Akkermansia terminal electron acceptors

Ouwerkerk JP, van der Ark KCH, Davids M, Claassens NJ, Finestra TR, de Vos WM, Belzer C. Adaptation of Akkermansia muciniphila to the Oxic-Anoxic Interface of the Mucus Layer. Appl Environ Microbiol. 2016 Dec 1;82(23):6983-6993. PMC free paper

Why this paper is so fantastic

This paper is being examined to start to answer the question of why Akkermansia mucinophila is such a good, health promoting probiotic.   If this microbe is an obligate anaerobe, what is the terminal electron acceptor?  Is it something bad like sulfate giving the product of hydrogen sulfide? Maybe it is just plain, boring TCA cycle intermediate fumarate. This Finnish and Dutch collaboration, with Dr Carol Velzer as the corresponding author, argues that if this microbe colonizes the mucus layer, the conditions are not truly anaerobic.  How do these bacteria compensate to the small amounts of oxygen?  What cassettes of normal anaerobic environment genes get turned off?  The sulfate reduction genes turned off during the transition from anaerobic to aerobic growth have far reaching implications not only for propionate production but also the use of sulfur containing compounds as preservatives.

What is mucin?

Mucin is a gel forming glycoprotein secreted by cells of the respiratory and gastrointestinal tracts. This takes the reader to a UniProt.org site that provides a variety of relevant information.

Let’s start at the top and work our way down this composite figure. Post translational modifications occur after the mRNA has been translated into amino acids in the growing protein chain. Glycosylation starts by adding the sugar mannose to serines or threonines. Glucosamine is added to the Nitrogens of asparagine. Theses are just starter sugars that can form polysaccharides. Continuing to the right, disulfide bonds are bonds between the sulfur groups of two cysteines. In the botom left we have the domains of MUC5, from UniProt. Mucin 5 has well over 5000 amino acids … a rather large protein to start with. Each cysteine rich domain contains about 100 amino acids

CGEKCLWSPWMDVSRPGRGTDSGDFDTLENLRAHGYRVCESPRSVECRAEDAPGVPLRALGQRVQCSPDVGLTCRNREQASGLCYNYQIRVQCCTPLPC

This is the single letter amino acid sequence of the first cysteine rich repeat. C’s are cysteines. At the bottom right there is a cartoon of a mucin mega protein complete with N- and O- linked polysaccarides. This substance has the potential to be a major food source for bacteria as well as a protector against bacteria.

What is a terminal electron acceptor?

Wikipedia has a good page on anaerobic respiration. that has a table of terminal electron acceptors and the products of reduction that include a comparison with aerobic respiration in which O₂ is the terminal electron acceptor and H₂O the product. The Wiki authors list two likely possibilities:

1. SO₄ ⇒H₂S … or more properly just sulfide HS- performed by the Desulfovibrionales E^o’ = -0.22 V
2. fumarate ⇒ succinate …. performed by the ever familiar gut microbe E coli E^o’ = +0.03 V

E^o’ is the reduction potential. The more positive, the more likely to acquire electrons from the environment. The reduction potential of O₂ is + 0.82 V. Without O₂ in the picture fumarate would be predicted to be the favored terminal electron acceptor.

This paper describes a massive reduction in transcripts for proteins involved in sulfate processing when Akkermansia muciniphila is exposed to O₂ compared to anaerobic growth. This post will come out of password protection when the corresponding author answer the question of which one it is.

Introduction, relevant points of the Ouwerkerk publication

• The outer layer of mucus is colonized with microbes that differ in composition from the luminal (fecal) microbiota.   Presence of A mucinophila may suggest some other event that dislodges them from the mucosa into the feces rather than a difference in overall numbers.
• Mucin is the source of nitrogen and carbon.  In turn the bacteria produce short chain fatty acids needed for host health.
• The  process of SCFA production is required to maintain the redox balance in the cell, as it can restore the NAD/NADH ratio.
• Aside from the brain/heart infusion medium used in other studies covered on this site,  Akkermansia muciniphila,can use mucin as a sole source of carbon and nitrogen according to Ouwerkert and coauthors.  
• A. muciniphila is associated with a healthy GI tract, as its abundance is inversely with several disorders.  Its presence in the mucosa stimulates the immune system and thickness of the mucosa itself.
• Initial reports described A. muciniphila as a strict anaerobe but later reports indicate that it can tolerate small amounts of oxygen that diffuses from GI epithelial cells.
• To survive in an oxygen containing environment microorganisms must many of the same enzymes as their mammalian hosts:  Catalase, superoxide dismutase,  thioredoxin,  glutaredoxin, and the peptide glutathione.
• Many of these enzymes require small molecule reducing equivalents NADH and NADPH as cofactors.

Figure 1 A. muciniphila can survive O₂

Based on CFU (colony forming units) after exposure to ambient air, 1% of the A. muciniphila were able to survive for up to 48. Note that the vertical scale of Panel 1A is in log scale. Each horizontal par represents a reduction of 100x. A gas tube assay with redox sensitive dye resazurin was used to detect possible coping mechanism.  Reasurin was used to test the oxygen reduction capacity. Resazurin is a dye that turns purple when oxidized by O2 in ambient air.  That A mucinoplilia reduces the thickness of the purple layer suggest that it is producing reducing equivalents to reverse this reaction in the adepts of the tube.

Oxygen really does kill this bug, but not completely. What are the implications for other bacteria that might go both ways.

Figure 2, of O₂ concentration and redox potential

T1 is the start of the exponential growth phase of A muciniphila in under anaerobic conditions. T2 is the mid-exponential phase.  T3 is the stationary phase.  The transcripts under anaerobic and low air were compared at these time points.

One of the two fermenters was switched to an ambient airflow of 0.2 liters/h (aerated fermenter) and the other fermenter was maintained under strict anaerobic conditions (anaerobic fermenter). Note that the Y-axis scahle is in mV. T1 is hovering around -0.25V where sulfate reduction looks favorable, i.e. -0.22V.

On the surface, almost nothing in Figure 2 makes sense. 

• 2A Why does the dissolved O₂ take over 500 minutes after ambient air was introduced at t=500 min? 
• 2B  Why does the redox potential rise so much more slowly than the dissolved O₂?
• 2C  In the presence of bacteria why does the redox potential spike, instead of slowly rising, when dissolved O₂ is introduced? 

Perhaps the bacteria themselves are functioning as a redox buffer.

Figure 3, other surprisingly small differences

Differences were at the p<0.05 level of significance when using the appropriate tests.  Are these small differences functional to the microbe or to the host? We don’t know because it appears that we are looking at the differences between anaerobic and aerobic respiration. The best guess so far is that fumarate is replacing O₂ in anaerobic respiration.

Ecoli strain COM4-AmuCytbd, that is capable of using O2 as an electron carrier and is is able to produce H₂O and NAD maintain the NAD/NADH ratios,.   the extra NAD production needs to be balanced by additional NADH regeneration. Therefore, the metabolism might shift from propionate,
where a net total of 2 NADH are oxidized to 2 NAD,

Parts of supplemental Figure 2 have been expanded. The deep blue lines were drawn to emphasize parts of this pathway in common with the TCA cycle that generates NADH for the electron transport chain. The bottom right is the furmarate to succinate reduction and the likely terminal electron acceptor event.

4. Transcriptome analysis

Transcriptome analysis pointed in the direction of the cytbd complex.  Another way of looking at things are the transcripts for sulfate reduction related genes that are very significantly reduced. The fold change in thse volcano plots is recorded on the X-axis. The Y-axis isthe -1.00e^-1, 1.00e^-2, and so on of the p value. The higher the point on the plot, the higher the probability of being different from the reference. We see a lot of “Assimilatory sulfate reduction” transcripts at the upper left hand corner of the plot.

This is a comparison of aerated mid log phase T2 with the anaerobic T2 phase. Both are rapidly respiring, The anaerobic culture is doing so without O₂ as a terminal electron acceptor. Sulfate would be the first pick.

At T3 we are comparing aerobic versus anaerobic during the stationary T3 phase. The bugs are not growing as fast so they don’t need as much ATP. The aerobic bugs have to deal with an accumulation of reactive oxygen species.

Figure 5, cytochrome bd

It is one thing to say that a transcript is increased. It is another thing to say that a functional protein is expressed. Absorbances of extracts of A. muciniphila and E coli expressing the Cytochrome bd complex were measured over the visible spectrum. The dithionate reduced spectra were subtracted from the oxidized spectra. The two spectra show similar patterns at 430 nm.

Figure 6, summary

In the mammalian electron transport chain, electrons and H⁺ enter the chain via NADH, a product of the TCA cycle. Under conditions of the biological niche, the H⁺ and electrons come from H₂. The incomplete TCA cycle components have been discussed.

Concluding remarks

The massive implosion of the assimilatory sulfate reduction transcripts, while not the focus of the study, cannot be ignored. Why is A muciniphila going to the trouble of producing these enzymes under strict anaerobic conditions?

• This first obvious reason to jettison such assimilatory sulfate reduction enzyme production is that doing so when they are not needed is a waste of resources .
• One thought is that sulfate assimilated into cysteine is not going to be turned into H₂S that could poison A muciniphila and the mammalian host.