Seminar 3 Inter-kingdom signaling

Question 1

What properties have the major classes of hormones in eukaryotes? How do these compare with QS molecules in bacteria?

Answer 1

3 categories of hormones: proteins, steroids, amio-acid derivatives (amines)

prots or peptides

• don’t cross cell memrbae, bind to cell surface receptors (receptor kinases, G-prot-coupled receptors)
• most hormones
• contain 3-200 amino acids
• usually pst-translationally processed
• ex: epidermal growth factor (EGF), insulin, glucagons

amino-acid derivatives:

• don’t cross cell memrbae, bind to cell surface receptors (receptor kinases, G-prot-coupled receptors)
• synthesized from tyrosine
• ex: adrenaline, noradrenaline, dopamine
• > compares to autoinducer 3 (AI-3) in bacteria, structure unknown, aminated aromatic compound (can propbabl not cross the cell membrane)

steroids

• steroids cross membranes, bind to intracellular receptors
• derived from cholesterol
• > compares to acyl homoserine lactone (AHL), conserved homoserine ring, variable acyl chain, can cross cell membrane

Question 2

What are the main features and differences between mammlian signaling via different types of cell surface receptors : receptor kinases, and G-protein-coupled receptors?

Answer 2

Signaling through receptor kinases:

• cell surface receptors kinase activity
• hormone binds -> recruitment+ phosphorylation of downstream protein -> signal cascade

example: EGF receptor (EGFR): binding EGR ->signal transduction ->DNA synthesis and cell proliferation
- lEGFR signaling required for cell-fate specification, growth and survival

Signalling through G-Protein coupled receptor
7 transmembrane domaine, coupled to guanine binding proteins (G proteins)
-hormone binding causes conformation change -> receptor interacts with Gprot -> stimulates release of GDP from GTP ->activation of intracellular target (ion channel or enzyme)
GPCR specificity is controlled by G prot type ( 4 types, largest class mammalian adrenergic receptors, adrenaline or NA initiates signal cascade)

similarity both signal from outsided

Question 3

What evidence is there that bacteria respond to human hormones such as adrenalin and noradrenalin?

Answer 3

test: compare transcription of bacteria before and after you have added the homone

• NA induces bac growth, frimbria and toxin expression in e.coli, also maybe sideophore (mixed evidence)
• EHEC can sense host adrenaline and NA interchangable with AI3 (QS signal) to express virulence traits

Question 4

How does e.coli sense eukaryotic hormones?

Answer 4

with normal QS AI3 receptor QseC,

binds AI3, adrenaline and NA

QseC triggers TCS and regulates virulence genes

Question 5

Why might it be useful for bacteria to sense eukaryotic hormones?

Answer 5

learn about host to adapt their physiology

gauge metabolic state of host, exploit a weakned immune system (since adrenaline is a stress response)

Question 6

What evidence suggests that AHLs enter and influence mammalian cells?

Answer 6

AHL: acyl-homoserine lactones

respiratory epithelial cells: dose dependent increase in IL8 low concentrations of AHL impair immune response, high concentrations exacerbate them

AHLs promote apoptosis
AHLS elicit proinflamatory effects

Question 7

What were the aims and purpose of the mBio study? Why was it important to develop a novel biofilm model?

Answer 7

Figure out how comensal pneumococci turn into opportunistic pathogen, which signals, events varation promote biofilm dispersal

there was no model where pneumococci coexist with living human respiratory cells (for more than 24h)
in vivo it takes days weeks months

dispersed bacteria are different (in gene expression and virulence phenotype) from Bacteria from colonizing biofilms and broth grown planctionic bacteria (which is usually used in studies)

Question 8

What experimental evidence showed that viruses affected the biofioms? What host factors were involved in bacterial dispersal from the biofilm?

Answer 8

NE, ATP, glucose, cell lysate induced active biofilm dispersal and release of diplococci into the supernatant,
greatest effect at 38°C
so symptoms of virus infection cause dispersal

ex either with real virus or mimic infection (raise temp or add siehe oben )

Question 9

What is the evidence that pneumococci are phenotypically different in planktonic culture, in biofilm, and in dispersed biofilm cells?

Answer 9

biofilm formation:

• genes in competence upregulated
• virulence genes (capsule production, pneumolysin, adhesins) significantly downregulated

dispersed bacteria show higher expression of adherence proteins than broth grown planktionic controls–> planktonic bacteria originating from broth cultures and from biofilm release are phenotypically distinct

Question 10

What is the evidence that dispersed cells are more virulent?

Answer 10

• higher expression of virulence genes in dispersed bacteria
• dispersed bacteria showed higher internalization (into host cell) than both planktonic and biofilm pneumococci, higher internalization meant higher toxicity

Question 11

How did the different bacterial types behavior differ in a murine colonization model?

Answer 11

dispersed colonized less thightly in nasopharyngeal tissue
dispersed grew in lungs, the others didnt
dispersed migrated more to the middle ear

results for EF3030
all three populations colonized the nasopharynngeal tssue, but dispersed colonized less tightly (12,5% not coloized to 1,7% broth grown and 0,15% biofilm)

dispersed grew in lungs, the others didnt

all populations migrated to middle ear, but dispersed much more

histological exaamination
dispersed led to pronounced denudation of nasal epthelium
dispersed inflammatory infiltrates in middle earand lungs

results for D39
generally less severe (no infection of middle ear at all) but dispersed were always stronger than the others

Question 12

How did the different bacterial types behavior differ in a murine pneumonia model?

Answer 12

dispersed led to high bacterial burdens in lung

broth grown planktonic significantly lower

Question 13

What happened when the different bacterial types were given i.p.?

Answer 13

planktonic and biofilm healthy
dispersed EF3030 (usually not bacteremic strain) 2 mice died, 5/6 bacteremic

D39 (more virulent strain) : dispersed: rapidly moribound, broth and biofilm, not moribound but had high bacterial load

dispersed phenotype may play a critical role in survival and groth of pneumococcus during the development of acute infection in the vascular compartment

Question 14

What were the effects of virus infection after a pneumococcal carriage was established in the murine model?

Answer 14

more lethargy, huddling, ruffled fur
both strains: dissemination to both lungs and middle ears (without virus no infection of lungs) ATP release

EF 3030 continuous colonization for day with or without virus, but dissemination to lungs and ears increased only if virus present (pneumonia)
D39, ear infection could be clearded out but also pneumonia

Question 15

How did the authors test potential host signals for dispersion?

Answer 15

higher dispersal more pneumonia, release of ATP
cause and effect? virus lyses cells releases ATP
intranasal application of diff factors, see if it gets the same result

Question 16

What do you think might be the area to focus on in future research?

Answer 16

kjh