Lecture 7 - The intracellular bacterial pathogen part 1 Flashcards
What are some examples of cytosolic bacteria?
- Shigella flexneri (has T3 secretion system)
- Burkholeria psedomallei
- Literia monocytogenes (unpasterurised dairy products)
- Francisella tularenesis (biting organism)
- Rickettsia spp
What is the necessary pathway for cytosolic bacteria?
- gain entry by phagocytosis
- rapidly escape the vacuole by degrading the vacuolar membrane
- replicate (but cytosol not permissive to the growth of bacteria)
- avoid innate cytosolic immune responses and autophagy
What does listeria monocytogenes use for secretion?
Listeria has a sec secretory system (G+)
Uses sec pathway
SipZ (a Spase) permits the secretion of key virulence factors
- lecithinase phosphatidylcholine-specific phospholipases C (plcB)
- Hly (haemolysin e.g. lysteriolysin O)
What is listeriolysin-O, and what is the evidence of its purpose?
- Primary determinant fo listeria escaping the vacuole
- mutants lacking LLo fail to escape (shown using transposon intertions)
- complementation of transposon mutants with plasmids expressing LLO restores virulence
- Expression of LLO in bacillus subtilis enables bacteria to escape the vacuole
- Purified LLO encapsulated into a pH-sensitive liposome can lyse the liposome
What are the features of the LLO in listeria monocytogenes?
- listeriolysin-O is encoded by the hly gene
- related to a family of cholesterol dependent cytolysins e.g. strptolysin-O of streptococcus pyogenes and perfinoglysin of clostrisium perfringens
- alpha helix restrcutres into a beta sheet to insert into the membrane
- D4 domain binds membrane and cholesterol
What is the genomic structure of listeria monocytogenes?
- Continous genes on the chromosome: prfA, plcA, hly, mpl, actA, plcB
- Two phospholipase C genes are important for virulence:
- PlcA a phosphatidylinositol-specific PLC (PI-PLC)
- PlcB a phosphatidylcholine phospholipase (PC-PLC)
- mutant PI-PLC is 2 fold less virulent in mice
- mutant PC-PLC is 20 fold virulent in mice, defective in cell-cell spread but not vacuole escape
- combination of both mutants 500 times less virulent and fail to escape vacuole
How is cell-cell spread achieved in listeria?
Once listeria has entered the cell and degraded the internalisation membrane, actin polymerisation allows motility, then get bacterial protrusions into the neighboring cell, listeria is then engulfed into the neighboring cell and ends in a dissolution of the double membrane
How does listeria use actin polymerisation for motility?
And how can other molecules result in polymerisation?
- Host Arp2/3 acts as a nucleation point. In the presence of Host N-WASP Arp2/3 is activated and ACW domain on Host N-WASP is activated which results in actin polymerisation.
- Listeria ActA is similar to N-WASP - also has AWC domains (evolutionaryly conserved mechanism for polymerisation of actin)
- Monomers are added on to (+) end of the filament ((-) end attached to bacteria) Arp2/3 accessory complex increase the speed of formation
Other molecules
- Formin results in an extension of the filament with the help of profilin - FH1 uses profilin to bind the actin monomers and FH2 causes addition by phosphorylation
How does the remodelling of cell junctions (tight junctions) allow bacterial spread cell-cell?
- N-WASP and tuba provide tension.
- listeria secretes InIC binds to tuba protein displacing N-WASP displacing the cortical tension - easier to move cell-cell
- Has preference for tri-juntion. Motility provided by actin polymerisation
- Membrane curvature is provided by clatherin and epsin-1 in the neighboring cell, membrane scission is acheives by dynamin 2
What are common mechanisms required for escape from the vacole in cytosolic bacteria? Why are there lots of common mechanisms?
- Bacterial genes e.g. LLO and type C phospholipases in listeria monocytogenes
- Host factors e.g. GILT activated LLO by a thiol reductase mechanism (l. monocytogenes)
- Specific vacuolar conditions e.g. acidic pH5.5 (l. monocytogenes)
- Specific escape kinetics e.g. 17 mins (l.monocytogenes)
- Use of actin based motility or not e.g. Yes ActA (l.monocytogenes)
Heterogenity within bacterial community as only a few that escape can enter cytosol and begin spreading
What is a critical host factor for listeria monocytogenes infection? And how was this experimentally determined?
GILT (gamma-interferon-inducible lysosomal thiol reductase)
Growth of L. monocytogenes is decreased in GILT-deficient mice and GILT-deficient macrophages
Take animal model affected with listeria and count CFU per gram of tissue
What occurs in GILT negative macrophages?
Phagosomal escape of L monocytogenes is delayed. If macrophages no longer hace thiol reductase, they don’t have GILT. GILT- macrophages don’t get as many bacteria or actin polymerisation. If no LLO no bacteria at all.
Used immunofluoresence with floidin which binds to actin. Listeria shown to be green, show actin tails.
Wt compared to GILT- cells, after 8 hours becteria were still surrounded by phagosomal membrane
What is the interaction between GILT and LLO?
LLO is activated by GILT reduction during infection and in cell free assays
GILT activates LLO by using the classical thiroredoxin reduction mechanism
LLO is a substrate for GILT through a physical interaction therefore GILT needs LLO to activate
How do you locate where bacteria are
Immunoprecipitation assay. CFU, western blot
Is the cytosol permissive for bacterial growth?
- some bacteria can surivive in the cytosol e.g. bacillus subtilis expressing LLO
- other species e.g. Yersinia when injected directly into epthelial cells fail to replicate
- Explanations: entry and escape from the vacuole may prime
- the bacteria to grow in the cytoplasm and/or
- the maytoplams may be affected by the invasion of the microorganism
- Growth can be dependent on the cell type
- generally the cell cytosol is pH 7.2, low Mg, high K, low Ca, reduced glutathione and iron is bound to carrier molecules such as ferritin
