Exopolysaccharides (EPS) are extracellular biopolymers that are produced by many species of microorganisms, including
In terms of structure, two main groups of EPS produced by bacteria are distinguished: homo- and heteropolysaccharides. Homopolysaccharides consist of one type of monosaccharide moiety, typically glucose or fructose, and have a simple structure. Differences between their various types are related to the characteristics of the primary structure, such as the type of monomer and bonds between the monomer subunits. Heteropolysaccharides have a more complex structure. They are composed of monosaccharides belonging to at least two different types. The structure of individual heteropolysaccharides may vary considerably. The common feature for most of them is the presence of glucose, rhamnose and galactose, found at different ratios (Welman and Maddox 2003).
There are two known pathways of EPS synthesis in lactic acid bacteria, i.e. the extracellular glucosyltransferase or fructosyltransferase (GT) dependent pathway, and the Wzy/Wzx-dependent pathway (Ryan et al. 2015). The substrate used for the synthesis of homopolysaccharides via the GT dependent pathway is sucrose or another oligosaccharide containing fructose for fructan synthesis (Galle and Arendt, 2014), and the energy used in this process derives from its hydrolysis. Glucosyltransferases carry out the synthesis of glucans, while fructosyltransferases are involved in the formation of fructans (Korakli et al. 2003). Many different types of GT are encoded in the genomes of lactobacilli and only a small part of them have been biochemically characterized (van Hijum et al. 2006). The GT pathway is a relatively simple enzymatic process, which requires low energy inputs (Ryan et al. 2015).
The Wzy/Wzx-dependent pathway is a more complex enzymatic process, with several enzymes and regulatory proteins involved in heteropolysaccharides synthesis and secretion. It initially takes place in the cytoplasm, where repeating units of heteropolysaccharides are assembled by the glucosyltransferases. The Wzx flippase translocates the repeated units into the periplasm, where polymerization by the Wzy polymerase takes place. In the last stage, polysaccharide is transported outside the cell by the OPX protein (Schmid et al. 2015).
Most stages of exopolysaccharide biosynthesis occur in the cytoplasm (Welman and Maddox 2003). Polymerization or EPS secretion into the environment takes place within the cell membrane. In some cases (e.g. dextran, levan) the synthesis of EPS can be entirely extracellular (Schmid et al. 2015).
The amount and type of synthesized heteropolysaccharides depend on the environment, in which the strain grows (De Vuyst and Degeest 1999). Depending on the composition of the medium, temperature, pH and oxygen content, lactobacilli can produce exopolysaccharides of different sugar monomers, interconnected by different types of bonds (De Vuyst and Degeest 1999; Furukawa et al. 2000).
The ability of lactic acid bacteria to produce EPS is commonly used in the dairy industry to improve texture and to stabilize the products (Patel and Prajapati 2013). The physiological role of EPS produced by lactic acid bacteria remains, however, little known. EPS affect the surface properties of bacterial cells. EPS play an important role in adhesion and colonization of the gastrointestinal tract by probiotics. They also promote the formation of the biofilm that facilitates colonization of the epithelium and increases the chance of bacterial survival in the gastrointestinal tract (Lebeer et al. 2009; Stack et al. 2010). EPS enhance the hydrophobicity of the bacterial cell surface, increasing the ability of bacteria to bind to the intestinal mucosa (Sun et al. 2007), and likely participate in enterocyte adhesion as ligands for epithelial surface receptors (Ruas-Madiedo et al. 2006). There are also reports that EPS protect against phage attack and phagocytosis (Gopal and Crow 1993; Comstock and Kasper 2006).
Exopolysaccharides may also act as prebiotics, i.e. non-digestible food ingredients that are beneficial for the host organism by selectively stimulation of growth and/or activity of one or more species of bacteria colonizing the gut. It has been shown that certain bacteria belonging to
The aim of our work was to determine the role of EPS on adhesion of
Composition of the Protein Deglycosylation Mix.
Enzyme | Hydrolyzed bounds | Number of enzymatic units added per 100 μl of reaction |
---|---|---|
Catalyzes the removal of core 1 and core 3 | 400000 U | |
PNGase F | Cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from | 5000 U |
Neuraminidase | Catalyzes the hydrolysis of terminal, non-reducing α2,3, α2,6, and α2,8 linked | 500 U |
β1-4 Galactosidase | Catalyzes the hydrolysis of terminal, non-reducing β1-4 linked D-galactopyranosyl residues from oligosaccharides and glycoproteins. | 80 U |
β- | Catalyzes the hydrolysis of terminal, non-reducing β- | 40 U |
Monosaccharides composition of the EPS produced by
Monomer | % of a monomer in EPS isolated from the strain tested | |
---|---|---|
Maltose | 0.71 | 0 |
Glucose | 30.9 | 15.7 |
Galactose | 53.7 | 31.4 |
Arabinose | 14.6 | 51.6 |
Ribose | 0 | 1.4 |
Many strains of intestinal microbiota exhibit the ability to degrade polysaccharides. The mechanisms they use may differ, but they always include the cell surface processing of a large glycan that precede the cellular import.
In this study we demonstrated for the first time that
In order to carry out experiments that required the inhibition of EPS synthesis and deglycosylation of lactobacilli, it was necessary to inactivate the bacterial cells synthesis during and after the experiments. The effect of inactivation on the adhesion of lactobacilli to Caco-2 cells has been thoroughly tested in our previous work (Markowicz et al. 2016). Formaldehyde inactivation was chosen due to the relatively low impact on the bacterial cell surface properties. It neither cause complete loss of protein activity nor reduce the adhesive properties, which was important for further analysis.
Incubation of bacteria with a mixture of deglycosylating enzymes resulted in a decreased efficiency of adhesion in
The deglycosylation of
Function of glycosidase inhibitors used to inhibit EPS synthesis in
GT inhibitor | The enzyme inhibited |
---|---|
Castanospermine | β-glucosidase |
Deoxymannojirimycin | α-(1→2)-mannosidase |
Kifunensine | |
Swainsonine | α-(1→3)-mannosidase |
Deoxynojirimycin hydrochloride | α-glucosidase |
To the best of our knowledge this is the first report on a new type of the bacterial interaction i.e. properties of one bacterial strain influence the activity of the other strain towards their host. Enzymatic deglycosylation, EPS synthesis inhibition and incubation with