Lecture 7 Flashcards
How was the T3S system discovered?
Signature tagged mutagenesis
What are the four challenges for cytosolic bacteria to overcome?
1) Gaining entry 2) Rapidly escaping the vacuole 3) Replicating in the non-permissive cytosol 4) Avoid innate cytosolic immune response
What type of secretory system does Listeria Monocytogenes have?
Sec secretory system
How are proteins recognised to be sent to the Sec secretory translocon?
Signal recognition pepitides - SecA and SecA2
What happens to proteins after going through the Secc translocon?
SPases cleave off the signal Sortases either anchor the protein into the cell membrane or cause it be released
What are the key proteins secreted by L. monocytogenes that allow it to escape the phagosomal membrane?
Phospholipases C and Haemolysins
Which Haemolysin was shown to be most important for L. monocytogenes?
Listeriolysin-O -Mutants lacking LLO fail to escape - Complementation with plasmid carrying LLO gene restores virulence - Expression of LLO by Bacillus subtilis enables escape - Purified LLO causes lysis of liposomes
How to Listeriolysin-O work?
Binds to cholesterol as an alpha-helix and then reorganises into a beta-sheet spanning the membrane. at low pH, these sheets then bind together and circularise to form a pore
Which two phospholipase C’s were shown to be most important for virulence?
PlcA - Phosphotidylinositol specific PLC
- Loss of PlcA causes 2 fold loss in virulence
PlcB - Phosphotidylcholine specific PLC
- Loss of PlcB causes 20 fold loss in virulence
Loss of both causes 500 fold loss in virulence
How does Listeria move intracellulary?
Host actin based motility
Polymerisation of actin pushes the bacteria forward
What does actin based motility allow?
Cell-to-cell spread
What can cause actin polymerisation?
Phosphorylation of N-WASP activates the complex allowing it’s a and c subunits to bind to the Arp2/3 complex. Arp2/3 complex then binds to actin filaments and acts as the nucleation point with the w subunit of N-WASP adding actin monomers
Formin homology domains 1 bind profilin which binds actin monomers. Formin homology domain 2 then causes polymerisation of these monomers onto actin filaments
How does Listeria cause actin polymerisation?
Has ActA - a N-WASP like protein
How else does Listeria act to allow cell-cell spread?
Produces IniC which will displace N-WASP from it binding to Tuba and release cortical trension - making spread easier
Which host factor was shown to be important for Listeria infection?
Who by?
Singh
GILT - gamma-interferon inducible lysosomal thiol reductase
What stain can be used to visualise actin?
Phalloidin
What is the role of GILT?
Reduces LLO - activating it
Outline immunoprecipitation
- Inert matrix of sepharose of agarose bead associated to antibody
- Spin down lysate with the antibody
- Can pull down protein and then run on gel
Why is the cytosol difficult to live in?
- Reducing environment - and bacteria are used to oxidising environments
- Low Magnesium
- Low Calcium
- High Potassium
- Low glutathione
- Low iron
What are the two main limiting products of the cytosol?
Aromatic amino acids and nucleotides
Therefore bacteria use pyruvate and hexose phosphates as carbon sources for their biosynthetic pathways to make them
What is autophagy?
Self-cannibilisation
Delivery of organelles and cytoplasm to the lysosome for nutrient release
When does a cell use auophagy?
During periods of starvation
What are the possible outcomes of loss of autophagy?
- Amino acid defficiency
- Aggregate formation
- Apoptosis
- Nrf2 hyperactivation
- ROS production
- Chronic infection
What two proteins are important for autophagy?
ATG8 and LC3
What protein does shigells use to avoid autophagy?
IcsB
When would green and red staining appear yellow?
When the stained targets are less and 200µm away from each other
Outline immunogold labelling
- Fix cells with formaldehyde
- Use antibody associated with gold
- Gold is electron dense and hence you can see where the gold is
- Different sixes of gold allows localisation of antibody target
Which of shigella’s proteins are involved in autophagy?
VirG (IcsA)
Atg5 binds VirG to target autophagy around the bacteria.
IcsB will bind the VirG instead to stop autophagy
