Lecture 4

Question 1

what makes sure that the microbiota in the small intestine doesn’t overgrow?

Answer 1

rapid flow through the small intestine and immune factors

Question 2

what are people usually referring to if they say ‘gut microbiota’?

Answer 2

colonic microbiota.

Question 3

why is there more microbiota within the colon?

Answer 3

transit slows in the colon so bacteria have time to divide.

Question 4

we have a mutualistic relationship with our microbiota, what does this mean?

Answer 4

it is a relationship where both partners benefit.
we provide them with nutrients in the form of complex dietary (and host) glycans, and they play a key role in maintaining health and nutrition.

Question 5

why are carbohydrates metabolised into SCFAs by microbiota?

Answer 5

because its done in anaerobic conditions.

Question 6

what has dysbiosis in the microbiota been linked with?

Answer 6

inflammatory bowel disease, obesity, diabetes (T1+T2), colon cancer, heart/kidney/liver disease, autoimmune disease (e.g. allergy, asthma, neurological disease: anxiety, depression, autism, Parkinson’s. Infectious diseases by cholera and salmonella.

Question 7

why may non-communicable chronic diseases be on the rise in the western world?

Answer 7

due to a high-fat, low-fibre diet which negatively impacts our microbiota.

Question 8

what did the human microbiome project use to analyse the microbiome?

Answer 8

16SrRNA and Metagenomics sequencing to study microbiota of 242 healthy individuals at 5 main body sites.

Question 9

what bacteria is found in live yoghurts?

Answer 9

Bifidobacteria.

Question 10

what was discovered in a European metaHIT study about Bacteroides and Firmicutes?

Answer 10

Bacteroides and Firmicutes were the main phyla but Firmicutes were much more diverse below the phyla level than Bacteroides which was mainly Bacteroides spp.

Question 11

what were the main findings of the human microbiome project?

Answer 11

aggregate microbiome ~1000 bacterial species
each person has ~100-150 different species
these are largely different between individuals and even at genus level only ~30% overlap.
does mot appear to be a ‘keystone’ group of essential species that everyone shares.

Question 12

what is l-fucose?

Answer 12

a common terminal component of many mammalian cell glycans which the microbiota can use as an energy source.

Question 13

why is Bacteroides thetaiotaomicron used as a model?

Answer 13

its genetically tractable and a prominent member of the normal microbiota.

Question 14

what was discovered about germ-free mouse and wild type mouse fucose levels?

Answer 14

wild-type mice had epithelial cell surface with complex glycol structures with a fucose terminal sugar, germ free mice had no fucose on their cell surfaces.

Question 15

what happened when germ free mice were colonised with Bt?

Answer 15

fucosylation of glycans on intestinal enterocytes was similar to mice with normal microbiota.

Question 16

how did they check that bt was responsible for the fucosylation?

Answer 16

they isolated a mutant strain of bt using transposon mutagenesis that can’t use fucose or signal fucosylation. (Fu4).
when sequencing either side of the transposon, they found a fucose utilisation locus, consisting of FucR, FucI, FucA, FucK and FucP, all of the gene products of the locus encode the metabolic pathway that allows bt to import fucose and metabolise it down to useful metabolites. so they found that it was the fucose utilisation locus that the transposon went into that knocked out the ability to use fucose and signal fucosylation.

Question 17

why did FucR look like a regulator?

Answer 17

because it had a putative DNA binding domain at the N-term.

Question 18

how does fucR repress transcription of the locus?

Answer 18

fucose binds to fucR bound to the promoter, causing it to fall off and RNA polymerase can arrive and transcription of locus is initiated and leads to the production of protein, leading to the metabolism of fucose.

Question 19

how is fucose metabolism regulated?

Answer 19

with the presence of fucose, when fucose is present, metabolism is switched on, when its absent its switched off.

Question 20

if you knock out fucR, fucosylation can still be induced, what does this suggest?

Answer 20

suggests that fucR must also act as a repressor of a different locus involved in signalling host to make fucosylated glycans.

Question 21

what is the summary of the whole fucose experiment?

Answer 21

shows that a normal member of the gut microbiota signals the host telling it to provide a nutrient source and coordinates this with its metabolic capacity to use this carbon source.
it provides a competitive advantage to bt and provides an example of how the host can shape the composition of its microbiota.

Question 22

what are the main SCFAs?

Answer 22

butyrate, acetate and propionate.

Question 23

what receptor recognises SCFAs when they cross the epithelium?

Answer 23

GPR43 receptors on Treg cells.

Question 24

what happens when SCFAs bind to the GPR43 receptors on Treg cells?

Answer 24

IL-10 is production is induced, which blocks inflammation and allows colonisation by microbiota.

Question 25

beneficial microbes also have PAMPs, how are they distinguished from pathogenic microbes?

Answer 25

Bacteroides fragilis (gut mutualist) produces polysaccharide A (PSA), PSA is recognised by innate immune cells which leads to indiction of Treg cells to produce the tolerogenic cytokine IL-10, which promotes B. fragilis as its recognised as a mutualist.

Question 26

what receptor was it found that PSA signals through, and why was it unexpected?

Answer 26

it signals through toll-like receptor 2 to promote immune tolerance (balanced th1 and th2 cells). was very unexpected because TLR2 signalling typically associated with immune activation and bacterial clearance.

Question 27

What happened in mice inoculated with B. fragilis which didn’t have PSA?

Answer 27

there was an imbalanced immune response which led to inflammation.

Question 28

what is so special about polysaccharide a?

Answer 28

PSA is composed of several hundred repeating units of tetrasaccharide, a zwitterionic polysaccharide, very few species produce molecules like this.
it gives PSA a very distinct shape and may be what TLR2 recognises.