Gut microbiota functions - metabolism of nutrients and other food
components
1
Ian Rowland
Department of Food & Nutritional Sciences
University of Reading
Co-authors
• Glenn Gibson (University of Reading)
• Kieran Tuohy (FEM-IASMA)
• Jeremy Nicholson (Imperial College London)
• Karen Scott (Rowett Institute/University of Aberdeen)
• Jonathan Swann (Imperial College London)
2
Gut microbiota interaction with host
Digestive
function
Cancer
Obesity
AAD
Metabolism
Metabolic
syndrome/Diabetes
IBS/IBD
Immune
system
4
• Firmicutes
– Clostridium
– Roseburia
– Faecalibacterium
– Blautia
– Dorea
– Lactobacillus
– Peptostreptococcus
– Eubacterium
– Streptococcus
– Staphylococus
– Butyrivibrio
• Archaea
– Methanobrevibacter
• Fusobacteria
Human gut microbiota – Main phyla & genera • Bacteroidetes
– Bacteroides
– Prevotella
• Verrucomicrobia
– Akkermansia
• Proteobacteria
– Escherichia
– Klebsiella
– Desulfovibrio
• Actinobacteria
– Bifidobacterium
– Collinsella
90% of bacteria are in
Bacteroidetes/Firmicutes phyla
Human vs mouse microbiota
5
• Extrapolating results from mice with regard to
microbiota metabolism and human health is not
straightforward
– Many differences at the level of specific genus/species
abundances (Nguyen T et al Disease Models & Mechanisms 8:1-16. 2015)
– Distribution of microbiota different
• But – much of the evidence that gut microbes play a
role in metabolism and human health comes from
studies in mice especially comparing germ free vs
conventional mice
Aims of the review
• To review the mechanisms and pathways involved in
gut microbial metabolism of dietary components
• To review the literature on the types of
microorganisms involved in GI metabolism of dietary
nutrients and non-nutrients
• To consider the methodologies for investigating gut
microbiota metabolism
6
Dietary components reviewed
• Carbohydrates
• Proteins
• Fats
• Vitamins
• Phytochemicals/polyphenols
• Bile acids
7
Methodologies
• Isolated cultures
• Gut microbial enzyme activity
• Mathematical modeling
• System biology approaches
– Metagenomics
– Metatranscriptomics
– Metaproteomics
– Metabonomics
8
Carbohydrate fermentation
Polysaccharides Oligosaccharides Mucin Microbial metabolism
Breath Flatus
Transported in
blood to
peripheral
tissues,
lipogenesis
Gluconeo-
genesis
in the liver;
satiety
signalling
Major energy
source
for human
colonocytes;
Protects
against cancer
Faeces
H2, CO2, H2S, CH4
Acetate
Propionate Butyrate
Biomass
© Glenn Gibson/
Karen Scott
Fermentation by main bacterial phyla
Numerical Functional importance Example importance Firmicutes 60% Cluster XIVa (25%) butyrate production E.rectale Cluster IV (25%) polysaccharide utilisation F. prausnitzii Roseburia bromii Cluster IX (10%) propionate production Megasphaera Bacteroidetes 25% wide range of substrates utilised acetate, succinate, propionate Bacteroides produced Actinomycetes 10% Oligosaccharide users Bifidobacteria prime/modulate immune response lactate production Others 5% lactic acid bacteria Lactobacilli sulphate reducing bacteria Desulfovibrio © Karen Scott mucin degraders
Sulphate-reducing bacteria
• Occur in 45% of healthy persons
• Reduce sulphate to sulphide, in
conjunction with an electron donating
substrate
• Compete with methanogens for
hydrogen
• Commonest genus is Desulfovibrio
• Pathological role in the gut remains
unclear
© Glenn Gibson
Methanogens
• Archae that produce methane as a
metabolic byproduct
• Obligately require hydrogen
• There are over 50 species of
methanogens, but only a few isolated
from the gut so far
• Very difficult to cultivate
© Glenn Gibson
Protein fermentation
Microbial metabolism
Blood Faeces
Urine
Blood Faeces Urine
Ammonia Amines Phenols
Branched-chain fatty acids (i-Val,
i-But, i-Cap)
clostridia
peptostreptococci
peptococci
COLON
LIVER
GUT mucosa
tyrosine
4-cresol
4-cresyl
sulfate (4CS)
phenylalanine
phenylacetate
phenylacetylglutamine (PAG)
glutamine
Microbiome / Genome
© Jon Swann
Proposed pathways of LA metabolism by bacterial species
isolated from the human gut.
Devillard et al. J. Bacteriol. 2007;189:2566-2570
Linoleic acid metabolism
Metabolite Species
OH 18:1 FA Lact. reuteri, lactis; Prop.thoenii,
(HFA) Bif. adolescentis, infantis; F. praunitzii;
Roseburia intestinalis*, faecis*
CLA Prop. freudenreichii; Bif. breve
VA Roseburia inulinivorans*, hominis*;
Butyrivibrio fibrisolvens
* 76-100% conversion in vitro
Isolated strains did not produce stearic acid, only faecal slurries
17
Devillard et al. J. Bacteriol. 2007;189:2566-2570
Vitamin synthesis
• Studies in GF & CV animals and in Ab treated humans
indicate microbiota can synthesize certain vitamins
• Metagenomic studies show vitamin synthesis genes are
common esp.vitamin K and B group vitamins - biotin,
cobalamin, folate, nicotinic acid, panthotenic acid,
pyridoxine, riboflavin and thiamine
• For riboflavin & biotin, all tested microbes in Bacteroidetes
Fusobacteria and Proteobacteria phyla had required
pathways, fewer Firmicutes and Actinobacteria had the
pathways
• Bacteroidetes seem to be most important phyla for vitamin
synthesis
• Many of vitamins are utilized by other bacteria
18
Bile acids
Bile salt hydrolase
• BSH genes have been identified in the main bacterial
genera including Bacteroides, Bifidobacterium,
Clostridium, Lactobacillus, and Listeria
• Most hydrolyze both glyco and tauro-conjugates.
• Reduces toxicity of bile acids and releases nitrogen,
sulphur and carbon atoms
19 Jon Swann
BILE ACIDS
7a-dehydroxylation
Deconjugation by bacterial
bile salt hydrolases (BSH)
Ursodeoxycholic acid
Epimerization
Lipid digestion:
– Deconjugation reduces efficiency of bile acids for emulsification
of dietary lipids and micelle formation
– CA greater than CDCA and DCA at emulsifying lipids
– Gut microbial BSH expression altered plasma bile acid signature
and transcription of genes involved in fat metabolism and
metabolic signaling pathways (Joyce 2014)
© Jon Swann
Polyphenols
• Often poorly absorbed in small intestine
colon
• Studies in GF and human microbiota-
associated animals and in vitro faecal
incubations show parent polyphenols are
extensively metabolized by the microbiota,
(deglycosylation, ring fission, dehydroxylation)
- can impact bioactivity
23
Gut microbiota & inter-individual
variation in polyphenol metabolism
• Differences in composition of microbiota between
individuals can have significant effects on extent of
metabolism, metabolite profile & bioavailability
• Examples:
– Isoflavonoids (daidzein to equol)
– Naringin
– Anthocyanins
– Lignans
– Tea catechins
– Rutin
24
Polyphenol metabolism –
microbes involved
Often requires involvement of a range of
microbes.
Example: secoisolariciresinol diglucoside
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Methodologies
• Isolated cultures
• Gut microbial enzyme activity
• Mathematical modeling
• System biology approaches
– Metagenomics
– Metatranscriptomics
– Metaproteomics
– Metabonomics
27
Isolated cultures
• Equol production: Serially dilute faecal sample from
equol producer onto agar, test single colonies for
equol production – yielded a 4-member consortium
(Decroos et al 2005)
• Enrichment culture – used for isolating CHO
degraders from ruminants. Continuous culture
fermenters containing medium with eg xylan as sole
CHO source, run for 8 weeks then samples plated
onto xylan agar. Identified complex communities
capable of metabolism (Ziemer 2014)
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Enzyme activities
• Measure specific enzyme activity in faecal samples eg
glucosidases, polysaccharidases, reductases. Ignores
individual organisms.
• Assay enzymes in gut bacterial isolates representing
major microbiota types to identify main organisms
involved. Glucuronidase activity mostly associated
with clostridial clusters, glucosidase with bifids and
bacteroides
• Limitation – activity in vitro may not reflect in vivo
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Gut microbiota - metabolism
• Reduction – C=C bonds
– Azo links
– NO3/NO2
– Aldehydes
– sulphates
• Hydrolysis – Glycosides
– Glucuronides
– Sulphates
– Esters
– Amides
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• Dehydroxylation (C & N)
• Methylation
• Demethylation
• Nitrosation
• Deamination
• Ring fission
• Aromatization
• oligomer breakdown
• Polysaccharide fermentation
Mathematical modeling
• Muñoz-Tamayo 2011: Kinetic modelling showed the
role of bacterial cross-feeding in conversion of lactate
to butyrate by two human gut bacteria, Eubacterium
hallii and Anaerostipes coli.
• Kettle 2014-15: Minimal model of the intestinal
ecosystem, 10 bacterial functional groups. Used to
estimate the effect of removing entire functional
groups (or single strains within a functional group) on
the community profile and activity. Potential for
assessing consequences of dysbiosis on development
of disease.
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Summary of interactions between
functional groups in model
From Kettle et al 2014. Env Microbiol doi:10.1111/1462-2920.12599
• Multivariate
• Hypothesis-
generating
• Model-building to
understand complex
systems
Top-down approach
Bottom-up approach • Hypothesis-led • Uni- or bi-variate
Systems Biology aproaches
Systems biology approaches
are ideally suited to study
the microbiome due to its
complexity and its
interactions with host © Jon Swann
Systems biology approaches
• Metagenomics – study functional genes associated with
specific microbial types,
• Meta-transcriptomics – monitor active bacteria, reveals
functional roles (eg CHO metabolism) info on functional
dysbiosis,
• Meta-proteomics – confirming microbial function (faecal
meta proteome is subject-specific and stable, protein
biomarkers)
• Metabonomics – pathway analysis, metabolic biomarkers of
disease risk eg high fecal bile acid, lower bc SCFA in IBS
(Gall et al J Proteome Res 10, 4208 2011)
35
Metagenomics applications
• Used to study changes in microbial function in disease
states
• Distribution of BSH genes in microbiota, high level of
redundancy
• Distribution and diversity of carbohydrate active
enzymes (CAZymes) in microbiota
36
Metatranscriptomics applications
• Identification of the most metabolically active microbial
groups
• Most active Firmicutes : Lachnospiraceae and
Ruminococcaceae
• Most active Bacteroidetes: Bacteroidaceae,
Prevotellaceae, Rickenellaceae
• Main activities :carbohydrate transport and
metabolism, energy production and conversion,
synthesis of cellular components
• Less important: amino acid and lipid metabolism, cell
motility, secondary metabolite biosynthesis
37
Metaproteomics applications
• Few studies, small subject numbers (~3)
• Some evidence that faecal metaproteome is subject
specific and stable over 1 year. Core of ~1000 proteins
• Main functional categories - metabolism of
carbohydrates, nucleotides, energy, and vitamins (B12
and folic acid).
• Can link proteins to taxonomic groups insight into
microbes at species/strain level involved in specific
catalytic functions and pathways i.e. genotype-
phenotype linkages
38
Metabonomics
• Measure metabolites in host samples that derive
directly from the microbiome a direct read-out of gut
microbial activity.
• If absorbed from the gut, microbial products can
interact with host metabolic processes resulting in
downstream metabolic perturbations and the
generation of microbial-host co-metabolites, all of
which can be captured by metabolic profiling.
40 © Jon Swann
Limitations
• Methodology – need for harmonization/standardization
to provide reference protocols to enable comparisons
of different studies
– Identification of critical points influencing results ‘HACCP’
– Sample collection & storage
– Macromolecule extraction and processing
– Analytical methodologies
• Bioinformatics – complex and developing, especially
for integrating data from different omics
• Reference databases limited
42
Conclusions
• Gut microbiota metabolism extends metabolic flexibility of
host to process wide range of substrates
• CHO metabolism major function of microbiota – pathways
well studied
• Large amount of functional redundancy so composition
variation may not result in functional variation
• Need for consensus on best ‘markers’ of microbial
metabolism
• ‘Omics’ provide insight into microbiota function at high
resolution
• In future important to integrate omics datasets to fully
exploit potential and develop mathematical models
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Integration of omics
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• sample processing & preservation: homogenization with RNAlater followed by centrifugation steps before
snap-freezing
cryomilling of cell pellets and solvent extraction of the intracellular polar and non-polar metabolite fractions
Physicochemical biomacromolecular isolation
Roume et al ISME 7,110-121