Lecture 8 - Gut Microbiota

Question 1

Broad change in prevalence of diseases between 1950 and 2000

Answer 1

Reduction in incidence of infectious diseases

Increase in autoimmune, allergic, inflammatory disease

Question 2

Hygiene hypothesis

Answer 2

Increased cleanliness associated with increased levels of autoimmune disease.
Reduced exposure to infectious agents leads to immune dysregulation

Question 3

Hypotheses for why autoimmune diseases are increasing in prevalence in the Western world

Answer 3

1) Hygiene hypothesis
2) Microbiota hypothesis (human gut evolved to have bacteria)
3) Diet hypothesis (human microbiome changes according to diet)

Question 4

Factors leading to altered diet in developed world

Answer 4

Economic
Demographic
Epidemiological

Question 5

Aspects of Western diet that could lead to dysbiota

Answer 5

High in fat, sugar
Highly refined and processed
Low in fibre (important)

Question 6

Factors potentially affecting microbiota composition

Answer 6

Host genetics
Maternal transfer, early colonisation 
Antibiotics, medications
Infection
Inflammation
Stress
Hygiene
Age

Question 7

Is the microbiome uniform through the GIT?

Answer 7

No. Increases in bacterial density/g from the stomach to the colon.

Question 8

Human vs microbial genome in human body

Answer 8

Human genome has ~23,000 genes.

Microbiome has over 100,000 genes

Question 9

Advances in studying GIT microbiome 
1)
2)
3)
4)

Answer 9

1) Generation of germ-free mice
2) High throughput DNA sequencing
3) Transcriptional and metabolomic tools, with which to measure microbiota impact on host physiology, immunity, development
4) Development of tractable experimental systems (C. elegans, drosophila)

Question 10

Germ-free mice

Answer 10

Mice raised in a germ-free environment. Can be from different genetic lines, clonal, non-clonal.
Can selectively restore bacteria in microbiome

Question 11

High throughput DNA sequencing effect on studying microbiome

Answer 11

Can generate metagenomic analyses to compare metabolic and taxonomic data from different microbiota

Question 12

What is the human microbiome project?
1)
2)
3)

Answer 12

1) NIH
2) First phase (2007-2012). Characterised composition and diversity of human microbiome in skin, nose, mouth, GIT, UGT. Evaluated metabolic potential.
3) Second phase (2013-2015). Create the first integrated dataset of biological properties of host and microbiome from cohort studies of microbiome-associated diseases

Question 13

What is a healthy microbiome?

Answer 13

1) Varies a lot between individuals
2) Predictions can be made loosely based on country, breast feeding, level of education
3) No core microbiotal genome, but broad metabolic characteristics are shared

Question 14

Study into the diversity of microbiota
1)
2)
3)

Answer 14

1) 16S rRNA sequences compared in mono- and dizygotic twins
2) No core genome shared. High variability in species/phylum composition
3) Analysis of genes showed a similarity in metabolic capacities

Question 15

Two major phyla in GIT microbiome

Answer 15

Firmicutes

Bacteroidetes

Question 16

Study into diet-induced changes in GIT microflora
1)
2)
3)
4)
5)

Answer 16

1) Microbiota of children from Burkina-Faso and Florence were compared.
2) Bacteroidetes dominated in Burkina-Faso
3) Firmicutes dominated in Italy
4) Diet has a larger role in shaping microbiome than ethnicity, climate, sanitation, hygiene
5) More pathogenic bacteria, lower SCFA levels in Italian children

Question 17

Functional members of non-pathogenic GIT microbiome

Answer 17

1) Probiotics
2) Autobionts
3) Pathobionts

Question 18

Probiotics

Answer 18

Transiently-present bacteria.
Can be beneficial.
EG: bifidobacterium spp, lactobacillus spp

Question 19

Autobionts

Answer 19

Permanent, symbiotic, immunomodulatory.
Part of normal microbiome.
Direct influence on host immune function
EG: Bacteroidetes fragilis, clostridium XIV

Question 20

Pathobionts

Answer 20

Permanent, parasitic/infectious.
Don’t cause disease normally, unless microbiome is perturbed.
EG: Clostridium difficile, helicobacter

Question 21

How are autobionts adapted to life in the GIT?

Answer 21

Express polysaccharide utilisation loci
Allows them to digest polysaccharides from plants.
Necessary for humans to be able to get nutrition from certain sugars (humans can only digest starch, maltose, sucrose).

Question 22

Example of a polysaccharide utilisation locus in bacteroidetes ovatus

Answer 22

Xyloglucan PUL

Question 23

Digestion of dietary fibre

Answer 23

1) Gut bacteria digest dietary fibre into short-chain fatty acids.
Bacteroidetes make acetate (2C), propionate (3C)
Firmicutes make butyrate (4C).
2) Short-chain fatty acids diffuse across lumen, are taken up by host transporters or bind to GPCR

Question 24

Roles of short-chain fatty acids in GIT
1)
2)
3)
4)
5)

Answer 24

1) Stimulate mucus production by epithelial cells
2) Stimulate B cells to make IgA
3) Promote differentiation of Treg, leading to tolerance
4) Maybe lead to inflammosome activation, IL-18 release. This promotes epithelial integrity
5) Inhibition of inflammaroty NF-kB

Question 25

Only source of nutrition for colonic cells

Answer 25

Short-chain fatty acids

Question 26

``` Importance of the GIT microbiome 1) 2) 3) 4) 5) ```

Answer 26

1) ~10% of calorific intake 2) Vitamins B and K 3) GIT epithelial development 4) Immune system development and tolerance 5) Competition for pathogenic bacteria

Question 27

Results of metagenome-wide association studies into microflora in T2DM patients 1) 2)

Answer 27

1) Decreased microbiome diversity associated with weight gain, insulin resistance, fatty liver, low-grade inflammation 2) Reduced butyrate products, increased pathogenic bacteria

Question 28

``` Evidence for GIT microflora influencing adiposity 1) 2) 3) 4) ```

Answer 28

1) Gut microflora from obese/lean human twins transferred to mice 2) Tranfer of 'obese' microbiome resulted in greater adiposity 3) Presence of lean mice with a high-fibre diet or 'lean' microbiome led to decreased adiposity in obese mice (from coporophagy) 4) Transfer of microbiome from first trimester/third trimester pregnant women to mice. Mice that received first trimester remained lean. Mice that received third trimester became obese.

Question 29

Modulation of host metabolism by short-chain fatty acid synthesis 1) 2) 3)

Answer 29

1) GPRC41 and GPRC43 stimulated. GPRC41 stimulation leads to peptide YY release. GPRC43 stimulation leads to hormone glucagon-like peptide release 2) Intestinal gluconeogenesis uses short-chain fatty acids as a carbon source 3) Inhibits fasting-induced adipose factor (FIAF).

Question 30

Fasting induced adipose factor role

Answer 30

Inhibits lipoprotein lipase. Lipoprotein lipase leads to VLDL and chylomicron release of free fatty acids, leading to their storage in adipocytes. Inhibiting FIAF might lead to increased adiposity

Question 31

GPRC41

Answer 31

GPRC that binds short-chain fatty acids. | Binding leads to release of peptide YY, which regulates gut motility and intestinal transit rate

Question 32

GPRC43

Answer 32

GPRC that binds short-chain fatty acids. | Binding leads to release of hormone glucagon-like peptide (GLP-1), which increase insulin-sensitivity

Question 33

Critical GPRC regulators of obesity

Answer 33

GPRC43-deficient mice are obese on a lean diet. Overexpression of GPRC43 in adipose tissue leads to leanness, regardless of calorie intake. These effects were dependent on gut microbiome composition (absent in germ-free mice)

Question 34

``` Effects of diet-induced dysbiosis 1) 2) 3) 4) ```

Answer 34

1) Local and systemic inflammation and inappropriate immune response 2) Change in Treg/Th17 balance 3) Increase in pathobionts, release of endotoxin (LPS) 4) Breakdown in gut barrier function, increased gut bacteria in blood

Question 35

``` Immunosuppressive immune dysregulation from dysbiosis 1) 2) 3) 4) ```

Answer 35

1) Treg/Th17 ratio increased 2) Butyrate promotes formation of Treg 3) Increase in IL-10 4) Th17 activation suppressed

Question 36

``` Inflammatory immune dysregulation from dysbiosis 1) 2) 3) 4) 5) ```

Answer 36

1) Treg/Th17 ratio reduced 2) Pathobionts promote Th17 differentiation 3) Associated with chronic autoimmunity 4) Induced by segmented, filamentous, G+, anaerobic bacteria 5) Segmented filamentous bacteria monocolonised mice don't mount inflammatory response or colitis

Question 37

Healthy microbiotal GIT response to a pathogen 1) 2) 3))

Answer 37

1) Commensal bacteria kill pathogen by competing for resources, reducing oxygen levels, suppressing virulence factors 2) B cells are stimulated to make IgA by SCFA release form commensals 3) IL-1b release from B cells leads to neutrophil entry into GIT, killing pathobionts, pathogenic bacteria

Question 38

Dysbiotic GIT response to a pathogen 1) 2) 3)

Answer 38

1) Depleted autobionts leads to overgrowth of pathobionts 2) Epithelial damage from lack of SCFA 3) Pathogenic bacteria enter blood

Question 39

``` Potential immunotherapy using microbiome 1) 2) 3) 4) ```

Answer 39

1) Less fat, more fibre in diet 2) Pre/pro biotics (pro - beneficial bacteria, pre - beneficial bacteria + undigestible food) 3) Faecal transplant 4) Targeted manipulation

Question 40

When is the risk of immunoallergenic disorders highest for a migrant form a low-risk country to a high-risk country?

Answer 40

Risk decreases with age. | Greatest risk is when you move at a young age from a low-risk to a high-risk country

Question 41

Do pre/probiotic supplements work?

Answer 41

Only when taken long-term

Question 42

Successful treatment for refractory C. difficile infections

Answer 42

Faecal transplant from a healthy donor

Question 43

Alternatives to a faecal transplant 1) 2) 3)

Answer 43

1) Use of cultured autobiotants 2) Inclusion of microbiota small molecules (such as acetate, butyrate) 3) Identify, administer antimicrobials produced by gut autobionts (EG: thuricin from bacillus thuringiensis kills C difficile)