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University of Groningen

Novel physiological and metabolic insights into the beneficial gut microbe FaecalibacteriumprausnitziiKhan, Muhammad Tanweer

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Citation for published version (APA):Khan, M. T. (2013). Novel physiological and metabolic insights into the beneficial gut microbeFaecalibacterium prausnitzii: from carbohydrates to current. s.n.

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Chapter 8

General discussion and future perspectives

Chapter 8

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In recent years, it has become increasingly clear that there is an intricate

connection between human health or disease conditions and the gut microbiota.

Accordingly, today's 'concept of health' includes the indigenous microbiota as an

essential component12. Nevertheless, the definition of a 'healthy microbiome' is

still imprecise due to the fact that the microbial community in the human gut and

its functional characteristics are both complex and dynamic. Importantly, previous

research that was aimed at unraveling the functional interactions between gut

microbial communities and their habitats were hampered by the fact that the large

majority of gut microbes could not be cultured 3-5. Such unculturable microbes

included even some of the most abundant members of the gut microbiota, like F.

prausnitzii, to which important health benefits have been attributed as is reviewed

in Chapter 1 of this thesis. The inability to culture potentially beneficial microbes

is a major hurdle in the development of probiotics that could be applied to treat

patients with a disturbed gut microbiota 6-8. For this purpose, such potentially

beneficial gut microbes need to be isolated, cultured, physiologically characterized

and formulated. In line with these needs, the overall objective of the PhD research

described in this thesis was to obtain novel physiological and metabolic insights of

the important beneficial gut bacterium F. prausnitzii.

To start the intended research, the main hurdle that had to be overcome was the

isolation of representative F. prausnitzii strains, which is notoriously difficult 9.

Chapter 2 of this thesis describes how this hurdle was overcome by devising a

new strategy for the isolation of F. prausnitzii from the freshly voided human fecal

samples. Previously, media enriched with undefined components like rumen fluid

or fecal extract were employed for the isolation of butyrate-producing microbes,

including F. prausnitzii, 10. However, incorporation of such non-standardized

components makes the isolation procedures laborious and unpredictable and this is

exacerbated by their oftentimes problematic accessibility. The results showed that,

for isolation and cultivation of F. prausnitzii, the undefined ingredients can be


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replaced with commercially available materials, such as acetate, flavins and hemin.

However, it is important to note that medium enrichment with rumen fluid or fecal

extract can still be useful to simulate a native gut habitat, which can help to

increase the efficiency of retrieving certain gut microbes in pure culture11.

Specifically, the present studies show that the previously developed YCFAG

medium that contains yeast extract, casitone, fatty acids and glucose does not only

allow the culturing of F. prausnitzii 12-14, but that it can also be successfully

employed for the direct isolation of F. prausnitzii from fecal samples. For this

purpose, the fecal material was processed without the use of liquid dilution series,

but directly plated onto a YCFAG agar medium. This strategy was based on the

rationale that this would help maintaining native microbial associations, which can

be important for cross-feeding between different microbes and their initial

adaptation to the in vitro growth regimen. Although, no directly comparable data

are available on the efficiencies of retrieving particular gut microbes in pure

culture by either employing liquid serial dilutions or solid media, the present data

show that the direct plating approach was very effective in isolating different F.

prausnitzii strains as well as various other gut microbes that are also notoriously

difficult to culture. However, it is interesting to note that, also for the isolation of

different soil microbes, it has been reported that the culturing on solid media was

significantly more efficient than a serial dilution strategy 15.

Generally, anaerobic bacteria cannot cope with the lethal effects of molecular

oxygen in ambient air, and they require negative redox potentials for their survival

and growth. The negative redox potential is in fact typical for oxygen-deficient,

reducing environments. Accordingly, the gut lumen has a redox potential of ~-300

mV, and it thus provides an ideal niche for anaerobic microbial growth 16.

Therefore, the use of reducing agents has been frequently reported for the isolation

of anaerobic gut commensals 10,17. For instance, in YCFAG medium cysteine was

included to reduce the dissolved oxygen levels and to achieve the required negative

redox potential 13,14. The studies in Chapter 2 highlight the significance of sample

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handling and maintenance under anaerobic conditions during the entire isolation

procedure. In fact, the results suggest that even a brief exposure of freshly voided

fecal samples to ambient air causes a steep redox gradient between the sample

surface (~-0.15 V) and the center zone (~-0.26 V). This suggests a potentially

oxidative process at the sample surface that may be lethal for the oxygen-sensitive

microbes residing there. This process may actually be responsible for the

previously reported observation that only ~50 % of the gut microbes retain

viability in freshly voided fecal samples 18, and the depressingly low recoveries of

gut microbes in pure culture. Also, it provides an explanation for the fact that the

present recovery rate of F. prausnitzii (~1%) in pure culture was still relatively low

compared to its abundance in stools of healthy individuals (~20%), despite the

minimal delay of ~5 min between sample collection and processing. Importantly,

the studies described in Chapter 2 show that YCFAG medium can also be

employed to isolate other common gut microbes, which is consistent with the fact

that it is a non-selective and enriched growth medium. The presence of yeast

extract, hemin, fatty acids and other components simulate the luminal contents and

clearly support the growth of several other microbes. Besides F. prausnitzii, the

other isolates retrieved on this medium belonged to the genera Bacteroides,

Prevotella, Mitsuokella (Clostridium cluster IX), Megamonas (Clostridium cluster

IX), Dorea (Clostridium XIVa), Roseburia (Clostridium XIVa), unclassified

Lachnospira (Clostridium XIVa) and Bifidobacterium. Species of these genera are

commonly identified through metagenomics analyses, but seldom cultured.

The isolates of F. prausnitzii retrieved during the studies presented in Chapter 2

as well as previously reported isolates or strains that were identified only through

16S rRNA sequencing belong to two broad phylogroups within the family of

Ruminococcaceae as is shown through the studies presented in Chapter 3 10,19,20.

These phylogroups include the previously reported isolates M21/2, ATCC 27766,

ATCC 27768T (phylogroup I), A2-165 and L2-6 (phylogroup II) 21,22. The F.

prausnitzii isolates from the present work (Chapter 2; designated as HTF strains)


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belong to the phylogroup II. Importantly, the two phylogroups cover 97% of the F.

prausnitzii 16S rRNA sequences that have been detected by direct amplification

from human fecal DNA 21,22. Metabolically, the strains from phylogroups I and II

that were isolated from healthy individuals did not reveal any significant

differences. However, previous studies indicate a more severe reduction in the

phylotypes related to isolate M21/2 (phylogroup I) as compared to phylotypes

related to isolate A2-165 (phylogroup II) in biopsy specimens 20 and fecal samples 19 obtained from CD patients.

Despite the fact that some F. prausnitzii strains were able to grow on inulin, the

overall metabolic profiles of isolates from both phylogroups showed a limited

ability to utilize polysaccharides that can be frequently encountered in the gut

lumen. These common polysaccharides include arabinogalactan, xylan and soluble

starch 23. Interestingly, in vivo studies on healthy human volunteers employing

different prebiotics revealed a clear stimulation of F. prausnitzii 24

,25

,26 .This

indicates that F. prausnitzii is well adapted to the gut environment and is possibly

cross-fed by the other members of the gut microbiota, such as Bacteroides spp.

Interestingly, the “Carbohydrate-Active enZYmes Database”

(http://www.cazy.org/) suggests that a pectin lyase is the only polysaccharide lyase

expressed in F. prausnitzii. The idea that this enzyme is expressed is supported by

the finding that most of the isolates grew well on apple pectin (Chapter 3).

Notably, only few groups of human colonic bacteria can exploit pectin as a

substrate for growth, despite the fact that pectin is well fermented within the

human colon 27. In general, the Bacteroides spp. are more efficient pectin utilizers

than the Gram-positive anaerobes 28. Furthermore, in vitro competition studies

including the known pectin utilizers B. thetaiotaomicron and E. eligens suggest

that, under physiological conditions, F. prausnitzii can play a vital role in pectin

fermentation and that it can compete successfully with other gut microbes for this

substrate (Chapter 3). F. prausnitzii strains were also shown to be capable of

utilizing the host-derived sugar N-acetylglucosamine. This indicates that during

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fasting conditions, F. prausnitzii has the ability to switch between diet- and host-

derived substrates, which seems to be a characteristic feature of dominant colonic

species 29. On the other hand, F. prausnitzii was unable to grow in vitro on amino

acids or gastric mucin, suggesting that this bacterium does not contribute to the

ammonification of the gut lumen30 (Chapter 3).

Gut pH and bile salt tolerance can be regarded as the important physiological

factors that can play a role in determining the ability of an organism to survive in

the gut environment. Additionally, these traits possibly contribute to the

temporal/spatial organization of different gut microbes 31. At low pH values the

growth of F. prausnitzii was generally inhibited, but this phenomenon was found

to be strain-dependent (Chapter 3). Similarly, the bile salt tolerance differed

among isolates and, at physiological concentrations between ~0.05% and 0.5%, the

average inhibition varied from 76% to 97%, respectively. In contrast, other species

of intestinal bacteria, such as Bacteroides spp. and Enterococcus faecium can

withstand bile salt concentrations of up to 20% or even 40% respectively 32,33,34.

Thus, both the local pH and bile salt concentrations in the gut are likely to

influence the distribution of individual faecalibacterial strains. However, it should

be noted that no statistically significant evidence for consistent differences

between the members of two phylogroups could be obtained. Altogether, the

findings presented in Chapter 3 provide a plausible explanation why

faecalibacteria exhibit a reduced abundance in Crohn’s disease patients, because

such patients often have acidic stools with elevated bile salt concentrations 35. On

top of that, an aberrant mucosa, thiol depletion plus oxidative stress are highly

likely to lead to a significantly altered microbiota, which is believed to contribute

to the pathogenicity of Crohn’s disease 36,37.

In a healthy gut, the redox status of the gut mucosa is well-controlled via a thiol-

redox couple 37. However, as shown by studies in rats, a deficiency of dietary

sulfur-containing amino acids can result in a shift of the thiol/disulfide redox status

to the oxidized state in the gut mucosa and plasma. Such an alteration of redox


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pools is associated with oxidative stress and contributes to the onset or progression

of several pathological conditions 38. Notably, there is a continuous influx of

oxygen from adjacent blood capillaries into the gut mucosa, which generates a

partial oxygen pressure that would be sufficient to restrict the growth of oxygen-

sensitive bacteria, such as F. prausnitzii 16,39,40. The studies described in Chapter 4

provide novel insights into the physiology of F. prausnitzii and explain how these

oxygen-sensitive bacteria can survive in the moderately oxygenized gut mucosa.

Using a gas tube system, it was shown that F. prausnitzii can exploit extracellular

electron transfer (EET) to survive and thrive in moderately oxygenized

environments. A growth rim, presumably due to enhanced growth of F. prausnitzii,

was observed at the oxygenized end. The presence of flavins and thiols in the

growth medium was shown to be critical for this behavior. Notably, in the presence

of oxygen, the free thiols of cysteine are chemically oxidized to form cystine,

which can subsequently act as an electron acceptor. In the case of F. prausnitzii, it

was shown that riboflavin served as an electron shuttle between the bacterial cells

and cystine, and that the regenerated cysteine was oxidized again to cystine by

oxygen. This flavin-dependent electron shuttling in gut microbes is probably a

rather specialized phenomenon, since abundant microbes such as Bacteroides

species were shown to lack this ability (Chapter 4). On the other hand,

Bacteroides species and Escherichia coli are capable of producing riboflavin, but

unable to exploit it as a redox mediator for EET 41. Importantly, riboflavin and

thiol-containing compounds that are required for EET by faecalibacteria are

generally abundantly present in the healthy gut. Microbial or common dietary

sources, such as dairy products, plant foliage, fruits and fibers are the main

contributors to the flavin pool in the gut, while thiols can be obtained from dietary

sources, such as egg yolk, dairy products and grains42. Additionally, the

considerable amounts of thiols that colonocytes secrete as antioxidants can also

serve as electron acceptors for EET 36,43. Altogether, the results presented in

Chapter 4 indicate that flavins and thiols may be exploited by ‘anaerobic’ bacteria

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in niches where a relatively high oxygen tension exists, such as the gut mucosa 44.

As such these compounds would promote 'gut health'. If so, the oral

supplementation of flavins together with thiols may allow a modulation of the

redox conditions in the gut, which could perhaps be applied to induce a potentially

beneficial increase of the numbers of faecalibacteria in the gut of patients with

colitis 45,46. Intriguingly, the findings presented in Chapter 4 also seem to provide

new insights into clinical conditions, such as Crohn’s disease, where an aberrant

mucosa, thiol depletion, and oxidative stress lead to an altered microbiota. Clearly,

the absence of thiols alone could be sufficient to impair EET by F. prausnitzii

which, in turn, would limit the growth of this bacterium. This would then again

have a negative impact on pathogenesis due to a reduced production of butyrate

and potentially anti-inflammatory compounds by the normally abundant

faecalibacteria. Thus, the present findings highlight the flavin and antioxidant

status of the human diet as a parameter that can have a major influence on

beneficial bacteria and their spatial distribution in the gut and, hence, on human

health.

The studies described in Chapter 5 provide further insights into the riboflavin-

mediated EET by F. prausnitzii. In these studies, a microbial fuel cell (MFC)

system was employed in which glucose, resting bacterial cells and riboflavin were

respectively used as electron donor, bio-catalyst and redox mediator. The bacterial

oxidative metabolism in the anode chamber was coupled to the reduction of

ferricyanide in the cathode chamber 47. When riboflavin was introduced into the

anode compartment containing faecalibacterial cells that were pre-energized with

glucose, an immediate current wave was generated. This indicates that glucose was

metabolized by the F. prausnitzii cells, generating electron carriers such as NADH

or reduced Ferredoxin (Fd red), which can then participate in EET. Furthermore, it

was shown that chemical oxidation of NADH was facilitated by riboflavin, which

is consistent with the fact that the redox potential of riboflavin (Eo’= -0.21V) is

more positive than that of NADH (Eo’= -0.32V). Additionally, Chapter 5


191 | P a g e

highlights the ability of F. prausnitzii to use glucose as an electron donor in

contrast to other well-studied electrogenic bacteria, such as Shewanella spp. and

Geobacter spp. that are unable to use glucose as an electron donor 48,49. Through

cyclic voltammetry of spent growth medium it was shown that, unlike other

bacteria such as L. lactis or Shewanella spp 50,51, F. prausnitzii did not actively

secrete riboflavin or other redox mediators into the growth medium (Chapter 5).

This observation seems to be indicative of the adaptation of F. prausnitzii to the

colonic environment, where sufficient amounts of flavins and an oxidative

environment are generally available 42.

The studies in Chapter 6 were aimed at demonstrating possible links between

metabolic pathways and EET in F. prausnitzii. Using chronoamperometry,

bacterial cells were cultivated in a MFC at different anodic potentials and their

metabolic profiles were assessed. The growth profiles showed that, at oxidizing

potentials, the initial current-producing or electrogenic phase was followed by a

fermentative growth phase. The catalytic current that was generated during the

electrogenic phase indicates that a portion of the reducing power produced during

glucose metabolism was utilized for EET. To provide better insights into EET,

resting cells were employed, which were fed with either glucose or pyruvate, with

or without acetate. These experiments with resting cells clearly demonstrated that

the portion of the reducing power that was generated during glycolysis and via the

activities of the pyruvate:ferredoxin oxidoreductase system can indeed be

exploited for EET. This suggests that, during the metabolism of glucose or

pyruvate to acetyl-CoA, F. prausnitzii will respectively respire 8 or 2 moles of

electrons to external electron acceptors. Accordingly, the glucose-fed resting cells

can generate a catalytic current that is ~4 fold higher than the catalytic current

generated by the pyruvate-fed cells (Chapter 6). Possibly EET can also lead to the

generation of ATP via proton pumping and an F-type ATP synthase, thereby

increasing the net ATP gain per mole of substrate. Similarly, it has been reported

previously that Shewanella spp. can generate 4 moles of electrons during the

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oxidative metabolism of lactate to acetate 51. This in turn indicates that during the

production of an oxidation current of 35 µA, Shewanella can produce ATP at a rate

of ~9.6 mol ATP mg.protein-1.h-1. Notably, the studies in Chapter 6 revealed that

F. prausnitzii produces substantial amounts of extracellular polymeric substance

(EPS) during the electrogenic phase. The results suggest that carbon flux is

regulated during the electrogenic phase with a part of the glycolytic flux being

utilized for the biosynthesis of EPS. Altogether, the data imply that the metabolic

flux in F. prausnitzii is modulated towards EPS production in response to

oxidizing environments and that the internal redox balance of this bacterium is

maintained via EET. Translated to the situation in the human gut, the present

findings seem to suggest that F. prausnitzii uses EPS and EET to shield itself from

the oxidative environment that can be experienced at the gut mucosa. A model for

the coupling between the faecalibacterial metabolism, EPS production and EET is

presented in Figure 1. Lastly, a major hurdle for the potential application of F.

prausnitzzi as a probiotic lies in the fact that this important producer of butyrate

and anti-inflammatory compounds is highly oxygen-sensitive. This has so far

precluded the administration of faecalibacteria to patients with low numbers of

these potentially highly beneficial bacteria. Building on the observation that

riboflavin and cysteine are highly effective in sustaining the growth F. prausnitzii,

the studies described in Chapter 7 show that this highly oxygen-sensitive

bacterium can be kept alive at ambient air for 24 h when formulated with cysteine

and riboflavin plus the cryopreservant inulin and the bulking agents corn starch

and wheat bran. These findings thus pave the way for the biomedical exploitation

of this and possibly other oxygen-sensitive gut microbes in the treatment of major

disorders of the human gut.


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Figure 1. Proposed metabolic pathway of F. prausnitzii showing the connections between metabolic flux biosynthesis of extracellular polymeric substance (EPS) and extracellular electron transfer (EET) as inferred from the studies in Chapters 4 to 6 of this thesis. PEP, phosphoenol pyruvate; RB, Riboflavin; R-SS-R, oxidized thiols; R-SH, reduced thiols; F-6-P, Fructose 6-phosphate; G-1-P, Glucose 1-phosphate; G-6-P, Glucose 6-phosphate; M-6-P, Mannose 6-phosphate; M-1-P, Mannose 1-phosphate; GDP-M, GDP-Mannose; UDP-G, UDP-glucose; UDP-GA, UDP- glucouronic acid; IDP-G, Isoprenoid pyrophosphate glucose.

RBred

RBoxd

Glucose

G-6-P F-6-P

M-6-P

M-1-P

GDP-M

G-1-P

UDP-G

UDP-GA

PEP

Pyruvate

Building blocks

for EPS

IDP-G

2 NAD(P)H.H+

2 Acetyl-CoA

Butyrate

Butryl-CoA

NAD+

Fd oxd Fd redAcetate

Acetyl-CoA

Acetate

EtfH2

Acetoacetyl-CoA

NADH

Crotonyl-CoA

Etf

NAD+NADH

Formate

Fd oxd

Fd red

CO2

ATP

ADP

NAD+

NADH

ADP

ATP

ATP

ADP

ATPADP

Fd red

NAD+

Fd oxd

F1FoATP

ATPase

H+H+

H+ H+

Rnf

complex

Acetate

ATP

ADP

R-SS-R

R-SH

O2

H2O

RBred

RBoxd

RBred

RBoxd

R-SS-R

R-SH

O2

H2O

In the Gas Tube System

Possibly in Colon at Gut Mucosa

In the Microbial Fuel Cell

Ex

tra

cell

ula

r p

oly

me

ric

ma

trix

Chapter 8

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Concluding remarks and perspectives

The microbiota in our gut can, in many respects, be regarded as an ‘organ’ of the

human body. It is a multicellular entity with specialized cells that have

unambiguous roles in the digestion of ingested food and a major impact on

systemic and mucosal immunity. As is the case for other organs in our human body,

like the heart, liver and lungs, maintaining our gut microbiota in a good condition

is of crucial importance for our health and well-being. The bacterium which was in

the focus of this PhD research, Faecalibacterium prausnitzii, is a key determinant

of the gut microbiota; it is abundantly present and contributes to our health by the

production of butyrate and potentially anti-inflammatory compounds. To do so, it

has co-evolved with other gut microbes from which, if necessary, it can derive

important nutrients in the form of carbohydrates and flavins. F. prausnitzii can

then employ the flavins from fellow gut microbes or ingested food for EET and

survival in the gut mucosa. The presently obtained insights into the phylogeny,

metabolism, physiology and adaptations to the colonic environment are likely to be

helpful in the development of a robust F. prausnitzii-based pro- or prebiotic

formulation. The next logical step will be to perform pre-clinical trials to assess the

potential efficacy and safety of such formulations as described in Chapter 7 of this

thesis. In this context, the identification, functional characterization and large-scale

purification of the as yet somewhat ill-defined anti-inflammatory compounds

produced by F. prausnitzii will also be of importance, since such compounds can

be beneficial in the treatment of patients with, for example, colitis. Specifically,


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any anti-inflammatory compounds produced by F. prausnitzii could be added to

pro- or prebiotic formulations to enhance their efficacy. Intriguingly, the present

findings suggest that F. prausnitzii can also be employed in completely different

ways to the benefit of mankind. For example, now that we know how to culture F.

prausnitzii, this bacterium is also a potential candidate for the bio-transformation

of industrial waste into butyrate or electricity. In this context it is noteworthy that

the catalytic currents generated by F. prausnitzii in the applied MFC setting with

glucose as electron donor are amongst the highest that have been reported in the

literature 47,50,52. For sure, if F. prausnitzii were not only applicable for the benefit

of human health and well-being, but also for the sustainable production of energy,

this would give a completely different meaning to the title of this thesis:

“Novel physiological and metabolic insights into the beneficial gut microbe

Faecalibacterium prausnitzii - from carbohydrates to current”.

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