Convince the expert 1

Convince the expert 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)

• 20%
• Acetate: converted in liver to fatty acids + triglycerides (related to obesity)
• 60%

o Propionate: inhibits cholesterol synthesis in liver

• 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 →

• Exacerbates hepatic insulin signalling and glucose tolerance
• 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)

• Major component of the outer membrane of Gram-negative bacteria
• 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

• AKA murein

o Gram+ bacteria: thick peptidoglycan that acts as the cell wall

• Gram – bacteria: thin peptidoglycan, but have an outer membrane that both acts as the

cell wall

• 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

• Ranges in molecular mass: 30.000-60.000 Dalton 1 / 4

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:

• Deoxycholate
• Ursodeoxycholate
• 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.

• Summarize the different antimicrobial peptides produced by intestinal epithelial cells, indicate

which specific cells produce these peptides and what their function is?

AMPS: antimicrobial peptides hydrophobic interactions

• Weak → can’t bind
• Negative charge inside (multicellular animal cells)
• Strong → bind to bacterial cytoplasmic membrane → opsonization → cell-lysis 2 / 4

• Negative charge on outside (bacteria)

• Absorptive epithelial cells/enterocytes:

• Defensins

▪ β-defensins: colon → penetrating a microbe's cell membrane and cause

microbial death

• Paneth cells (small intestine):

• 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

• C-type lectins (REGIIIγ s, regenerating islet-derived proteins): block bacterial

colonization of the epithelial surface

• Lysozyme C: damages Gram-positive bacterial cell walls by cleaving peptidoglycan
• sPLA2: rapidly degrade bacterial phospholipids, thereby destroying cell integrity
• ANG4: retains ribonuclease activity that makes it effective against various Gram-

positive and Gram-negative bacteria • Goblet cells

• 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

• 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) 3 / 4

- Gut ILC1:

• Activated trough IL-12,18
• Defenses against infections with viruses and

certain bacteria

- Gut ILC2:

• Activated by neuropeptides (neuromedin U +

vasointestinal peptide) produced by enteric neurons

• 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
• 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

• 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

• / 4