Lecture 7 Flashcards

1
Q

How was the T3S system discovered?

A

Signature tagged mutagenesis

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2
Q

What are the four challenges for cytosolic bacteria to overcome?

A

1) Gaining entry 2) Rapidly escaping the vacuole 3) Replicating in the non-permissive cytosol 4) Avoid innate cytosolic immune response

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3
Q

What type of secretory system does Listeria Monocytogenes have?

A

Sec secretory system

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4
Q

How are proteins recognised to be sent to the Sec secretory translocon?

A

Signal recognition pepitides - SecA and SecA2

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5
Q

What happens to proteins after going through the Secc translocon?

A

SPases cleave off the signal Sortases either anchor the protein into the cell membrane or cause it be released

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6
Q

What are the key proteins secreted by L. monocytogenes that allow it to escape the phagosomal membrane?

A

Phospholipases C and Haemolysins

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7
Q

Which Haemolysin was shown to be most important for L. monocytogenes?

A

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

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8
Q

How to Listeriolysin-O work?

A

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

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9
Q

Which two phospholipase C’s were shown to be most important for virulence?

A

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

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10
Q

How does Listeria move intracellulary?

A

Host actin based motility

Polymerisation of actin pushes the bacteria forward

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11
Q

What does actin based motility allow?

A

Cell-to-cell spread

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12
Q

What can cause actin polymerisation?

A

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

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13
Q

How does Listeria cause actin polymerisation?

A

Has ActA - a N-WASP like protein

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14
Q

How else does Listeria act to allow cell-cell spread?

A

Produces IniC which will displace N-WASP from it binding to Tuba and release cortical trension - making spread easier

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15
Q

Which host factor was shown to be important for Listeria infection?

Who by?

A

Singh

GILT - gamma-interferon inducible lysosomal thiol reductase

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16
Q

What stain can be used to visualise actin?

A

Phalloidin

17
Q

What is the role of GILT?

A

Reduces LLO - activating it

18
Q

Outline immunoprecipitation

A
  • 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
19
Q

Why is the cytosol difficult to live in?

A
  • Reducing environment - and bacteria are used to oxidising environments
  • Low Magnesium
  • Low Calcium
  • High Potassium
  • Low glutathione
  • Low iron
20
Q

What are the two main limiting products of the cytosol?

A

Aromatic amino acids and nucleotides

Therefore bacteria use pyruvate and hexose phosphates as carbon sources for their biosynthetic pathways to make them

21
Q

What is autophagy?

A

Self-cannibilisation

Delivery of organelles and cytoplasm to the lysosome for nutrient release

22
Q

When does a cell use auophagy?

A

During periods of starvation

23
Q

What are the possible outcomes of loss of autophagy?

A
  • Amino acid defficiency
  • Aggregate formation
  • Apoptosis
  • Nrf2 hyperactivation
  • ROS production
  • Chronic infection
24
Q

What two proteins are important for autophagy?

A

ATG8 and LC3

25
Q

What protein does shigells use to avoid autophagy?

A

IcsB

26
Q

When would green and red staining appear yellow?

A

When the stained targets are less and 200µm away from each other

27
Q

Outline immunogold labelling

A
  • 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
28
Q

Which of shigella’s proteins are involved in autophagy?

A

VirG (IcsA)

Atg5 binds VirG to target autophagy around the bacteria.

IcsB will bind the VirG instead to stop autophagy