Convince the expert 1
1. Specify the origin of the following metabolites and components of the commensal microbiome
(Basic Challenge 1)
• Short-chain fatty acids: Bacteroides, Lachnospiraceae and Ruminococcaceae → ferment
complex plant-derived undigestible carbohydrates (dietary fibers) → into pyruvate → into
Acetyl-CoA → short-chain fatty acids (SCFA):
o Butyrate: fuel for colonocytes (gets oxidized)
o 20%
o Acetate: converted in liver to fatty acids + triglycerides (related to obesity)
o 60%
o Propionate: inhibits cholesterol synthesis in liver
o 20%
• Trimethylamine N-oxide (TMAO): microbiome (C. sporogenes, Anaerococcus hydrogenalis,
Providencia rettgeri) metabolizes choline + L-carnatine into trimethylamine (TMA) which is
absorbed in the gut → converted by FMO1, 3 (flavin monooxygenases) into trimethylamine-N-
oxide (TMAO) in the liver →
o Exacerbates hepatic insulin signalling and glucose tolerance
o Promotes adipose tissue inflammation, atherosclerosis and cardiovascular diseases
= Microbial conversion of dietary choline is an emerging metabolic hallmark of
cardiovascular diseases
= Choline → TMA → TMAO
• LPS (lipopolysaccharides): is a PAMP (pathogen associated molecular pattern) that gets
recognized by PRR (pathogen recognition receptors) such as TLR (Toll-like receptors)
o Major component of the outer membrane of Gram-negative bacteria
o AKA endotoxin → potent stimulator of the innate immune response
o Contain both lipid and carbohydrate and consist of three parts:
▪ Lipid A: contains two glucosamine sugar derivatives, each with fatty acids and
phosphate attached → endotoxin activity
▪ Core polysaccharide: constructed of 10 sugars → joined to lipid A
▪ O-side chain/ O-antigen: has unusual sugars → extends outwards from the core
• Peptidoglycan: a polysaccharide consisting of sugars and amino acids that forms a mesh-like
peptidoglycan layer in bacteria that covers entire plasma membrane
o AKA murein
o Gram+ bacteria: thick peptidoglycan that acts as the cell wall
o Gram – bacteria: thin peptidoglycan, but have an outer membrane that both acts as the
cell wall
o Structure: composed of many identical subunits in which each subunit contains two
sugar derivatives →
▪ NAG (N-acetylglucosamine)
▪ NAM (N-acetylmuramic acid)
• Flagellin: a globular protein (subunit) that arranges itself in a hollow, rigid cylinder to form the
filament in a bacterial flagellum
o Ranges in molecular mass: 30.000-60.000 Dalton
, o Structure: helical shape → important for its proper function
o Flagellum function:
▪ Mobility
▪ Attachment to surfaces
▪ Virulence factor (contribute to the ability of the bacterium to cause disease)
• Indole: microbiome (tryptophanase-expressing bacteria) catabolized tryptophan, 5% gets
metabolized, (one of the nine essential amino acids that can’t be synthesized by the body) into
indole and indole derivatives (IPA and I3A)
o Indole:
▪ Acts as a ligand for AhR (aryl hydrocarbon receptor) → prevent the infection of
Citrobacter rodentium and Candida albicans by snatching of metal ions (restore
epithelial barrier function and normalized dysbiotic bacterial population)
▪ Signaling molecule to intestinal L cells to produce glucagon-like protein 1 (GLP-
1) Acts as a ligand for AhR (aryl hydrocarbon receptor) →
▪ Gets metabolized by the liver to indoxyl sulfate, where an excess is detrimental
to human health
o IPA (indole-3-propionate):
▪ Acts on intestinal cells via pregnane X receptors (PXR) → maintain mucosal
homeostasis and barrier function
▪ Act on the brain → neuroprotective effects
o I3A (indole-3-aldehyde):
▪ Acts on the AhR found on intestinal immune cells → increases interleukin-22
(IL-22) production
▪ Activation of AhR → crucial role in gut immunity (maintaining the epithelial
barrier function and promoting immune tolerance to promote microbial
commensalism)
• Secondary bile acids: anaerobic bacteria of the genera Bacteroides, Eubacterium and
Clostridium metabolizes (deconjugation) bile acids by bile salt hydrolase into secondary bile
acids:
o Deoxycholate
o Ursodeoxycholate
o Lithocholate
= Passively absorbed (95%) in the colon and are transported back to the liver & the rest
is deconjugated and excreted through faeces
= Pathological effects of deconjugation by 7αβ-dehydroxylation of bile salts: obesity
and cancer
= Primary bile acids: stimulate germination of C.difficile spore
= Secondary bile acids: inhibit spore germination as well as vegetative growth of C.
difficile
2.
a. Summarize the different antimicrobial peptides produced by intestinal epithelial cells, indicate
which specific cells produce these peptides and what their function is?
• Absorptive epithelial cells/enterocytes:
o Defensins
▪ β-defensins: colon → penetrating a microbe's cell membrane and cause
microbial death
• Paneth cells (small intestine):
o Defensins
▪ a-defensins: small intestine → inactive pro-peptides and need proteolytic
cleavage to gain antimicrobial activity
• HD5 (human defensin 5): bind to glycosylated proteins and neutralize
bacterial exotoxins
• Trypsin: activate a-defensins
o C-type lectins (REGIIIγ s, regenerating islet-derived proteins): block bacterial
colonization of the epithelial surface
o Lysozyme C: damages Gram-positive bacterial cell walls by cleaving peptidoglycan
o sPLA2: rapidly degrade bacterial phospholipids, thereby destroying cell integrity
o ANG4: retains ribonuclease activity that makes it effective against various Gram-
positive and Gram-negative bacteria
• Goblet cells
o Mucins (extensively glycosylated proteins) → form a viscous physical barrier that
prevents microbes from contacting the epithelial lining of the gastrointestinal tract
• Upper crypt epithelial cells
o Cathelicidins: colon
▪ LL-37: exerts microbicidal effect (neutralizes lipopolysaccharide + pore
formation which induces lysis) on both Gram-negative and Gram-positive
bacteria
b. Summarize the function and classes of ILCs
= Innate lymphoid cells: respond to local cytokines produced by epithelial cells in response to injury or
microbes and serve as an alarm for the immune system. T-cell, without TCR → can’t recognize
antigens (react to cytokines)
, - Gut ILC1:
o Activated trough IL-12,18
o Defenses against infections with viruses and
certain bacteria
- Gut ILC2:
o Activated by neuropeptides (neuromedin U +
vasointestinal peptide) produced by enteric
neurons
o Produced rapidly after intestinal helminth
infection
o Secrete:
▪ IL-5: activates eosinophils, which
secrete enzymes that degrade the
outer integument of helminths
▪ IL-13: Increases mucus production,
contributing to expulsion of the worms
(by stimulating the differentiation of
mucus-secreting goblet cells and more
tuft cells from intestinal crypt stem
cells)
- Gut ILC3
o Produced after a bacteria or fungi infection (alarmin IL-1)
o Secrete:
▪ IL-17: promotes acute inflammatory response to the microbes + enhance
intestinal mucosal barrier function by stimulating production of defensins +
enhancing epithelial tight junction function
▪ IL-22: enhance intestinal mucosal barrier function by stimulating production of
defensins + enhancing epithelial tight junction function
3. Intestinal epithelial cells play an important role in orchestrating the host-microbial interface.
Explain how signals from the commensal microbiota might mediate (Include at least the
following: PAMPs, PRRs, TLRs, GPR109A, SCFAs, NLRP6 inflammasome and the central role of
IL-18)
• The release of antimicrobial peptides the production of mucus by epithelial cells
When a PAMP (pathogen-associated molecular pattern), such as LPS (lipopolysaccharide) gets
recognized by PRR’s (pathogen recognition receptors), such as TLR’s (toll-like receptors) or NOD1,2
(nod like receptors) on the intestinal epithelial cells, a direct coupling gets assessed where
antimicrobial peptides (RegIIIγ, RegIIIβ, Ang4 and Itln1) and mucus are produced.
(In detail) when:
- TLRs recognizes PAMP
- G-protein-coupled receptor GPR109a is activated
→ Transcriptional activation of pro-IL-18 → is a pro peptide, thus must be cleaved through Caspase-1
to be effective → IL-18 → induces IL-18-dependent antimicrobial peptides production and mucus by
epithelial cells
Commensal bacteria also induce IL-18
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