Biofilm Matrix Polysaccharides

One of the most distinctive features that distinguishes biofilms from planktonic populations is the presence of an extracellular matrix embedding the biofilm bacteria and determining mature biofilm architecture (Sutherland 2001; Starkey et al. 2004). Along with expression of proteinaceous adhesins, production of this matrix is essential for maturation of the biofilm structure. The biofilm matrix is a complex milieu essentially composed of water (97%), but it also includes exopolysaccharide polymers, proteins, nucleic acids, lipids/phospholipids, absorbed nutrients, and metabolites (Ghannoum and O'Toole 2001).

3.2.1 Role of the Biofilm Matrix

Although the matrix is a hallmark of bacterial biofilms, its role is not fully understood. The biofilm matrix offers a constantly hydrated viscous layer protecting embedded bacteria from desiccation or from host defenses by preventing recognition of biofilm bacteria by the immune system. The matrix may also play a significant protective role as a diffusion barrier and a sink for toxic molecules (antimicrobials, hydroxyl radicals, and superoxide anions). The biofilm matrix could also inhibit wash-out of enzymes, nutrients, or even signaling molecules that could then accumulate locally and create more favorable microenvironments within the biofilm (Redfield 2002; Welch et al. 2002; Starkey et al. 2004). All these aspects of the putative roles of the matrix could contribute to development of phenotypic resistance of pathogenic E. coli biofilms and lead to persistent infections (Anderson et al. 2003; Justice et al. 2004).

In addition to its protective role, one of the main functions of the matrix is probably also a structural one. The adhesive properties of the matrix enable the bacteria to remain in proximity to the surface and to adhere to each other. Moreover, the interactions between polysaccharides and the other components of the matrix, such as those between cellulose and curli, may participate in three-dimensional growth of the biofilm (White et al. 2003).

Due to biofilm heterogeneity, analysis of the extracellular polymeric substance (EPS) is progressing slowly, and little is yet known about the composition of the biofilm matrix (Sutherland 2001). Several exopolysaccharides found in the E. coli biofilm matrix (cellulose, PGA, colanic acid) are key components of the biofilm matrix, while others such are lipopolysaccharides and capsular polysaccharides may not accumulate significantly in the matrix, but still play an important indirect role in biofilm formation. While these components may coexist in the matrix, our current knowledge seems to indicate that they are subject to very distinct regulatory pathways, the coordinated expression of which remains to be clarified.

3.2.2 Polysaccharides Secreted in the Biofilm Matrix

Secreted polysaccharides have been recognized as key elements that shape and provide structural support for the biofilm (Sutherland 2001). These polymers are very diverse and are often involved in the establishment of productive cell-to-cell contacts that contribute to the formation of biofilms at liquid-solid interfaces, pellicles at air-liquid interfaces, cell aggregates and clumps in liquid cultures, and wrinkled colony morphology on agar plates. Evidence for a structural role of some of these matrix polysaccharides is accumulating, and the regulation of production of these exopolysaccharides is now actively being investigated in different bacteria (Kirillina et al. 2004; Branda et al. 2005; Simm et al. 2005). To date, three exopolysaccharides, P-1,6-N-acetyl-D-glucosamine polymer (PGA), colanic acid, and cellulose, have been detected in the biofilm matrix of E. coli and have been shown to be important for biofilm formation. Poly-P-1,6-N-acetyl-glucosamine

P-1,6-N-acetylglucosamine (P-1,6-GlcNAc) is a polysaccharide polymer known to participate in biofilm formation in Staphylococcus aureus and Staphylococcus epidermidis, where it contributes to their virulence (Mack et al. 1996; Rupp et al. 2001; Gotz 2002; Maira-Litran et al. 2002). P-1,6-GlcNAc, or PGA, was recently identified in E. coli K-12, where the expression of P-1,6-GlcNAc exopolysaccharide polymer is involved in both cell-cell adhesion and attachment to surfaces (Agladze et al. 2005). Moreover, PGA depolymerization by treatment with metaperiodate or a P-hexosaminidase from Actinobacillus actinomycetem-comitans (DspB), which degrade the P-1,6-GlcNAc, leads to nearly complete disruption and dispersion of the biofilm (Wang et al. 2004; Itoh et al. 2005). PGA production depends on the pgaABCD locus (Wang et al. 2004). The E. coli pgaABCD (or ycdSRQP) operon encodes proteins involved in the synthesis (the PgaC glycosyltransferase), export and localization of the PGA polymer. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, it has been proposed that P-1,6-GlcNAc serves as an adhesin that stabilizes biofilms of E. coli and other bacteria such as A. actinomycetemcomitans and Actinobacilluspleuropneumoniae (Kaplan et al. 2004; Wang et al. 2004). Cellulose

Cellulose, the main component of plant cell wall, is a homopolysaccharide composed of D-glucopyranose units linked by P-1^4 glycosidic bonds. Outside of the plant kingdom, cellulose has primarily been thought to be produced only by a few bacterial species such as the model organism Gluconacetobacter xylinum (Czaja et al. 2006). The ability of cellulose to bind fluorescent chemical dyes such as cal-cofluor has provided a convenient screen for cellulose-producing bacteria, showing that cellulose production is a widespread phenomenon in Enterobacteriaceae, including Salmonella enterica serovar Typhimurium, S. enterica subsp. Enterica serovar Enteritidis, and commensal and pathogenic strains of E. coli, Citrobacter spp. and Enterobacter spp. (Zogaj et al. 2001; Solano et al. 2002; Romling et al. 2003; Zogaj et al. 2003; Romling 2005; Da Re and Ghigo 2006; Uhlich et al. 2006). In these bacteria, cellulose production is clearly associated with the ability to form a rigid biofilm at the air-liquid interface; however, these characteristics vary between strains and serovars and are highly dependent on environmental conditions.

Genetic analysis performed in Salmonella serovar Typhimurium and Salmonella serovar Enteritidis showed that cellulose synthesis genes are organized as two divergently transcribed operons, bcsABZC and bcsEFG, which are constitutively expressed and composed of genes sharing homologies with genes of the bacterial cellulose operon of G. xylinum (Gerstel et al. 2003; Romling 2005).

Although these genes are present in most enterobacterial genomes, including Salmonella, E. coli, Shigella, Enterobacter, and Citrobacter (Zogaj et al. 2003), little is known about the function and localization of the bacterial cellulosome. BcsA is a cytoplasmic membrane protein whose cellulose synthase activity is allos-terically controlled upon binding of a small molecule called cyclic-di-GMP (c-di-GMP), a ubiquitous second messenger produced and degraded by diguanylate cyclase and phosphodiesterases, respectively. In Gluconacetobacter xylinus, it has been suggested that c-di-GMP binds to BcsB, promoting an allosteric change in the protein conformation that leads to its activation (Mayer et al. 1991). Recently, a PilZ domain believed to be part of the c-di-GMP binding protein has been identified in several bacterial cellulose synthases, including BscA, although direct evidence for c-di-GMP binding is still missing (Amikam and Galperin 2006). c-di-GMP is now known to antagonistically control the motility and virulence of single, planktonic cells, on the one hand, and cell adhesion and persistence of multicellular communities on the other (Jenal and Malone 2006; Romling and Amikam 2006).

Genetic analyses performed mostly in S. Typhimurium revealed that the combined and coregulated syntheses of cellulose and curli fimbriae lead to a distinctive pheno-type on Congo red agar plates, the red dry and rough (rdar) morphotype (Zogaj et al. 2001; Solano et al. 2002; Romling et al. 2003; Da Re and Ghigo 2006). While most of what is known of the regulation of cellulose production has been learned from Salmonella, cellulose has also been found in E. coli, where cellulose synthesis is correlated with biofilm formation and expression of multicellular behavior (rdar morphotype), and where treatment with cellulase totally disperses existing biofilms (Zogaj et al. 2001, 2003; Romling 2002; Da Re and Ghigo 2006). Colanic Acid

Colanic acid is a negatively charged polymer of glucose, galactose, fucose, and glucuronic acid that forms a protective capsule around the bacterial cell under specific growth and environmental conditions (for example, colanic acid is not produced in rich medium at 37°C). Colanic acid has a structure and assembly pathway very similar to that of the group I capsule and is therefore often included in that category. However, in contrast to most capsular types, a significant portion of the colanic acid produced is released into the extracellular medium.

Colanic acid synthesis involves 19 genes located in the same cluster, named wca (formerly known as cps) (Stevenson et al. 1996). It is induced by the three-component system RcsC/RcsD/RcsB and requires an auxiliary positive transcription regulator RcsA (Majdalani and Gottesman 2005). Although the signal for sensor kinase RcsC remains uncharacterized, RcsC seems to respond to complex cues such as desiccation, osmotic stress, the level of periplasmic glucans, and growth on a solid surface (Ophir and Gutnick 1994; Sledjeski and Gottesman 1996; Ferrieres and Clarke 2003). A recent observation also indicates that colanic acid is induced by near-lethal levels of a subset of b-lactam antibiotics that may exacerbate the formation and persistence of a biofilm (Sailer et al. 2003). Although colanic acid has been reported to impair initial bacterial attachment, its synthesis is consistently upregulated within biofilms, and its production plays a role in the development of the mature biofilm architecture (Prigent-Combaret and Lejeune 1999; Prigent-Combaret et al. 1999; Danese et al. 2000b; Hanna et al. 2003). Interestingly, several groups have reported that expression of the colanic acid capsule may also have an inhibitory effect upon the biofilm ability of E. coli strains by masking autotransporter adhesins such as Ag43 and AidA (Hanna et al. 2003; Schembri et al. 2004).

3.2.3 Cell Surface Polysaccharides

Cell-surface glycoconjugates play a critical role in interactions between bacteria and their immediate environment. Besides released polysaccharides that have been identified as part of the biofilm matrix, surface polysaccharides can also contribute to the biofilm phenotype. Most E. coli isolates produce a complex layer of serotype-specific surface polysaccharides: the lipopolysaccharide (LPS) O antigen and capsular polysaccharide K antigen. Variations in the structure of these polysaccharides give rise to 170 different O antigens and 80 K antigens that enable typing of most enterobacteria. Lipopolysaccharides

The lipopolysaccharide (LPSs), also known as endotoxin, is a glycolipidic polymer that constitutes the main component of the outer leaflet of the outer membrane of Gram-negative bacterium. Constitutively expressed, LPS consists of three parts:

lipid A, which is the toxic component and to which the core region is attached, which can be divided into an inner and an outer part; and finally the O-antigen polysaccharide, which is specific to each of the 170 E. coli serogroups.

The sugar residues in lipid A and the core region are decorated to varying extents with phosphate groups and phosphodiester-linked derivatives, ensuring micro-heterogeneity in each strain. The lipid A part is highly conserved in E. coli, while the core contains six different basic structures, denoted R1-R6. The O-polysaccha-ride is linked to a sugar in the outer core. The O-antigen, absent in rough strains such as E. coli K-12, usually consists of 10-25 repeating units containing two to seven sugar residues. Thus, the molecular mass of the LPS present in smooth strains will be up to 25 kDa. Finally, in some serotypes, the core can be bound to group 1 capsule, forming KLPS (Raetz 1996).

More than 50 genes are required to synthesize LPS and assemble it at the cell surface. Some of these genes are clustered in large operons or are isolated on the E. coli chromosome. Mutations affecting LPS synthesis have been shown to affect E. coli ability to adhere to abiotic surfaces (Genevaux et al. 1999), suggesting a role of LPS in adhesion processes. Studies investigating E. coli mature biofilm formation also reported that LPS mutation could lead to a significant decrease in biofilm capacity. Inactivation of waaG, coding for a LPS core glycosyltransferase that resulted in a truncated LPS core structure and a deep rough phenotype, completely abolished biofilm formation of uropathogenic strain 536 without affecting its growth rate in liquid culture, suggesting that an intact LPS core is a major factor in adhesion to abiotic surfaces in E. coli strain 536 (Landini and Zehnder 2002; Beloin et al. 2006). However, since alteration of LPS synthesis can also impair Type 1 pili and colanic acid expression as well as bacterial motility, the phenotype of LPS mutants could still be attributed to indirect effects.

In contrast, like capsule masks the function of short membrane adhesins, it has recently been reported that the reduction in LPS expression caused by an rfaH mutation could unmask E. coli adhesins and therefore allow initial adherence and/ or biofilm formation (Beloin et al. 2006).

These results suggest two distinct mechanisms by which LPS either promotes or inhibits biofilm formation, mainly by interacting with cell-surface-exposed adhesion factors.

  1. 2.3.2 Capsules
  2. coli capsules are surface-enveloping structures comprising high-molecular-weight capsular polysaccharides that are firmly attached to the cell (see, however, the discussion in this section below). They are well-established virulence factors, often acting by protecting the cell from opsonophagocytosis and complement-mediated killing (Whitfield 2006). The 80 different capsular serotypes in E. coli were originally divided into more than 80 groups based on serological properties. Despite the diversity of bacterial capsule glycoconjugates and the complexity of their synthesis and assembly processes, later revisions classified capsules into four groups. E. coli group 1 and 4 capsules share a common assembly system, and this is fundamentally different from that used for group 2 and 3 capsules (Whitfield and Roberts 1999; Whitfield 2006).

As seen for colanic acid and LPS, the E. coli capsule has also been shown to play an indirect role in biofilms by shielding of bacterial surface adhesin (Schembri et al. 2004). While capsular polysaccharides are linked to the cell surface of the bacterium via covalent attachments, capsule can be released into the growth medium as a consequence of the instability of phosphodiester linkage between the polysaccharide and the phospholipid membrane anchor (Roberts 1996; Whitfield 2006). Recently, group II capsular polysaccharides were shown to be significantly released into the culture supernatant and to display antiadhesion activities toward both Gram-positive and Gram-negative bacteria, therefore antagonizing biofilm formation by a mechanism distinct from steric hindrance of surface adhesin (Valle et al. 2006). Capsule-mediated biofilm inhibition is widespread in extraintestinal E. coli, suggesting that the anti-biofilm property of group II capsular polysaccharides could also play a role in the biology of these pathogens. Group II capsule may contribute to competitive interactions (bacterial interference) within bacterial communities, or to modulating E. coli's own adhesion to surfaces encountered during the intestinal or urinary tract colonization process. Analyses showed that group II capsular polysaccharides affect biofilm formation by weakening cell-surface contacts (initial adhesion), but also by reducing cell-cell interactions (biofilm maturation). Interestingly, direct treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens, which may be used in the design of new anti-biofilm strategies (Valle et al. 2006).

We have seen that E. coli biofilm initiation and maturation can involve many different factors. None of them, however, are strictly required. Indeed, E. coli's ability to form a biofilm depends considerably on environmental conditions, and even well-demonstrated adhesion factors can be replaced by others. For instance, expression of conjugative pili totally overcomes the need for curli, type 1 pili of flagellar expression (Reisner et al. 2003). These results not only suggest that many different pathways can be used during E. coli biofilm formation, but also that regulatory mechanisms could coordinate the biofilm adhesion and maturation processes.

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