Quantitative Measurement of Mucolytic Enzymes in Fecal Samples

Background

The gastrointestinal tract (GI) is home for trillions of microorganisms which play diverse functions in the physiological processes (Sommer and Backhed, 2011). Commensal gut microbiota process undigested food, provide energy, nutrients and vitamins, activate the immune system, and prevent pathogens from infecting the intestinal mucosal tissue (Round and Mazmanian, 2009; Pickard et al., 2017). Despite these beneficial roles, gut commensal microorganisms may act as opportunistic pathogens when they get the opportunity to colonize intestinal epithelial barrier and invade into the mucosal tissue. However, a gel like mucus layer above the apical surface of the epithelial cells throughout the intestinal tract ensures physical separation of commensal microbes from the intestinal mucosal tissue and helps maintain intestinal homeostasis (Pullan et al., 1994; Linden et al., 2008; Atuma et al., 2011; Johansson et al., 2011; Juge, 2012). In the large intestine, mucus barrier is very thick, about 700 nm, and can be divided into two distinct layers – a thick outer layer and a thin inner layer (Johansson et al., 2008 and 2011). While the outer layer is nutrient rich, easy to be dislodged, and often colonized with anaerobic bacteria, the inner layer is firmly attached to the epithelial layer and is mostly sterile (Johansson et al., 2008 and 2011).

The mucus is primarily composed of glycoprotein called mucin, produced by goblet cells which are a type of columnar epithelial cells in the intestinal tract. Upon synthesis, mucin proteins are O-glycosylated or N-glycosylated with oligosaccharides and transported to the cell surface or secreted outside (McGuckin et al., 2011). Secretory mucins are heavily O-glycosylated and are homo-oligomerized via inter-molecular disulphide bond formed between the cysteine-rich D domain at the C and N terminus (Thornton et al., 2008). The major mucins in the outer layer that oligomerize to form the matrix are MUC2, MUC5AC, MUC5B, MUC6, and MUC19 (Thornton et al., 2008; McGuckin et al., 2011). The mucus is embedded with many antimicrobial peptides and immunoglobulins, which also keep the inner mucus layer sterile (McGuckin et al., 2011). On the other hand, oligosaccharides of the mucin serve as ligands and a source of nutrients for many anaerobic bacteria. Thus, several intestinal commensals as well as pathogens produce mucolytic enzymes, such as sulphatase, proteases, neuraminidases, α-glycosidase, β-glycosidase, β-galactosidase, fucosidase, β-N-acetylglucosaminidase, α-N-acetylgalactosaminidase, etc., to degrade mucins (Corfield et al., 1992; Linden et al., 2008; Johansson et al., 2011; Desai et al., 2013). Based on the diversity and complexity of mucin oligomers, cooperative actions are required from a number of enzymes as mentioned above for the degradation of mucins (Lombard et al., 2014). The major mucosa-associated bacteria belong to the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Verrucomicrobia (Derrien et al., 2010; Tailford et al., 2015).

Enzymatic degradation of the mucus layer allows gut commensal bacteria or pathogen to breach the mucus barrier (Khan et al., 2020). Therefore, increased abundance of mucolytic bacteria facilitates enteric infection and is associated with inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis (Prizont, 1982; Carroll et al., 2010; Png et al., 2010; Hansson, 2012; Sheng et al., 2012). Thus, the level of mucus degrading enzymes in the colon could be a predictive marker for IBD. Measurement of mucolytic enzymes is also very useful in studies aimed at dissecting the mechanism of IBD pathogenesis in experimental or clinical settings.