EXAM Lecture 14

Question 1

Barrier defence mechanisms: mucus production

Answer 1

three barrier defence mechanisms:
- first was previous apical junctions barrier
- second is this one: mucus
- third is antimicrobial proteins (AMPs)

mucus composition: mucins, primarily comprised of O linked glycans
- primary mucin of the outer mucus layer in colon is Muc2
- other mucins include Muc1 - 17
- Muc1 and 3 bound to epithelial cell and part of the inner mucus layer in the colon

recap (important):
- Mucins are glycoproteins that are post translationally modified
- majority are O linked glycan, rest are M linked glycan

Fcgbp (FC gamma binding protein) functions as a mucus cross linker: forms a mucin lattice work that binds water and becomes a gel (mucus)

inner thin stratified mucus layer:
- compact firmly attached to the epithelium
- no or very few bacteria
- thinner than outer

outer thick mucus layer:
- loose structure, thicker layer
- degraded by microbiota, glycans are used by the bacteria for energy and metabolite production

Question 2

Muc1 Muc2 Muc3

Answer 2

Muc1 and Muc3
- membrane bound (inner mucus layer)
- O linked glycans AND N linked glycans

Muc2
- secreted mucin (outer mucus layer)
- O linked glycans ONLY

O linked glycans:
- core protein with monosaccharides attached via O glycosylation reactions to the oxygen atoms in SERINE and THREONINE residues in the muc protein

N linked glycans:
- core protein with oligosaccharides attached to the nitrogen atom within the amide group of ASPARAGINE residues in the muc protein via N linked glycosylation reactions

Fcgbp cross links all mucins together
- then binding of water to the protein complex makes gel

Question 3

Post translational modification of mucus

Answer 3

Muc2
- secreted mucin
- O glycosylated; carbs added to an oxygen via glycosylation
- packed into granules
- has to transverse inner mucus layer to get to outer
- after granule release, water binds to mucins; expand in size, form gel and need Fcgbp to cross link

Muc1/Muc3
- membrane bound
- O and N glycosylated at nitrogen
- goblet and epithelial cells will have both embedded into the apical plasma membrane
- form fenceline

Question 4

what happens when a bacteria gets through the mucus layer

Answer 4

a) secretion (protection mechanism)
- pathogen comes into contact with epithelial cell apical membrane (has reached inner mucus layer where it shouldnt be) which stimulates the release of anti microbial proteins (AMPs) stored in granules (need aMPs ready for rapid release)
- AMPs are the first back up after pathogen has gotten to inner layer

b) signal transduction (consequence)
- pathogen comes into contact with the epithelial cell apical membrane and stimulates either; apoptosis or stimulates host secretion of inflammatory mediators that break down tight junctions = leaky barrier
- happens if AMPs dont get rid of pathogen but not the best solution becase it creates gaps in barrier

c) shredding of cell surface mucins (protection mechanism)
- shed extracellular domains of Muc1 and Muc3 to coat the pathogen and prevent its function
- release of the fence line to coat and cover pathogen

d) steric hindrance (protection mechanism)
- inner mucus layer mucins (muc1 and 3) form a physical barrier that results in steric hindrance of the pathogen from physically contacting the host cell surface receptors
- prevention of bacteria interacting with host receptors

Question 5

How do pathogenic bacteria disrupt barrier defence

Answer 5

• bacteria secrete mucus degrading enzymes that destroy the mucus layer
• bacteria can use the monosaccharides that make up mucus for energy and short chain fatty acid production

many pathogenic bacteria secrete toxins that can diffuse through the mucus:
- can disrupt tight junctions (leaky gut)
- induce epithelial cell apoptosis
- disrupt mucus production
- reduction in mucus will allow pathogens to reach cell surface

Question 6

Mucus layer structure and size differs throughout the GI tract

Answer 6

small intestine mucus layer is diffuse (not continuous) and has small clusters of mucus
- allows for increased absorption of nutrients etc
primary mucins produced: MUC5AC and MUC2 ?

mucus associated bacteria degrade mucus layer which provides energy for the bacteria; can be used to make SCFA, etc
- mucus layer is constantly replenished by the host

Question 7

Anti microbial proteins/peptides (AMPs); third line of defence

Answer 7

produced primarily by Paneth cells and epithelial cells
- also produced by immune cells like APCs
- typically stored in granules so that theyre ready for rapid release when needed

defensins
- alpha and beta defensins
- post translational activation
- still stored in granule just not rdy yet

regenerating islet derived proteins (REG)
-a,b,y subfamilies

cathelicidins
- in humans, CAMP gene encodes LL-37 peptide
- LL-37 promotes wound healing and can modulate the adaptive immune response against bacteria (influences BOTH microbiota and host) DUAL FUNCTIONING
- LL-37 expression is induced by calcitriol

lysozymes
- lyse open and kill bacteria

immunoglobulin A (IgA)
- anti inflammatory.anti bacterial released by host immune system (b cells) NOT epithelial

Question 8

Balance between host defenses and microbial defenses

Answer 8

bacteria secretes/releases into the lumen:
- enzyme inhibitors: a1 protease inhibitor and a1-anti-trypsin as their defense mechanism

host secretes/releases into the lumen:
- pro defensins; inactive
- proteolytic enzymes; needed to activate the defensins (trypsin and peptidase)
- –fine control over activation of defensins

the NET RESULT (bacteria death or survival) will depend on the interaction between host and bacterial signals

Question 9

how secretede anti microbial proteins attack mucosal associated bacteria

Answer 9

post translational processing of defensins
- secreted as a pro defensin (inactive)
- needs to be proteolytically cleaved by enzymes in the lumen (trypsins or peptidases)
- these enzymes will then convert the inactive pro defensin into the active form (defensin)

• host also has to secrete these proteolytic enzymes for defensins to work
• to prevent defensin activation, bacteria express and secrete enzyme inhibtors (a1 protease inhibitor, a1 anti trypsin) as their defense mechanism

host defensin release from granules can be:
- constitutive (happening at low levels all the time)
- induced by:
– bacterial components (LPS, PGN)
– bacterial metabolites
– host growth factors
– host inflammatory cytokines (IL-1, TNFa)