Lecture 7 - Intracellular bacteria 2 Flashcards
What are the steps of infection for cytosolic bacteria?
1 Gain entry
2 Rapidly escape vacuole
3 Replicate
4 Avoid innate cytosolic immune responses and autophagy
What species of bacteria are cytosolic?
Shigella, Listeria, Rickettsia, Burkholderia, Francisella
How are proteins transported into the lumen of the ER?
From ribosome, Signal peptide binds to SRP, this binds to receptor and protein goes through translocon.
What is the secretory system of Listeria called?
Sec secretory system
What is SipZ?
A type 1 signal peptidase involved in the Sec secretory system
What does SipZ do?
Permits secretion of key virulence factors such as PlcB and Hly (haemolysin or Listeriolysin-O)
Features of Listeriolysin-O (LLO)
- Primary determinant for Listeria escaping vacuole
- Secreted by Sec system
- Mutants of LLO fail to escape
- Rescue restores virulence
- Expression of LLO in Bacillis subtilis enables escape
- Purified LLO escapsulated into a pH-senstive lipsome can lyse it
Other than acidic pH what is LLO also dependent on for its function?
Cholesterol
What other LLO-like proteins are there and what bacteria produce them?
Streptolysin-O from Streptococcus pyogenes
Perfingolysin-O from Clostridium perfringens
How can you visualise the formation of lysing pores on vesicles?
Biotinylated them.
Add biotin (small chain)
Lots of proteins strongly bind to biotin (Strepavadin or avadin)
Extra protein allows you to see contrast in electron microscopy and see ring like structures (as well as horseshoes)
Which domain of LLO binds cholesterol on the membrane?
Domain 4
What does domain 3 of LLO do when it all binds to membrane?
6 alpha helices rearrange to form a beta sheet which inserts and crosses both layers of the bilayer
What are the two phospholipases important to Listeria’s virulence?
A - phosphatidylinositol specific (PI-PLC)
B - phosphatidylcholine specific (PC-PLC)
What effect does each phospholipases and both on Listeria’s virulence?
A - 2 fold less virulent in mice
B - 20 fold less virulent in mice
Both - 500 fold less virulent, failed to escape vacuole
How do Listeria, Shigella and Rickettsia move in the cytoplasm?
Actin nucleation behind it
What else does actin nucleation allow?
Movement from cell to adjacent cell (pushes its way through)
Features of actin polymerisation
- ATP dependent
- Limited by actin monomer avail
- Arp2/3 complex binds to side of filament to branch off (after N-WASP activates arp with WCA domain)
- Formins FH1 and 2 and profilin promote polymerisation
What is ActA similar to and how?
N-WASP - has a WCA domain
What is Sca-2
Formin-like protein in Listeria
What does Tuba do?
In the presence of N-WASP creates cortical tension on the membranes of cells creating bTJs and tTJs (bi and tri tight junctions)
How does Shigella do to reduce cortical tension?
Procudes InlC which binds to tuba and displaces the N-WASP so it can push its way through tTJs.
What are the 3 proteins involves in endocytosis and their functions?
Dynamin 2 - pinches off membrane
Clathrin - recuits proteins for endocytosis
epsin-1 - creates a curved end with its bar domain
How long does Listeria take to escape?
17 minutes
What is GILT?
Gamma interferon-inducible lysosomal thiol reductase
What was Singh et al (2008)’s hypothesis about GILT?
It is the only serine reductase in macrophages so it must be involved in activating LLO
What experiment did Singh et al (2008) perform on GILT and what were the results?
Made GILT deficient mice and GILT deficient macrophages and showed that growth of Listeria is reduced
What were Singh’s other results?
In a GILT defective mouse with LLO that had artificially had the serine reduced there was no reduction as LLO is already activated with out the need for GILT
What is immunoprecipitation?
- Have an inert bead covered in antibodies to a particular protein (GILT in this case).
- Lyse cells and dip bead in, GILT binds.
- If GILT binds anything, it will bring it with it (LLO)
What kind of environment is cytosol for bacterial growth?
- Highly reducing environment
- Most iron is bound to protein
- Low calcium and magnesium
- High potassium
- Limiting for aramatic aa and nucleotides
- Not very good.
What is a heterotrophic bacteria?
If you get rid of a pathway that bacteria can replace it with another
What can bacteria utilise to grow in the cytosol?
Pyruvate and hexose phosphates
What is autophagy?
Self-cannibalisation
What are the proteins involved in autophagy?
Atg5 and LC3
What happened when icsB Shigella mutants were made by Ogawa et al?
They were trapped by autophagy
What are the 3 inhibitors of lysosomal fusion Ogawa et al added?
Wortmannin, 3-MA, Baf-A1
What is icsB
Virulence factor in Shigella
What did Ogawa et al show with staining?
icsB binds onto LC3 to escape autophagy
What did Ogawa show by inhibiting lysosomal fusion?
Number of bacteria associating with LC3 became reduced
What happened when Ogawa knocked out atg5 (inhibiting autophagy)
Don’t see any LC3
What happened when they expressed autophagy markers in absence of icsB?
Saw a capping on the bacteria. VirG (regulator of actin polymerisation) bound Atg5.
What happens with out VirG?
Atg5 did not associate with the bacteria and so autpphagy is very low
What did knocking out icsB do?
A much higher amount of autophagy
What did Ogawa find when they blotted Atg5 with and with out IcsB?
In the presence of IcsB, VirG did not bind Atg5
What did Ogawa’s blotting show?
IcsB and Atg5 compete for the same binding site
So how do Shigella evade autophagy?
Produces IcsB which binds to VirG instead of Atg5 preventing autophagy
