfriendly for bacterial settlement, such as mineral surfaces. These pre-condition the target surface by adsorbing to it hence making it more attractive for bacterial attachment. In Gramnegative bacteria, lipopolysaccharides represent a significant component of the outer leaflet of the outer membrane. As a result, these lipopolysaccharides are also likely to have a profound influence over the adhesive behaviour of the microorganism (Fletcher, 1996). In Escherichia coli, the lipopolysaccharide core and O-antigen have been identified as key components that mediate bacterial binding with inorganic surfaces. Hydrogen bonding is an important factor in controlling O-antigen adhesion to inorganic molecules such as Si3 N4 , TiO2 , SiO2 , and Al2 O3 (Strauss
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et al., 2009), and they also facilitate aggregation with other cells (Sheng et al., 2008). The length and heterogeneity of the O-antigen may also contribute to the adhesive interactions between the microorganism and its environment (Abu-Lail and Camesano, 2003; Strauss et al., 2009). Equally, the ability of a bacterium to attach and adhere to biotic targets such as host tissues and other microorganisms is also strongly influenced by the secretion of EPS. The temperature, solution pH, electrolyte and macromolecule concentration, and adsorbent surface chemistry will directly influence the chemical composition and structure of the EPS substances that are responsible for the surface conditioning (Cheng et al., 1994; Ong et al., 1994).
13.2 Bacterial Polysaccharides Chemistry and Structures Bacterial polysaccharides are composed of repeating monosaccharide units that are joined by glycosidic linkages. They can take form of homo- or heteropolymers, with heteropolysaccharides generally comprised of oligosaccharide repeating units. Bacterial cell surface polysaccharides, such as lipopolysaccharides and capsular polysaccharides, are characterised by enormous structural complexity. The efforts to understand their biosynthesis and export are driven by their importance in host– bacteria interface biology, their strong association with bacterial pathogenicity, and their important role in microorganism adhesion and biofilm formation (Woodward et al., 2010). Such factors as the solution chemistry, abundance of nutrients, and the cell growth phase exert a significant effect on the nature and the distribution of the bacterial polysaccharides (Omoike and Chorover, 2004). Bacterial capsular polysaccharides are generally linear and comprised of regularly repeating subunits of one to six monosaccharides. Their molecular weight ranges between 100 and 1000 kDa. Lipopolysaccharides are comprised of the lipid A which is embedded into the bacterial membrane, a core oligosaccharide, and a polysaccharide, otherwise known as O-antigen, with repeating units of two to greater than 100 (Strauss et al., 2009). The chemical structure of lipid A is highly conserved, with the classical backbone of lipid A comprising of a β-1’,6-linked disaccharide of 2-amino-2-deoxy-D-glucose (D-glucosamine, D-GlcN) to which fatty acids, typically 3-hydroxyalkanoic acids, are bound via ester or amide linkages. More variable compared to the lipid moiety, the inner region of the core oligosaccharide is typically constructed from 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (LD-Hep). Kdo and phosphate, and less frequently hexuronic and other sugar acid residues, contribute to the anionic nature of both the inner core region and of lipid A. The phosphate groups frequently act as a bridge to an amino alcohol (ethanolamine, Et3 N), to 4-amino-4-deoxy-L-arabinose (L-Ara4N), or to other amino sugar residues. The O-antigenic side chains consist of polymerized oligosaccharide units, and are highly variable in terms of their chemical structure and composition. In addition to the vast potential for complexity and diversity available with the common hexoses alone, the scope of variation within extracellular polysaccharides is often further enhanced by the presence of less common enantiomers, other stereoisomers,
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monosaccharides of different chain length (C5 –C10 ), ketoses, aldoses and branched monosaccharides (Caroff and Karibian, 2003). In addition, the diversity of these polysaccharides is further enhanced by other structural modifications (e.g. positional and cumulative permutations of deoxy, amino and carboxyl functions; esterification, etherification and amidation) and the presence of non-carbohydrate residues (e.g. polyols, carboxylic and amino acids). Furthermore, a wide variety of configurations may be adopted between any two structural units due to the high number of hydroxyl groups in each monosaccharide, each of which may form a glycoside bond. The configuration of O-antigen is flexible and depends on the immediate environment of the bacterium. The preferred conformation of lipopolysaccharides seems to have the O-antigen positioned flat on top of the saturated fats and phospholipids of the lipid A, and possibly on the non-polar sites of the cell surface (Fletcher, 1996). Since the presence of certain terminal sugars within the structure of the O-specific side chain confers immunological specificity of the antigen, the degree of O-chain polymerisation can be used to define biologically distinct serotypes within a species. Depending on the degree of the O-antigen attenuation, a species with a complete O-antigen is classified as smooth (S-type), whereas loss of the O-specific region by mutation renders the strain rough (R-type), or deep-rough (core-defective R-type) if the core oligosaccharide is incomplete. In addition to having a direct effect on the virulence of the strain and its susceptibility to various chemicals, such mutations also significantly influence the surface properties of the microorganism, and hence the nature of the bacterium-host and bacterium-abiotic target interactions.
13.3 Biological Specificity of Extracellular Polysaccharides Extracellular polysaccharides are highly hydrated, generally containing more than 95% water (Costerton et al., 1981). The biosynthesis of extracellular polysaccharides is hierarchical, with the biological repeating unit polymerised from an already-assembled oligosaccharide. In most structural studies, only the “chemical” repeating unit has been determined, whereas the “biological” repeating unit may be any cyclic permutation of that structure (Jansson, 1999; Kenne and Lindberg, 1983). Biological specificity is achieved through chemical diversity, through variations in composition and structure. Hence, it is not surprising that bacterial populations within the same species can express distinctly dissimilar capsular polysaccharides. For instance, over 80 distinct capsular serotypes have been indentified for E. coli (Nesper et al., 2003), 91 capsular serotypes indentified for Streptococcus pneumonia (Weinberger et al., 2009), and 78 for Klebsiella pneumonia (Pan et al., 2008). On the other hand, chemically identical capsular polysaccharides have been identified in a taxonomically diverse range of bacterial species, including the group B capsule of Neisseria meningitidis homopolymer of α2,8-linked N-acetylneuraminic acid (NeuNAc), which is identical to the K1 capsule of E. coli (Grados and Ewing, 1970), while Pasteurella multocida type D repeating disaccharides of glucuronic acid linked to N-acetylglucosamine are identical to the E. coli K5 antigen (DeAngelis and White, 2002).
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13.4 Factors Influencing Bacterial Interactions with Surfaces Extracellular polysaccharides play a crucial role in the bacterial colonization of surfaces by facilitating cell adhesion to biotic (i.e. epithelial and endothelial cells) and abiotic surfaces (i.e. mineral surfaces or medical implants), and to each other (Beveridge and Graham, 1991; Bruno and Yves, 2002; Decho, 1990; Flemming, 1995). Adhesion of the cells to solid surfaces involves a combination of electrostatic interactions, hydrogen bonds, and London dispersion forces. This may result in the formation of biofilms and EPS-mediated interspecies co-aggregation within biofilms that can boost their individual potential for colonisation of various ecological niches (Costerton et al., 1987; Mayer et al., 1999). For bacterial cells, the biofilm can also act as a defence mechanism against predation by phagocytic protozoa, and can serve as a permeability barrier against antimicrobial agents (Costerton et al., 1999). It also confers certain nutritional advantages over the planktonic state, which can be essential for the survival of bacterial populations, by acting as a sorptive sponge that binds and concentrates organic molecules and ions close to the cells (Decho, 2000). A biofilm can comprise bacteria, algae, fungi and protozoa embedded in a dynamic aggregation of polymeric compounds, mostly polysaccharides, with the addition of proteins, nucleic acids, lipids, and humic substances. The composition and quantity of the extracellular polymeric materials that form the matrix of the biofilm will change with the type of microorganism, the age of the biofilm and the environmental circumstances – viz the level of oxygen and nitrogen, the extent of desiccation, temperature, pH, and availability of nutrients (Mayer et al., 1999). This highlights how bacteria can respond to their rapidly changing environment by adapting their capsular polysaccharides (Ahimou et al., 2007). It also explains how bacteria can successfully occupy a diverse range of ecological niches (Costerton et al., 1987). The degree of colonization and the stability of the attachment to an abiotic surface vary with the properties of that surface. Adhesion and settlement improve with surface roughness due to the associated increase in surface area available for colonisation. In addition, the “valleys” on the surface supply the microorganisms with a protected habitat, with reduced shear forces (Donlan, 2002). Factors influencing the rate and degree of attachment to the surface include the surface energy of the structure, the hydrophobicity of the bacterial cell, the presence of fimbriae and flagella, the degree of EPS production and the type of polymeric materials being produced by the cell (Donlan, 2002). Bacterial cells attach more favourably and rapidly to hydrophobic, non-polar rather than hydrophilic surfaces (Flemming and Wingender, 2001). Certain polysaccharide–surface combinations result in irreversible attachment. In these instances, the binding forces between the individual cell and the abiotic surface improve the overall stability of the biofilm matrix (Romaní et al., 2008). Charged non-carbohydrate components such as uronic acids or ketal-linked pyruvates present in the EPS further enhance the anionic nature of the surface polysaccharides of Gram-negative bacteria, thus allowing the association of divalent cations (i.e. calcium, magnesium) to increase the binding forces within biofilm (Sutherland, 2001). These non-carbohydrate components also strongly influence the tertiary structure and the physical properties of EPS.
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13.5 Role of Extracellular Polymeric Substances in Adhesion and Biofilm Formation Capsular polysaccharides and free EPS are present in the outermost layer of a cell. As a result, they form an additional barrier between the membrane of the bacterium and its environment. They have a significant role in the bacterial colonization of surfaces by facilitating cell adhesion to host cells, other surfaces, and each other (Camesano and Logan, 2000; Cescutti et al., 2010). Mineral surfaces and other harsh environments can be pre-conditioned for settlement by the bacterial cell using components of free EPS that adsorb to the target surface, making it more suitable for attachment. The nature of the polymeric substances involved in the conditioning step will be dependent on such environmental factors as temperature, solution pH, electrolyte and macromolecule concentration, and adsorbent surface chemistry (Cheng et al., 1994; Ong et al., 1994). In cell suspensions, EPS are distributed between the cell surface (i.e. capsular polysaccharides), the aqueous phase (i.e. free EPS), or a hydrated matrix in biofilm (biofilm EPS) (Omoike and Chorover, 2004). The distribution of the extracellular polymeric substances is also influenced to a great extent by the nature of the cells’ ambient conditions, such as solution chemistry, abundance of nutrients, and the growth phase of the cells.
13.5.1 Extracellular Polysaccharides of Clinically Relevant Microorganisms For pathogenic bacteria, cell adhesion to host tissues is strongly associated with the presence of extracellular polysaccharides, particularly capsular polysaccharides. For many years, capsular serotyping was the primary method used to classify strains of many encapsulated pathogenic bacteria, since their pathogenicity is affed significantly by the type of capsular polysaccharide. During invasive infection, the interactions between the host and the bacteria are directly mediated by extracellular polymeric substances (Tomlinson, 1993). For example, in S. pneumonia, the capsular serotype is closely associated with its propensity to cause diseases such as otitis media, pneumonia, bacteraemia, and meningitis, or to be carried by the host asymptomatically (Bogaert et al., 2004; Lipsitch and O’Hagan, 2007). Capsular polysaccharides of S. pneumonia are often a primary target of the immune system (Janeway et al., 2001). Consequently, they are used for vaccination (Black et al., 2000) and passive immunization. There is considerable diversity in the types of capsular polysaccharides that exist, thereby assisting the pathogen to elude host immune mechanisms (Shu et al., 2009). Similarly, capsular polysaccharides present on the surface of N. meningitidis have been shown to be a key virulence factor (Caugant et al., 2007), as almost all strains that cause meningococcal disease are encapsulated (Dolan-Livengood et al., 2003; Yazdankhah and Caugant, 2004). Along with averting detection and recognition by immune mechanisms of the host, the capsule may help meningococcal shedding from mucosal surfaces in the host, thus enhancing transmission of the bacteria from
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one individual to another (Dolan-Livengood et al., 2003). The presence of the highly hydrated capsule and “slime” provides protection against desiccation, which ensures host to host transmission and survival of encapsulated bacteria under harsh environmental conditions. Mucoid strains of E. coli, Acinetobacter calcoaceticus, and Erwinia stewartii have higher levels of resistance to desiccation than their corresponding isogenic acapsular mutants, with survival rates decreased from 35 to 5% in mucoid and nonmucoid cells, respectively (Ophir and Gutnick, 1994). Reduced humidity affects the osmolarity in the cells’ immediate environment, as shown by induction of β-galactosidase in a genetic fusion experiment (Ophir and Gutnick, 1994). The expression of capsular polysaccharides in alginate EPS of Pseudomonas aeruginosa and Salmonella typhi also increases with increased osmolarity in the environment (Berry et al., 1989; Pickard et al., 1994). Streptococcus pyogenes (or GAS) is the cause of many important clinical infections, ranging from mild superficial skin infections to systemic diseases such as acute rheumatic fever, streptococcal pharyngitis, streptococcal toxic shock syndrome, and necrotizing fasciitis (Hoge et al., 1993; Kaul et al., 1997). Apart from being a vital stage in the life cycle of S. pyogenes, colonisation of the pharynx by these cells also functions as a reservoir for strains of GAS associated with such infections as necrotizing fasciitis and pharyngitis. Several studies have demonstrated the importance of the hyaluronic acid capsule in the colonisation of the pharynx keratinocytes in vivo (Cywes et al., 2000). CD44, a hyaluronic acid-binding protein that mediates human cell-cell– and cell-extracellular matrix–binding interactions, acts as a receptor for attachment (Ashbaugh et al., 2000). Wessels and Bronze (1994) demonstrated that GAS capsule confers a powerful selective advantage in this environmental niche by injecting mice with an acapsular mutant of the strain that reverted to its encapsulated state at a low frequency (
