A gutful of microbes

Bacteria make up half of the mass of our colonic content, says Harry Brumer, professor of chemistry at the University of British Columbia in Vancouver, Canada, who is an expert in carbohydrate-degrading enzymes, particularly those from gut bacteria.

The human genome only allows us to produce enzymes to degrade starch and simple sugars, such as sucrose. Fortunately, bacteria degrade most of the dietary fibre from the fruits and vegetables that we consume, which amounts to about 10% of our daily caloric intake, explains Brumer. ‘The bacteria collectively have a very large metabolic capacity that is estimated to be as significant as that of a small organ such as a kidney,’ he adds.

Long-term dietary intake is known to influence the bacterial community – the gut microbiota – but recent research suggests that it can also respond rapidly to dietary changes. A team led by Laurence David at Harvard University, US, gave nine volunteers an animal-based diet consisting largely of meat and cheese for five days, followed by a month off, and then five days of a plant-based diet.¹

Analysis of DNA extracted from faecal samples from the volunteers revealed significant changes to the gut microbiota with diet. The animal-based diet had a greater impact, increasing the abundance of bile-tolerant bacteria, such as Bilophila, and reducing levels of bacteria that specialise in metabolising plant matter. In particular, the [animal-based] diet increased levels of a sulfite-reducing bacterium, Bilophila wadsworthia, which has been linked to IBD. The study backs the hypothesis that diet-induced changes to gut microbiota may contribute to IBD, suggest the researchers.

The gut’s microbial composition may also be linked to certain types of cancer. A team led by Jiyoung Ahn from New York University School of Medicine, US, analysed faecal samples from healthy people and those with diagnosed colorectal cancer. Those with cancer had less diverse gut microbes. Because of the ‘potentially modifiable nature of the gut bacteria’, the findings may have implications for colorectal cancer prevention, predict the researchers.²

Gut microorganisms may also help to determine how effective cancer treatments are, according to a study by researchers at the National Institutes of Health, US. They have shown that mice treated with antibiotics and ‘germ-free mice’, which have not been colonised by microorganisms, respond poorly to cancer therapy.

Gut microbiota affect inflammation not only in the gut but also in other body systems. This raises the question whether the gut microbiota affect inflammatory processes that contribute to cancer and its treatment, say the researchers. The team now intends to work out the molecular signalling by which gut bacteria can send messages to other organs to influence inflammation levels. They also plan to characterise the role that gut bacteria play in the way that tumours respond to therapy. They hope to gain insight into molecular processes linked to inflammation by studying healthy volunteers given antibiotics to alter their gut microbiota.

The researchers are also interested in microbes associated with the mucous layer that lines the GI tract, says joint project leader Giorgio Trinchieri from the NIH’s Center for Cancer Research in Maryland, US.³

‘Mucus has always been there but for a long time people chose to ignore it,’ says Natalie Juge, a research leader at the Institute of Food Research (IFR) in Norwich, UK. At IFR, scientists are working on Gut Health and Food Safety (GHFS), a collaborative research programme that brings together teams from the University of East Anglia, Imperial College London and IFR.

Juge’s research is already revealing the invaluable role this mucus plays in helping the GI tract to populate with essential healthy bacteria.^4,5

‘It’s a field that has exploded’, says Juge. For a long time, immunologists and microbiologists paid little attention to the mucous layer. As recently as five years ago, ‘it was still convenient to ignore it,’ she says. Part of the problem was the sheer complexity of the structures within the mucus, combined with difficulties in studying its gel-like structure, which rapidly dries up in samples removed from the GI tract. Now, techniques are available to study the mucous layer in rodents, and the availability of germ-free mice allows scientists to study the impact of different bacteria.

In the colon, the final section of the digestive system, the mucus is composed of two layers. The loose outer layer is home to gut bacteria while the inner layer is firmly attached to the colon wall and protects it from bacteria.

The mucus is rich in antimicrobial peptides and immunoglobulins, as well as lipids and electrolytes, but what interests Juge most are mucins. These are glycoproteins – proteins with attached sugar (glycan) groups – comprising 20% protein and 80% sugar.

Mucins contain a wide range of glycan structures, which are thought to provide binding sites for gut bacteria. The gut bacteria produce binding proteins (adhesins) that attach to sugars in the mucus. A key mucin is the gel-forming MUC2. In 2006, a study at Erasmus MC and Sophia Children’s Hospital in Rotterdam, the Netherlands, reported that mice lacking MUC2 experienced severe colonic inflammation because gut bacteria were able to come in direct contact with cells that would normally be protected by mucus.

In another study published in April 2014, Juge and her colleagues investigated a mucus-binding protein (MUB) produced by Lactobacillus bacteria (L. reuteri) in the GI tract. Using X-ray crystallography, the team discovered that the MUB contains repeated structural arrangements, much like ‘beads on a string’. They suggest that MUB may be particularly good at sticking to the mucus because the large number of repeats enables multiple interactions with the glycans.

The thickness of the mucus changes along the GI tract, as do the types of mucins, says Juge. ‘We also know that the microbiota change along the GI tract so it may well be that the two are related,’ she suggests. ‘If you have bacteria with a set of adhesins that allows them to recognise certain types of sugars, that’s why they stay in a particular area.’

Juge is working on a GHFS project that focuses on mucins and bacteria in people with IBD. The belief is that IBD patients may have different distributions of sugars or different ‘mucin glycosylation patterns’, says Juge. ‘We will collect the mucus from these patients and then use mass spectrometry [MS] to profile the type of sugars that are present in the mucins and compare them with those of healthy people.’

The plan is to relate the mucin findings to the types of bacteria present in the mucus of the IBD patients. ‘We know that IBD patients have a different microbiota,’ says Juge. It’s quite possible that the difference in microbiota relates to the glycosylation profile, she suggests. Bacteria such as Lactobacilli, with their sets of adhesins, may not recognise the sugars anymore and ‘get lost’, she says.

The story gets more complicated. While some bacteria bind to mucus, others can also use the mucin sugars as nutrients, making it difficult to say for sure if a different glycosylation patterns attract different bacteria. ‘You could also say that those patients have a different microbiota and that the bacteria are more adapted to utilise the mucin sugars so that they will start to nibble them, giving a change in mucin glycosylation,’ she adds.

One interesting fact to emerge from Juge’s work is that pathogenic or ‘bad’ bacteria also produce proteins similar to adhesins. The difference is that the pathogenic bacteria pass through the mucus to ‘invade’, says Juge.

When a pathogen such as Clostridium difficile repeatedly invades, antibiotics can fail. Faecal transplants [containing good bacteria] have had some success here but raise other health concerns. Recently, Canadian researchers have been working on a project to ‘repopulate’ the gut. As the name implies, this involves creating a ‘stool substitute’ by purifying intestinal bacterial cultures from a healthy individual faecal sample.

In a proof-of-principle study, a team led by Elaine Petrof from Queen’s University, Canada, ‘infused’ a stool substitute into the colons of two patients who had failed at least three courses of antibiotics to treat a C. difficile infection. Following the stool substitute treatment, both patients reverted to their normal bowel pattern and were symptom free after six months. The study ‘demonstrates that a stool substitute mixture comprising a multi-species community of bacteria is capable of curing antibiotic-resistant C. difficile colitis’, write the researchers in Microbiome.⁶

The team is currently working with Health Canada to deal with the ‘rather complex regulatory issues surrounding this treatment’, says Petrof. The scientists are also ‘figuring out mechanisms of action’, she adds.

In the future, her team plans to test how bacteria could be used to treat other diseases linked to a possible imbalance of the gut ecosystem. The mix of bacteria in the stool substitute is quite complex, says Brumer, who has visions of perhaps being able to ‘hand pick’ a few types of bacteria to do the job.

Meanwhile, Brumer is working with scientists from the US, and the University of York’s Structural Biology Laboratory, UK, to try to uncover how gut bacteria metabolise complex carbohydrates in fruits and vegetables. The team has located a gene sequence responsible for producing eight sugar-munching enzymes and two carbohydrate-binding proteins in a group of bacteria called Bacteriodetes.⁷

The team produces large amounts of the enzymes using recombinant DNA technology, inserting the bacterial DNA sequence that makes the enzyme into Escherischia coli, which is used as a production host. It then uses MS to find out exactly which sugar fragments are made by the bacterial enzymes. ‘If we think the enzyme is a xylosidase, we first use a colour-forming artificial substrate to test if it indeed cleaves at xylose. Then we want to know exactly how it works on natural carbohydrate, for which we make specific polysaccharide fragments for testing. We go through a number of yes/no questions,’ explains Brumer.

‘The carbohydrate-binding proteins recognise the complex carbohydrate and help the bacterium to sequester its food source to the cell surface,’ explains Brumer. Then the bacterial enzymes work together. A cell-surface enzyme cuts the polysaccharide chain into little fragments, which are immediately degraded into simple sugars. ‘It’s a really clever system,’ he enthuses. ‘Other bacteria just send enzymes out into the environment and mop up the sugars produced.’

From analysis of data collected from faecal samples, ‘it turns out that at least 90% people have bacteria with a variant of this locus [specific location of the DNA sequence], which says something about how important degrading xyloglucan is to our gut,’ he says. The figure could well be 100% but the data are not sufficient to confirm whether this is the case, he adds.

Knowledge so far is just the ‘tip of the iceberg’, says Brumer. ‘Some of the mechanisms by which many complex carbohydrates in the diet are broken down are not known. For example, it’s really not known who the players are and how they work together.’ The way forward is to take the huge datasets that are available and ‘drill down to understand the fundamentals of how enzymes and proteins and so on are enabling different cells and different bacteria to have niche roles in the gut’, he says.

The work is also likely to have applications outside of gut health, for example, in biomass and bio-based chemicals, says Brumer. He also thinks that enzyme companies could use the information from the gut study to develop new enzymes that cut xyloglucan. ‘You start characterising these families of enzymes and then you get tremendous predictive power for finding new enzymes,’ he says.

The complexity and strange beauty of the systems inside our guts are attracting scientists from all disciplines, from chemistry, to glycobiology and immunology. For Brumer, who trained as a chemist, ‘life is organic chemistry – that’s why this is of interest to me’. 

1 L. A. David et al, 2013, Nature, doi:10.1038/nature12820.
2 J. Ahn et al, JNCI, 2013, doi:10.1093/jnci/djt300.
3 N. Iida et al, Science, 2013, 342, 967.
4 S. Etzold et al, Environmental Microbiology, 2014, doi:10.1111/1462-2920.12377.
5 S. A. Frese et al, Plos Genetics, 26 December 2013, doi:10.1371/journal.pgen.1004057.
6 Elaine O. Petrof et al, Microbiome, 2013, doi:10.1186/2049-2618-1-3.
7 J. Larsbrink et al, Nature http://dx.doi.org/10.1038/nature12907.