Trypanosoma cruzi:Chagas disease

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Trypanosoma cruziChagas disease
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T. cruzi parasite-directed entry

• Enters survives in many cell types
• Does not require host actin cytoskeleton for
  invasion
• Actin cytoskeleton
• inhibitors enhance entry
• No host cell
• pseudopods observed
• Microtubule dependent

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T. Cruzi hijacks lysosomes

• Parasite oligopeptidase releases soluble agonist
• Receptor binding?IP3 and intracellular Ca release
• Lysosomes recruited to
• host cell plasma membrane
• Ca-regulated fusion of
• lysosomes with plasma
• membrane ?
• parasitophorous vacuole ?
• lysis ?parasite replication
• in cytosol

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Entry occurs through splash
Back to Shigella.

• Transient interaction w/membrane
• Bound bacteria elicit actin-rich host cell
  extensions
• Enriched in actin-binding proteins focal
  adhesion components

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IpaB, C, D have complex roles in Invasion et al
IpaB/C/D
IpaB/D

• 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
• IpaB is necessary sufficient for induction of
  apoptosis in mphages

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• IpaB C form translocation complex
• C-terminus of IpaC induces early actin
  polymerization events
• IpaB also involved in actin polymerization?
• IpaA C-terminus binds to vinculin

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• Filopodia ? lamellopodia ? macropinocytosis
• IpB/C ?Cdc42/Rac ?Cortactinezrin
• IpaA allows pseudopod maturation
• IpaA mutants show poor internalization despite
  actin polymerization
• IpaA binds to activates vinculin
• IpgD has PI phosphatase activity

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VirA destabilizes MT enhances Rac1-mediated
internalization
Yoshida et al, EMBO 2002
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Crossing the epithelial barrier

• BL Shigella entry more efficient
• Enter through M cells (ligated intestinal loop)
• Kill macrophages/dendritic cells via IpaB-induced
  apoptosis
• IpaB activates ICE ?IL-1b and IL-18
  activation/release

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Diverse strategies
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Life after invasion

• Three choices
• Get out of the vacuole (Listeria, Shigella)
• Modify the vacuole (Salmonella, TB, chlamydia)
• Put up and shut up inside the vacuole (Coxiella,
  Leishmania)

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Shigella

• Escapes vacuole by IpaB

IpaB- mutant
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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 or Vaccinia virus A36R
• Assembly occurs at old pole where IcsA
  concentration highest
• Lateral diffusion restricted by IcsP, which
  specifically cleaves IcsA

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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
• 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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The intracellular life cycle of Listeria
monocytogenes
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Escape requires LLO and PLC

• LLO
• LLO is necessary for escape
• LLO is sufficient
• Expression in Bacillus subtilis is sufficient for
  escape from MØ phagosomes and intracellular
  growth
• Cholesterol-directed pore-forming toxin
• LLO mutants greatly attenuated virulence in mice
• Phospholipases
• PI-PLC (phospho-inositol specific)
• PC-PLC (broad spectrum)
• Mutants defective in both show defective escape

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Why doesnt LLO destroy host cell membranes?

• Replace LLO with PFO host cell destroyed
• LLO has PEST sequence which allows 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
• Acidic pH optimum restricts activity
• Mutant LLO with neutral pH optimum has nl LLO
  activity and vacuolar escape but is 1,000-fold
  less virulent in mice
• Transcription not restricted to vacuole
• ? Role of potential nuclear localization signal

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How does Listeria solve the two-membrane problem?
J Cell Biol 109 1597, 1989
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How does Listeria manage the two-membrane problem?
Mol Micro 35 289, 2000 J Cell Biol 137 1390,
1997, and Infect Immun 68 999, 2000 (for an
alternative view)
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Listeria motility in the cytosol ActA

• ActA is necessary and sufficient for Listeria
  motility in the cytosol
• ActA mutants escape from vacuole but grow as
  microcolonies, dont spread cell-cell or form
  plaques in monolayers
• ActA is polarly localized

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ActA acts as scaffold to assemble host
cytoskeletal components

• N-terminus binds monomeric actin and stimulates
  Arp2/3 nucleation activity
• KKRKK sequence functions like WASP
• KKRKK mutants have Act-null phenotype
• Also has WASP-like acidic region
• 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

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• Central region
• 3-4 proline-rich repeats
• BIND EVH1 domain of ENA/VASP
• Mutants move slowly
• Ena/VASP binds profilin F-actin
• Cells expressing Ena/VASP lacking
  profilin-binding domain have decr rates of
  Listeria motility

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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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Listeria motility in the cytosol ActA and the
Arp2/3 complex

• Arp2/3 complex recruits G-actin but not F-actin
  to Listeria
• ActA Arp2/3 form an efficient actin-nucleating
  complex

Lm Arp2/3 TMR-labeled
G-actin
F-actin
Science 281 105, 1998
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ActA, Arp2/3, VASP/MENA, capping protein,
cofilin, phosphoinositides
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Listeria and Shigella 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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Reconstituting the minimal system in vitro
Loisel et al Nature 1999
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Vaccinia virus does it too
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So does Rickettsia
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Convergent Evolution
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Intravacuolar pathogens fail to replicate in the
cytoplasm
PNAS 9812221, 2001
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Replication in the cytoplasm requires
specialization
PNAS 9812221, 2001