What do you do once youve gotten in

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What do you do once youve gotten in

• Escape from the vacuole
• Listeria,
• Shigella
• Rickettsia
• Toxoplasma
• Trypanosomes

• Modify it
• Salmonella
• TB
• Chlamydia
• Legionella
• Brucella
• others
• Put up and shut up (ie, survive in the
  environment of an acidic lysosome)
• Coxiella
• Leishmania

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Escape requires LLO and PLC
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LLO

• Cholesterol-directed pore-forming cytolysin (gt22
  members)
• Bind to cholesterol-containing membranes
• Insertion
• Oligomerization (20-80 mers)
• Pore (20-30 nM) formation

Membrane binding
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LLO is necessary sufficient

• Necessary
• LLO mutants fail to escape
• Sufficient for vacuolar escape
• Expression in Bacillus subtilis is sufficient for
  escape from MØ phagosomes and intracellular
  growth
• Important in virulence
• LLO mutants greatly attenuated virulence in mice

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Phospholipases

• PI-PLC (phospho-inositol specific)
• PC-PLC (broad spectrum)
• Mutants defective in both show defective escape
• Working model LLO insertion into phagosome
  membrane
• Dissipates pH gradient and halt phagosome
  maturation
• Channel for passage of proteins (PLC)

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Why doesnt LLO destroy host cell membranes?

• Replace LLO with PFO bacteria escape phagosome,
  but host cell destroyed
• Screend for PFO mutants able to grown normally
  in host cells
• Dead enzyme
• Change in pH optimum (single mutation)
• Decrease protein stability

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More LLO-ology

• Acidic pH optimum restricts activity to the
  vacuole
• Vacuole pH5.9
• Inhibition of acidification inhibits Listeria
  escape
• Mutant LLO with neutral pH optimum (L461T mutant)
  
• has nl LLO activity and vacuolar escape
• Causes premature permeabliziation of infected
  host cells after 5 bacterial generations
• 1,000-fold less virulent in mice
• LLO has PEST sequence which may allow rapid
  proteasome-mediated destruction upon reaching
  cytoplasm
• PEST- LLO mutants nl LLO activity and vacuolar
  escape, permeabilize host cell membrane in vitro,
  10,000 fold less virulent in mice

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How does Listeria solve the two-membrane problem?
J Cell Biol 109 1597, 1989
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Escaping from a double membrane
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Pathogens move around

• Cytosolic pathogens
• Listeria monocytogenes
• Shigella flexnerii
• Rickettsia
• Burkholderia pseudomallei
• Mycobacterium marinum
• Extracellular pathogens
• EPEC
• Cell-cell spread
• Vaccinia virus

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Regulation of the actin cytoskeleon

• Actin nucleation is kinetically unfavorable
• Arp 2/3 NPF such as N-WASP, formins, Dyche
  Mullins new factor, or others
• Many conserved domains
• Bind actin monmers and Arp2/3 complex--gtconformati
  onal change--gtbinds to side of existing actin
  filament, initiates polymerization of new
  filament
• Many points
• of regulation

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Pathogen initiation of actin polymerization

• Express mimics of WASP (Listeria ActA or
  Rickettsia RickA)
• Recruit WASPs
• No known (yet!) examples of utilizing other NPFs

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N-WASP regulation
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Listeria motility in the cytosol ActA

• ActA is necessary for Listeria motility in the
  cytosol
• ActA is sufficient for bead motility in cell
  extracts
• ActA is polarly localized

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• ActA mutants escape from vacuole but grow as
  microcolonies, dont spread cell-cell or form
  plaques in monolayers

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ActA is a WASP wannabescaffold for assembly host
cytoskeletal components

• N-terminus binds monomeric actin and stimulates
  Arp2/3 nucleation activity
• KKRKK sequence functions like WASP-binds Arp2/3
• KKRKK mutants have Act-null phenotype
• Also has WASP-like acidic region
• Proline-rich domain binds Ena/VASP (which binds
  profilin and F-actin_
• ActA amino-terminal domain (aa 30-263) is
  essential for actin polymerization in cytosol,
  and is sufficient if anchored to particle
• ActA 30-263 does not directly interact with
  actin Arp2/3 is required
• Rho GTPase independent

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3 subdomains of ActA are required for cytoplasmic
motility of Listeria
J Cell Biol 150 527, 2000
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ActA, Arp2/3, VASP/MENA, capping protein,
cofilin, phosphoinositides
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Shigella

• 150 million cases/yr, 1 million deaths
• Very low ID50
• Only infects humans

• Bloody Diarrhea (dysentery)
• Intense inflammation
• Destruction of colonic mucosa
• Oral-fecal transmission

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IpaB, C, D have complex roles

• Secretion translocation
• IpaB/C/D req for initial secretion and are
  secreted into the medium
• IpaBD form translocation complex with ion
  chanelling activity
• Invasion
• IpaB or IpaC mutants defective in internalization
• Beads coated with IpaB/C are internalized
• IpaB is a ligand for 2 putative receptors
• CD44
• Integrin a5b1
• IpaB is required for escape from
• the vacuole

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Cytoplasmic Shigella has actin tails
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IcsA is Nec/Suffic for actin polymerization
IcsA
Actin
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IcsA is Nec/Suffic for actin polymerization

• Autotransporter localized to OM
• C-terminal 750 aa N/S
• No similarity to ActA, RickA, Vaccinia virus A36R
• Assembly occurs at old pole where IcsA
  concentration highest

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Whats in the tails?

• Protein Tail localiz Necessary
• N-Wasp
• Arp2/3
• Profilin Speed
• Vinculin ?
• Cofilin
• VASP ?
• Cdc42 - ?

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How does Shigella do it?

• IcsA-gtN-WASP-gtactiv ARP2/3
• Similar to what Cdc42 does reverses the
  autoinhibition of N-WASP
• Rapid elongation of filaments at barbed ends,
  x-linking
• Profilin brings in monomeric actin

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Whats next?

• Shigella spreads from cell-cell using cadherin

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Reconstituting the minimal system in vitro
Loisel et al Nature 1999
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Vaccinia virus does it too

• Cell-cell spread
• B5R-gtSrcactivation-gtA36R-P04-gtNck/Grb-gtNik-gtN-wasp
  
• Similar to EPEC

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So does Rickettsia

• Long filaments in parallel arrays
• Encodes WASP-homologous protein (RickA)
• Activates Arp2/3

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And Mycobacterium marinum
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Listeria, Shigella, Vaccinia differ in
dependence on N-WASP
Shigella flexneri
Listeria monocytogenes
Shigella cell- cell spread
Vaccinia
Nature Cell Biology 3 897, 2001
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Convergent Evolution
Goldberg, 2000
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Convergent Evolution
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Intravacuolar pathogens fail to replicate in the
cytoplasm
PNAS 9812221, 2001