EXAM Lecture 13

Question 1

Antibiotics impact microbial diversity /community structure

Answer 1

microbiota abundance and diversity with antibiotics decreases

some antibiotics will help maintain microbiota diversity whereas others will not
- broad spectrum antibiotic vs specialized targeted antibiotics

eg: C. difficile infection
- hard to get rid of
- antibiotics can kill C. difficule BUT also destroys the healthy microbiota community structure

Question 2

What does C. difficule do to the gut

Answer 2

24 hours after exposure:
- epithelial cells of the colon are normal

30 hours after exposure:
- C. difficult toxin has started damaging epithelial cells, triggering inflammation and fluid build up

36 hours after exposure:
- inflamed epithelial cells burst and die (necrotic cell death; releases contents, more inflammatory than apoptosis)
- C. difficule spores leave the colon via diarrhea and await the next host

reccurent infection with C. difficile is common (antibiotics usually dont get rid of all of it than any microbiota shift can activate it again)
- standard of care: patients with 3 recurrent C. difficile infections get fecal microbiota transplant (FMT) from a donor

Question 3

How do we study the microbiota: germ free (GF) mice

Answer 3

• no microorganisms or microbiota living in or on them
• raised under specific sterile environmental conditions
• test effects of bacteria strains or communities by innoculating GF mice (called gnotibiotic studies)

gut microbiota influences host barrier function
- mucus thickness in germ free mice is much lower untreated then increased with LPS and PGN luminally
*IDK WHAT THIS SLIDE IS SAYING SLIDE 53

Question 4

How do we study the microbiota: sequencing

Answer 4

16S: bacteria
18S: mammal

two types:
16S rRNA sequencing:
- PCR amplify 16S rRNA gene and sequenced
- expensive but affordable
- sequences put into databases -> identifies species and relative abudance

• data from Greengenes database assigns ID to each species based on taxa; called an operational taxonomy unit (OTU)
• determines percent and abundance
• through 16S sequencing we can learn WHICH bacteria are present and HOW MUCH of each
• 40-60% of bacteria sequenced have not been cultured

total microbiome DNA sequencing:
- total sequencing of DNA from microbiome
- filter host DNA and compare to databases -> identifies species, relative abundance, genes, functional information (the 3 importants)
- more expensive

Question 5

Relative abundance of bacteria at the phylum level

Answer 5

two dominant phyla:
- bacteroidetes and firmicutes
- though they remain dominant, actual amount of each varies

low abundance phyla:
- actinobacteria
- proteobacteria
- verrucomicrobia

% abundance decreases as you get more specific about the bacteria taxa level

low abundance bacteria can still have a big impact on the host physiological function

Question 6

SLIDE 59 OF MICROBIOTA PART 1

Answer 6

just write on cheat sheet

Question 7

Symbiotic relationship between microbiota and host

Answer 7

microbiota:
- diversity
- abundance
- function (gene counts)

host:
- epithelial barrier
- mucus barrier
- mucosal defenses: antimicrobial proteins and mucosal immune system

Question 8

Host Anti-Bacterial Defense and Barrier Integrity

Answer 8

protection from the gut microbiota
- intact bacteria
- portion of a bacteria that has been lysed (PGN, LPS)
- microbial derived metabolites

1. epithelial barrier: single cell barrier between microbiota and ALL host tissues
   - critical importance of tight junctions and adherens junctions (apical)
2. Mucus barrier: mucus production: secreted by goblet cells and covers the epithelial barrier
3. Anti-bacterial or anti microbial proteins (AMPs)
   - secreted into the lumen

without these defense mechanisms, bacteria crosses epithelial barrier and enters host tissues -> causes inflammation

Question 9

SLIDE 62 MICROBIOTA PART 1 PUT IT ON CHEAT SHEET

Answer 9

Eubiosis:
- diverse gut microbiota with beneficial commensals
- balance of pro inflammatory and anti inflammatory cytokines
- balance of tregs and T helper 17 cells
- primed APCs
- tight mucosal barrier
- thick mucous layer
- IgA secretion (anti microbial protein)

Dybiosis:
- low diversity of microbiota supporting growth of pathogens
- overwhelming inflammatory cytokine
- imbalance of T cell subsets with impaired immune responses
-APCs unprimed
- attenuated mucous layer with leaky mucosal barrier (decreased apical junctions)
- impaired IgA secretion ( decreased anti microbial protein secretion)

Question 10

Barrier defence mechanisms in the colonic microenvironment

Answer 10

• colonic environment refers to interface between microbiota and the host

layered like:
- microbiota
- secreted AMPs
- outer mucus layer: stratified so that mucus associated bacteria arent close to epithelial barrier
- inner mucus layer: connected to the apical membrane of cells in epithelial barrier)

outer and inner both have mucin, inner doesnt have any bacteria its just protection

• epithelial barrier: colonocytes aka epithelial cells secrete AMPs (at the top), goblet cells secrete mucin, tight junctions connect all

Question 11

Colon epithelial barrier structure

Answer 11

high cell turnover requires new tight junctions to be functional and intact, otherwise barrier permeability increases

moving from start at stem cell at base of crypt upwards, differentiation increases and proliferation decreases

Question 12

apical junction complexes review

Answer 12

tight junctions
- seals neighbouring cells together and prevents leakage between epithelial cells

claudins, JAMS, occludins
- anchor the tight junctions to zona occludins (ZO) which bind to the actin cytoskeleton to structurally hold the junctional complex together

RhoA and Cdc42 function as adaptor proteins

adherens junctions
- joins the actin bundle in one cell to the next cell; cadherins, catenins

gap junctions
- allows passage of small water soluble ions and molecules between cells

leaky barrier means LPS, etc can get through