Sialic acid may be key carbohydrate responsible for inflammation and dysbiosis in the gut

A surface mucous cell bordering on the stomach lumen secretes mucus (pink stain).

Our diet is full of various carbohydrates, composed of different monosaccharides and polysaccharides.  Many of these survive our own digestion and make it all the way to the colon where they modulate our microbiome.  Another source of saccharides for our gut bacteria is the mucous that we produce, which can be a rich source of fucose or sialic acid.  Sialic acid has been implicated in many inflammatory diseases, such as bacterial vaginosis.  Last week, researchers from Switzerland showed that sialic acid may play a critical role in colitis, at least in one colitis model commonly used in mice.  They published their results in Nature Communications.

One way to induce intestinal inflammation in mice is to feed them dextran sodium sulfate (DSS).  The reason this molecule causes colitis in this mice is unknown, but it is used in many models of the disease.  In order to understand the possible role of sialic acid in the colitis, scientists created mice that could not produce mucous with sialic acid.  They quickly realized that these mice were not as susceptible to the DSS-induced colitis as their normal counterparts.  After, they tested how various antibiotics affected colitis in the DSS—colitis mice and discovered that Escherichia coli abundance was directly associated with the severity of the disease. Putting these ideas together, they tested and discovered that E. coli used sialic acid as their main carbohydrate source in vitro.

Interestingly, the E. coli cannot actually access the sialic acid from mucins, but instead need other bacteria, such as Bacteroides vulgatus to cleave and release the sialic acid from the mucins in order to access it.  If sialic acid is indeed important to the human form of the disease there may be multiple approaches to combatting the disease.  First, by eliminating E. coli, and second by eliminating the free sialic acid.

A new way in which some gut bacteria rely on their hosts’ mucous for energy

It is often understood that our gut bacteria live off of the foods we eat.  However, many gut bacteria can actually metabolize the mucous that protects the lining of our gut.  In fact, many bugs have the ability to digest a specific sugar that is attached to our mucins, called sialic acid.  Interestingly, some bacteria have the ability to cleave this sugar from the mucins, others have the ability to consume this sugar once it is released, and others have the ability to perform both of these tasks.  Last week, a new method for how gut bacteria can transform sialic acid was discovered, that some bacteria can actually transform sialic acid before cleaving it, and that this may be clinically relevant for Crohn’s disease and colitis.  The authors published their results in Nature Communications.

The authors were testing a common commensal bacteria, Ruminococcus gnavus, and noted that it had the ability to both cleave and consume sialic acid from gut mucins.  When they identified the metabolites from this process they discovered that the sialic acid was actually being converted to a different form by these bugs.  After further experimentation they realized that a type of enzyme, called an intramolecular trans sialidase, which had never before been observed in gut bacteria, was responsible.  The researchers then compared the genes from R. gnavus to other bugs common in the gut and noted that a full 11% of human gut commensals had this enzyme, and that these bacteria were overrepresented in people with IBD.  The authors think that the bugs who code for this enzyme have an inherent advantage over other gut microbiota because after they transform the sialic acid they can still use it for energy, whereas other bugs cannot, leaving the sugar all to themselves. 

The paper did not discuss specific mechanisms as to why these bugs may be overrepresented in Crohn’s and colitis.  They did however test a few molecules that inhibited the activity of the enzymes.  Perhaps if these enzymes or the responsible bugs are the cause of IBD, than these inhibitors could be used as therapeutics to combat the disease. 

Yogurt may help combat colitis

Editor's note:  The following work comes out of Wendy Garrett's lab at Harvard.  Wendy will be an upcoming guest on the podcast.  If you have questions for her, feel free to email or call, and we will ask her on the show. 

A key signature of colitis is the build-up of reactive oxygen species (ROS) in the colon due to chronic inflammation.  One of major functions of ROS is actually to act as an antibiotic, and destroy any foreign bacteria that may exist at the site of inflammation.  However, ROS are known to be toxic to the host as well, and their high concentrations in colitis are likely a major contributor to the disease.  With this in mind, researchers at Harvard studied how probiotics from yogurt could ameliorate the disease by disposing the excess ROS.  They published their results last month in the Proceedings of the National Academy of Sciences.

The researchers had previously determined that a common yogurt that contained 5 strains of bacteria was helpful in decreasing symptoms in multiple mouse models of colitis.  In this study though, they identified Lactococcus lactis as being the most important of these strains in treating colitis symptoms.  They then compared the genome of L. lactis with the other strains in the yogurt and determined that a specific gene that codes for the enzyme, superoxide dismutase (SOD), which is capable of breaking down superoxide, an ROS, may be imparting L. lactis’ beneficial effects.  In order to support this hypothesis, the scientists showed that when this gene was removed from L. lactis the bacteria no longer reduced colitis.  They took this notion a step further and showed that superoxide levels were in fact decreased in vitro when combined with lysed L. lactis.  Finally, the scientists showed that the L. lactis must actually be lysed in the colons in order to release its SOD, destroy superoxide, and reduce colitis: when the scientists attempted to deliver SOD on its own to mice with colitis it was not as effective, and caused diarrhea.

This study is really interesting for two reasons.  The first is that it shows yogurt, like Activia, may be very helpful in dealing with colitis.  The second reason though, is that it shows a new system for deliverying SOD to a site of inflammation: via bacteria.  As they showed in the paper, simply using SOD was not effective, but using the bacteria as a vehicle for SOD, and then lysing it at the site was an effective means of drug delivery.  This has many important implications because ROS are important contributors to a variety of diseases.  

Specific bacteria implicated with microscopic colitis

Occurrence of Akkermansia muciniphila in healthy people and those with MC.
From:  Fischer et. al. 2015. Altered microbiota in microscopic colitis. Gut doi:10.1136/gutjnl-2014-308956

Microscopic colitis (MC) is a disease which is characterized by chronic diarrhea and an increase in the immune cells characteristic of inflammation in the colon, but an otherwise healthy looking colon based on a colonoscopy.  Interestingly, it most often afflicts middle aged and elderly women, and is highly associated with smoking.  Researchers in Sweden recently hypothesized that that MC was somehow related to the microbiome and designed an experiment to find out.  The results of that study were published this week in the journal Gut.

The scientists sampled the microbiomes of 10 women suffering from MC and 7 healthy controls.  They discovered that while the microbiomes in all the women were quite similar, there was one conspicuous difference.  The women with MC had much lower levels of Akkermansia sp. (100x to 1000x lower).  This genus is known to be important in healthy guts, where it breaks down the mucous lining of the gut.  It is associated with thicker mucous, which is likely a healthy trait, as well as glucose metabolism.

While the number of patients was low, the connection between MC and Akkermansia sp. certainly deserves further investigation.  Of course, these bacteria are only associated with the disease, and this study is in no way proof that the lack of this bug causes the disease.  Diseases like colitis are very complex, and there are likely many factors involved, but studies like this are certainly a step in the right direction in showing an association with the microbiome.

New info on fecal microbiota transplants for C. difficile and ulcerative colitis

"Fecal bacterial communities of recurrent [C. diff]  patients shift towards [healthy] fecal bacterial communities after FMT. Pre-FMT patient samples (red circle); post-FMT patient samples (green circles); trajectory of patient fecal communities after FMT (blue line)."
Image and caption from the C. diff paper: Weingarden et al. Microbiome 2015 3:10   doi:10.1186/s40168-015-0070-0

Two important papers regarding fecal microbiota transplants (FMTs) were published last week.  The first was an examination of a patient’s microbiome over time after he or she undergoes an FMT to treat C. difficile.  The second showed the results of clinical trials that used FMTs in an attempt to treat ulcerative colitis.   The FMT papers, which are described below, improve our understanding of this procedure, which holds promise to treat various microbiome-based diseases.

The C. diff paper, published in the journal Microbiome, attempted to answer the question: Do the microbiome changes that occur after FMT remain long after the procedure?  We know that FMTs are highly effective in treating C. diff because they install a healthy microbiome that can crowd out the infection.  However, it is unknown if these new bugs that take hold are transient, or if they become permanent members of the gut.  The researchers sampled the microbiomes of FMT donors and recipient patients before and up to 84 days after an FMT procedure to treat C. diff.  They discovered that the recipients’ dysbiotic microbiomes stabilized quickly, and after just one day they closely resembled the donors’ microbiomes.  Continued measurements showed that the microbiomes deviated over the next few weeks, but that they remained healthy.

The colitis clinical trial, published in the journal Gastroenterology, attempted to discover if FMTs could treat ulcerative colitis.  Ulcerative colitis is widely considered to somehow be related to a dysbiosis in the microbiome, so can FMTs from healthy donors treat this disease?  The study was a double blind randomized clinical in which 48 people suffering from ulcerative colitis either received stool from healthy donors (treatment) or just an FMT of their own stool (control).  7/23 patients who received stool from a healthy donor were in remission after 12 weeks, while 5/25 patients who received their own stool were in remission at that time.  Unfortunately, this is not a clinically significant result based on the number of patients involved.  The researchers measured the bacterial abundance in all of the patients microbiomes before and after treatment.  Before treatment the microbiomes all had some baseline similarity.  After treatment, though, the patients who responded to treatment from a healthy donor all had an increase in certain Clostridia, and the patients who responded to treatment from their own stool all had in increase in certain Bacilli, Proteobacteria and Bacteriodetes.  The researchers feel that this information warrants further study.

FMTs are an exciting new therapy that may be important in treating some really nasty diseases.  We do want to remind people, though, that it is still an unproven technique that should only be performed under the guidance of a doctor.  As we have written about before, the promise of the microbiome is what makes FMTs both attractive, but potentially dangerous at the same time.

Emulsifiers in food cause many adverse health effects in mice

Emulsifiers are often used in ice cream to keep it stable and to give it it's texture.

People often have spirited and impassioned views on the safety and consequences of adding ‘unnatural’ molecules to food.  An important aspect of this debate that everyone must keep in mind is the impossibility of testing every component of every molecule for its safety and long term impacts.  That being said, it should come as no surprise that new research can often teach us about the unexpected and overlooked safety issues regarding many food additives.  The newest class of compounds to come under scrutiny is emulsifiers, and a new paper published in Nature last week shows that these compounds may negatively impact the body through modulation of the intestinal lining and the microbiome. 

Emulsifiers are compounds that increase the stability of an emulsion.  They are often molecules like surfactants that have two parts, hydrophobic carbon chains and hydrophilic polar head groups.  Soap and egg yolks are common examples of emulsifiers.  There  are, of course, chemically produced emulsifiers as well that are often used in food.  Two examples of these, which were the emulsifying compounds used in the study, are polysorbate-80 (P80) and carboxymethylcellulose (CMC), and they are added to all sorts of foods like ice cream and pudding.  Evidence from this paper suggests though, that at least in mice these emulsifiers are wreaking havoc on the gut and microbiome.

A team of researchers from Israel, Cornell, and Emory did a variety of experiments on mice that were fed either of these emulsifiers in their water (at a concentration of 1%, similar to the levels added to human food.)  They first noticed that these mice had greatly compromised mucous layers on their gut, which allowed for bacteria to actually reach and be in contact with their epithelial gut cells.  In these mice gut permeability (leaky gut), inflammation, and incidences of colitis were all increased.  In addition, the inflammatory response and gut permeability were directly related to the average distance of bacteria to the actual epithelial layer; the closer the bacteria the more inflammation and permeability.

The researchers also measured the microbial populations of the feces in these mice and those that were eating emulsifiers had much less diverse microbiomes, which were enriched in Proteobacter, which are known to be associated with inflamed guts, and reduced in Bacteroidales, which are associated with healthy guts.  Also those eating emulsifiers had in increase in Ruminococcus gnavus which is associated with type 1 diabetes, as we have written about in the past. Interestingly, those mice that were given the emulsifiers tended to eat a lot more food than there control counterparts, and this led to weight gain and obesity amongst the mice drinking emulsifiers.  Moreover, these same mice had higher fasting glucose levels, indicative of impaired glycemic control and metabolic syndrome.  The scientists tested if these effects were seen in mice that were fed the emulsifiers in their food, rather than in their water, and the same outcomes were observed.  In addition, the scientists observed shifts in the production of certain short chained fatty acids and bile acids produced by the microbiome in mice fed emulsifiers (click on the tags below to read about the wide range of health effects that both these compounds have been implicated with).

The researchers then did a series of experiments that showed that it was the actual shifts in the microbiome populations and not just the change in mucous that was primarily responsible for the adverse health effects in the mice given emulsifiers.  For example, germ free mice that were given  emulsifiers did not have compromised mucous, and many of the negative health effects like inflammation were not observed.  On the other hand, performing a microbiome transplant from a mouse given the emulsifiers to a mouse that was not given the emulsifiers did result in these negative health effects, including the bacterial penetration of mucous to the epithelial lining.

This was a fantastic article that may, in time, prove to be immensely important.  Of course all the usual caveats apply, such as studies in mice are not indicative of human responses, and more studies must be performed in order to confirm these findings.  Still though, the introduction of emulsifiers into the mice’s diets resulted in many of the negative health impacts that are associated with the microbiome, something that we really haven’t seen in the literature before now.