Secreted Mucins and the Mucus Layer of the Gut

No discussion about the microbial ecology of the human gut would be complete without considering the importance of the ubiquitous mucus layer that protects the epithelial layer from the lumen contents. Immune system and epithelium wellbeing depends on mucus layer integrity and its ability to regulate the lateral and distal movement of gut contents. Large particles, organisms and chemicals, including undigested food, commensal and pathogenic bacteria, large viruses and toxic compounds, can be retained, rejected or neutralised by the mucus layer.

In a healthy person, mucus, which is produced in large quantities (~10 L per day for the entire body - Derrien2010), provides an important intervening layer between intestinal epithelial cells and the contents of the gut throughout the entire gastrointestinal tract. Mucus is a viscous, polymeric, hydrated gel (∼95% water) formed by large glycoproteins, called mucins. A number of other components involved in immune system defence, such as specialised proteins, lipids and salts, are also present (Derrien2010). The mucus can act as a lubricant, allowing the passage of food (digesta) and waste through the GI tract, as a selective barrier allowing small molecule absorption but preventing larger, potentially harmful molecules from reaching the epithelium surface, and as a niche environment for mucus-adapted mutualist bacteria (mucus bacterial consortium) to grow and thrive (Derrien2010).

Mucus is constantly being produced, but the amount, thickness and physical properties of the layer differs markedly from the start of the GI tract (oral cavity) to its end (distal colon). The thinnest mucus layer is found in the jejunem, which makes sense because this is where most of our food is digested and absorbed (Table 1). The thickest mucosal layers, on the other hand, are found in the colon, where gut bacteria concentrations are the highest (Atuma2001).

Table 1. Location and thickness of mucus layers throughout the gastrointestinal tract (Atuma2001, Derrien2010).

It should be noted that the mucus barrier consists of two, clearly demarcated layers: a firm, water-insoluble layer closest to the enterocyte surface, and a loose, water-soluble layer, which facilitates movement of digesta through the GI tract. The outer, more mobile layer is constantly being replaced as food waste and potentially dangerous agents, such as fungi, pathogenic bacteria and viruses, get trapped in it and are expelled from the gut. The inner, adherent layer is a more permanent fixture but still allows the movement and absorption of small molecules, gases and even virus-sized particles (200 nm - Bansil2006) (Derrien2010). Mucus thickness is greatest in the colon where a substantial loose layer (0.6 mm) overlays a firm inner layer (0.2 mm, Johansson2016, Atuma2001). Bacteria are unable to reside in this firm layer, whereas important consortia of microbes live amongst the structurally similar outer one. For example, the widespread bacterium, Akkermansia muciniphila, can use mucin as its sole source of energy, carbon and nitrogen.

The mucins

Apart from water, the major components of mucus are a variety of mucins (see Table 2). These are large glycoproteins ranging in size from about 2 to more than 50 million Daltons (MDa). To put this into perspective, the molecular weight of galactose, a key component of all mucins, is only 180 Da! The mucins come in two flavours: either they're membrane-bound forms expressed on the surface membranes of intestinal epithelial cells (and other tissues), or they're produced and secreted by various glands and tissues such as the salivary gland, stomach, colon and parts of the small intestine (Table 2).

Table 2. Some important human mucins relevant to the gut microbiome (highlighted). From Derrien2010, Johansson2016 and Sheng2022.

Mucin is produced by intestinal goblet cells (Figure 1) where it is slowly - albeit continously - released (basal secretory pathway) (Derrien2010). Copious and rapid release (aka compound exocytosis) can also occur in response to certain stimuli (Ca²⁺-dependent pathway), such as gut microbes (pathogens or commensals), inflammatory cytokines, bile salts, NO, LPS and other toxins (Liu2020e, Grondin2020).

Figure 1. Goblet cells are responsible for producing and secreting MUC2, the most important intestinal mucin. The protein backbone of mucin is produced in the endoplasmic reticulum and glycosolated occurs in the Golgi apparatus, before forming storage granules (goblet cell derived from Wikipedia: Illustration from Anatomy & Physiology, Connexions).

Mucin 2

The secreted intestinal mucin, mucin 2 (MUC2), is invariably the main structural component of mucus in the small and large intestines, and is the most important for the bacteria living in, or feeding on, the mucus layer. This glycoprotein mainly comprises a large number of oligosaccharides attached to a peptide backbone of many repeating amino acid sequences rich in proline, threonine and serine amino acids (Dekker2002, Derrien2010) (Figure 2).

For a more detailed discussion on MUC2 biosynthesis, structure and secretion, see the section on MUC2 Biosynthesis and Secretion.

Lack of mucus

A disturbance to the integrity of the protective mucus layer is implicated in a number intestinal diseases, including IBD, Crohn's disease and 'leaky gut syndrome' (to be added in the Health section, Derrien2010). In a telling animal experiment, a mucin 2 gene knockout (muc2-/-) breed of mice spontaneously developed colitis during development (VanderSluis2006). The pups failed to gain weight, developed diarrhoaea (7 weeks), and showed occult blood loss (8 weeks), clearly indicating a requirement for mucin for healthy growth and development. Interestingly, even the heterozygous mice (muc2+/-) were found to be more susceptible to colitis-inducing agents (dextran sulfate) compared to their wild-type relatives.

A dramatic reduction in mucin production is also observed in colon epithelial tissue that is progressing from precursor lesions (polyp, adenoma) to a carcinoma (Pothuraju2020). Carcinoma tissue produces no secreted mucus.

Figure 2. The MUC2 apoprotein is biosynthesised in the ER (left), where it undergoes dimerisation and N-glycosylation (McGuckin2011). Transfer to the Golgi apparatus allows further extensive O-glycosylation. The homodimer is encapsulated into secretory granules whereupon further trimerisation occurs under low pH and high calcium concentrations (Johansson2011) (picture modified from McGuckin2011).

MUC2 and Gut Bacteria

There are gut bacteria that specialise in binding to, degrading and consuming mucins. It is estimated that 1% of the anaerobic colonic microbiome is responsible for mucin breakdown, and this mucolytic activity appears independent of the number of facultative anaerobes present (Miller1981). In fact, the vast majority of healthy people possess at least one of these important microbes, but the ability of any species to utilise MUC2 in its entirety is rare (Tables 3 & 4, bottom of page). This is why other mutualistic and opportunistic bacteria coalesce around these mucin-degrading initiators, and together they form a metabolic microbial consortium able to fully metabolise the peptidoglycan (Figure 3). For example, Akkermansia muciniphila, a core species of the mucosa, can cleave and consume all monosaccharide residues of the O-glycan oligosaccharides and break down the peptide backbone that binds them.

Figure 3. Examples of competitive mucin-grazing commensal bacteria (bacteria at the bottom) and the opportunists that exploit the resources created by the grazers (top). It should be noted that commensals such as A. muciniphila and B. thetaiotaomicron can cleave more glycosidic substrates than those indicated.

Bifidobacterium bifidum strain PLR2010 has been shown to forage on MUC2 mucin as well (Turroni2010). Genomic analysis revealed the prerequisite glycolytic enzymes required to break down key core O-glycan structural motifs (Figure 4).

Figure 4. An example of a predicted metabolic pathway for the mucolysis of a Core-2 derivative of MUC2 by Bifidobacterium bifidum PLR2010 (Turroni2010).

Other saccharolytic bacteria, such as Bacteroides thetaiotaomicron, can initiate cleavage of the O-glycans by removing sialic acid and fucose residues and then consume most of the liberated sugars, although it cannot digest sialic acid. This process allows symbiotic microbes to either dine on the un-utilised sugars or attack the remaining accessible ones, like Gal (e.g. Escherichia coli) or GlcNAc. It has been shown that E. coli growth in mice intestines is limited when antibiotic "knockdown" of mutualist bacteria has occurred. This implies E. coli requires other mucin grazers to initiate O-glycan depolymerisation (Sonnenburg2005, Salyers1979). Other bugs are amino acid consumers and have the ability to degrade the protein backbone.

K-Strategists versus r-Strategists in Mucus Metabolism

Generally, bacteria that possess broad and efficient resource gathering strategies typically grow slower and are termed K-strategists (Andrews1986, Pettersen2021). They compete effectively in crowded environments, especially where resources are hard fought or elusive. r-Strategists, on the other hand, tend to pursue an opportunistic metabolic lifestyle, thriving under conditions that are uncrowded and resource-rich (Pettersen2021). In the context of mucus bacterial consortium, A. muciniphila would be considered a K-strategist, while E. coli would compete effectively (an r-strategist) with the slower growing bacteria when mucin has been sufficiently degraded (Figure 5).

Figure 5. K-Strategists are those organisms that compete well in crowded environments because they are efficient at gathering resources. The carrying capacity (K) tends to stabilise quickly for these organisms. r-Strategists prefer open spaces and rich, easily consumable food resources, resulting in boom-bust cycles. Opportunists, such as E. coli and V. cholerae, rely on initial breakdown of complex mucin glycans before proliferating to a point of population dominance.

Over reliance on mucin as the primary glycan source

In healthy people, the formation and breakdown of mucin is continous, but stable over time. However, it has been observed that UC patients have a thinner mucus layer, fewer goblet cells and increased levels of mucolytic enzymes have been detected (Derrien2010).

In the absence of dietary glycans, mucin-consuming mutualists like B. thetaiotaomicron signal to the epithelial layer to release more mucus in order to sustain itself. Signalling is also increased when the mucin foragers interact with other members of the metabolic consortium (Mahowald2009), and probably with opportunistic bacteria, such as E. coli, as well (see Triumvirate mutualism topic in Gut Microbe Symbiosis). So, in the event of a sustained limit of dietary glycans, one might expect rapid digestion of mucus and r-strategist blooming if there is an overabundance of mucolytic initiators relative to the availability of dietary glycans. It has been suggested that too many mucin-associated bacteria can compromise the integrity of the mucus layer and cause inflammation (Png2010).

Excess mucin breakdown generates nutrients attractive to opportunists. For example, the pathogenic enterohaemorrhagic E. coli strain picks up a trail of fucose in the lumen released as a result of B. thetaiotaomicron breakdown of mucins (Xu2003a). It detects fucose using a two-component signal transduction system, FusKR, and upregulates virulence and metabolic genes, which allows it to colonise and attack epithelial tissue (Pacheco2012).

An overabundance of Bacteroides fragilis has been demonstrated, in vitro, to encourage the growth of sulphate-reducing bacteria, which convert the sulfate hydrolysed from mucins to inflammation-inducing hydrogen sulfide (Willis1996). Using continous culture experiments involving the inoculation of faecal samples supplemented with mucin, a marked increase in Desulfovibrio sp., with concomitant toxic H₂S levels, was observed (Willis1996, Derrien2010). Mucin supplemented co-cultures of B. fragilis with the SRB, Desulfovibrio desulfuricans, confirmed this interdependency (Willis1996).

Enrichment of opportunistic Proteobacteria species - a case study

Raimondi and coworkers examined what happens when faecal samples (including mucosal bacteria) are subjected to repetitive enrichment on mucin media using this substrate as the only source of carbon, nitrogen and energy (Raimondi2021). After three consecutive colonisation enrichments of samples taken from five healthy volunteers they observed a marked increase in opportunistic Proteobacteria species and decreases in important mutualists such as members of the Bacteroidales order, and from the genera Faecalibacterium, Streptococcus, Blautia and Butyricicoccus spp. as compared to the original composition. Interestingly, all five samples enriched three Eubacteriaceae species not normally observed at high levels, namely Clostridium cocleatum, Clostridium disporicum and Paraclostridium benzoelyticum (Raimondi2021). These species were shown to only require mucin to grow. Other species of low-prevalence and not detected in the original slurries (<1% of the population) were also encouraged to grow under enrichment conditions. Theres include Eggerthella (lenta ?) (4/5 samples); Clostridium innocuum, Enterococcus durans, Erysipelatoclostridium (3/5 samples each); Angelakisella (massiliensis ?) and Eubacterium nodatum (2/5 samples each). On the other hand, A. muciniphila was enhanced in only two samples.

By artificially optimising this mucin-degrading bacterial community it is perhaps unsurprising to see that opportunistic bacteria pursuing an r-strategist lifestyle start to dominate. Mucinophilic organisms need only initiate the breakdown of mucin before other, more proliferative bacteria take over. Unfortunately, the researchers were only able to use commercially available pig stomach mucins (mainly MUC5AC and MUC6), which differ in their O-glycan profile to MUC2 (Raimondi2021).

Despite some limitations, these experiments are perhaps indicative of what occurs when the ecology of the mucosal flora is perturbed. When the population of a keystone species with inflammation-modulating abilities is altered it potentially allows inflammation-inducing species to proliferate and cause tissue injury.

Bacterial adhesion to mucin

MUC2 is an amphiphilic (possessing both hydrophobic and electrophilic parts) polymeric gel and has an overall negative charge due to the sulfate and sialic acid residues (Derrien2010). As such, it can attract and bind numerous types of substances (but not anionic structures) (Bansil2006). The array of O-glycosidic oligosaccharides of MUC2 are sufficiently conserved between people to suggest a role of mucin as a structurally supportive scaffold for beneficial bacteria. Having co-evolved with the host, these commensals most likely seek to bind with mucins to enjoy the benefits of reduced competition, increased resistance to being washed out of the system and use of the glycoprotein as a prebiotic nutrient source. In turn, there is no evidence that mucin is bacteriocidal, suggesting the host is deliberately encouraging mucus colonisation (Derrien2010). Furthermore, there is a suspicion the host may help select desirable types of bacteria through the modification of O-glycosyl binding site patterns (Arike2016, Juge2012).  There is also evidence to believe that the bugs need to bind to mucus in order to initiate colonisation (Juge2012, Etzold2014a).

In order to achieve binding it seems many microbes express specific adhering proteins, called adhesins, on their cell surfaces (Juge2012).

Mucus-binding proteins (MUB) and other methods of binding

Mucus-binding proteins (MUBs) are an important class of cell surface-expressed proteins that allow several Lactobacillus species to bind to MUC2 (Boekhorst2006). In particular, well known gut commensals, such as Lactobacillus gasseri, Lactobacillus acidophilus and Lactobacillus johnsonii, possess proteins with a high number (9-13) of MUB domains  (Boekhorst2006). These Gram-positive cell surface proteins contain a C-terminal sortase recognition motif that allows the organism to covalently bind to the the peptidoglycan (Etzold2014a). The non-motile Lactobacillus species are well known to have an association with mucus. Other commensals, such as Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Levilactobacillus brevis, Limosilactobacillus fermentum, Pediococcus pentosaceus and Lactococcus lactis subsp. lactis also possess this MUB domain protein but to a lesser extent (Boekhorst2006).

Lacticaseibacillus rhamnosus has been reported to use an adhesin-like protein expressed on the surface called mucin-binding factor (MBF). The organism uses this protein and pilus-mediated mucosal adhesion, to bind to MUC2. This organism is believed to only temporarily colonise the gut and is described as being transient (allochthonous) (vonOssowski2011).

Multifunctional proteins also play a role as potential microbe/mucus binding moieties (Juge2012). For example, Limosilactobacillus fermentum possesses mucus/mucin-binding proteins that are associated with an ATP-binding cassette transporter also known to bind to mucins.

Flagella and pili

Most Lactobacillus and other commensal mucin flora are nonmotile. Opportunists and pathogens, on the other hand, tend to be motile using flagella, pili or other methods of propagation. Being able to move independently allows pathogens, such as V. cholerae, Salmonella and E. coli, to penetrate into the mucus matrix and attack epithelial cells, but the extracellular appendages are also important for binding to mucins (Juge2012). L. rhamnosus has been shown to use mucus-binding pili to anchor itself to the mucins, allowing the probiotic to avoid rapid washout from the GI tract. Pili-mediated mucin binding has been implicated in Bifidobacteria species colonisation in the murine gut, and in bacterial autoaggregation in vitro by Lactococcus lactis (Etzold2014a).

Blood group antigen adhesins

The Lewis antigen system is one of over 40 human blood group systems that are used to determine the risk of antibody reactions to certain antigenic structures on the surface of red blood cells (RBC) in the event of a blood transfusion.  The Lewis system is based on the presence or absence of two fucosyltransferase genes, the Secretor gene (fut2, Se) and the Lewis gene (fut3, Le). Between 18-22% of the population have a recessive fut2 gene due to single nucleotide polymorphisms and these people who lack the functional enzyme are termed non-Secretors. In addition to RBC, FUT2 is also expressed by the intestinal epithelium (Wacklin2014) and people with a non-functional gene lack the ability to produce terminal fucose residues in their mucin. The absence of this terminal fucose has real consequences for how the microbiota interact with mucin and how it affects human health (Kashyap2013).

The downside to being a Secretor is that a number of enteric pathogens, such as Campylobacter jejuni, Helicobacter pylori, Vibrio cholerae and gastroenteritis-causing viruses (e.g., Norwalk virus), target human histo-blood group antigens expressed on mucins for the purposes of binding (Juge2012, Kobayashi2009). As mentioned previously, the pathogenic enterohaemorrhagic E. coli strain is chemotactically attracted to fucose produced at sites of mucolysis.

Conversely, non-Secretors are more likely to develop intestinal diseases associated with disturbance of the mucus layer (Wacklin2014), such as Crohn's disease and primary sclerosing cholangitis (Kashyap2013). Furthermore, a change in alpha diversity in faecal microbiota appears to be secretor status-related; healthy non-Secretor (sese) controls were shown to have significantly lower diversity compared to Secretor control subjects (SeSe or Sese) or Crohn's patients with either secretor or non-secretor status (Rausch2011). Loss of diversity was confirmed by Kashyap and coworkers who used fut2 knockout (gnotobiotic, humanised) mice with human faecal transplants (Kashyap2013). Non-terminal fucosylation due to the lack of fucosyltransferase 2 led to significant increases in Bacteroides, Parabacteroides, Parasutterella and Eubacterium species, at the expense of members of the Clostridales order when animals were fed a polysaccharide rich standard diet. However, a switch to a polysaccharide-deficient, high glucose diet showed no difference between the groups (Kashyap2013).

Table 3. Important mucin-degrading commensal gut bacteria (from Pu.biome, Derrien2010 and Tailford2015 - (highlighted)).

Table 4. Gut bacteria known (or predicted) to reside in the mucosal layer.