Apicomplexan host cell invasion II

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Apicomplexan host cell invasion II
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Different parasites use different mechanisms to
invade cells

• Trypansoma cruzi -- recruits endosomes and later
  escapes into the cytoplasm
• Leishmania -- induces phagocytosis and thrives in
  a mature lysosomal compartment
• Mycobacterium tuberculosis -- induces
  phagocytosis and blocks lysosomal maturation
• Toxoplasma -- resides in a specialized
  parasitophorous vacuole, how does it get in there
  and does this involve phagocytosis by the host
  cell?

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The parasitophorous vacuole is not fusing with
lysosomes

• Macrophages were incubated with life (A/B) and
  heat killed (C/D) parasites
• Note that only vacuoles containing heat killed
  parasites show staining for a lysosomal marker
  protein.
• Dead parasites go in by phagocytosis, living
  parasites enter differently

Joiner et al., Science 249641-6
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Invasion depends on parasite not host cell actin
Salmonella

• Host cell invasion by Salmonella and Toxoplasma
  can be inhibited by cytochalasin D, a drug that
  prevents actin polymerization
• In Salmonella, which is taken up by phagocytosis,
  invasion can be rescued by using a drug resistant
  host cell mutant (Cyt1)
• Toxoplasma invasion remains inhibited in the drug
  resistant host cell.
• (black bars drug resistant, white bars drug
  sensitive host cell)
• Generating a drug resistant parasite rescues
  invasion

Toxoplasma
Dobrowolski JM, Sibley LD. Cell. 1996 84(6)933-9.
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Invasion depends on parasite not host cell actin

• Cytochalasin treatment does not to appear to
  inhibit attachment (bar graph in c shows number
  of parasites bound to cells at different drug
  doses)
• CytD inhibits the movement of the parasite into
  the host cell.
• A parasitophorous vacuole (PV) is still set up,
  however the parasite can not move in, and the
  moving junction remains at the apical tip of the
  parasite

Dobrowolski JM, Sibley LD. Cell. 1996 84(6)933-9.
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Apicomplexan host cell invasion
Movies Dr. Gary Ward, Univ. of Vermont http//www.
uvm.edu/mmg1/videos_ward.php?id23
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Apicomplexan host cell invasion
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Invasion depends on sequential protein secretion
from three organelles

• Micronemes (Mn) secretion of micronemes brings
  protein to the parasite surface that provide
  traction
• Microneme proteins are required for gliding
  invasion, the cleanest example is the Plasmodium
  sporozoite protein TRAP (Mic2 is a T. gondii
  homolog that is functionally equivalent in the
  tachyzoite)
• Microneme proteins can bind to a variety of
  carbohydrates found on the surface of cells and
  other biological materials
• Microneme proteins are assembled into complexes
  in the ER, mature by proteolysis, are stored in
  micronemes and are secreted at the apical tip of
  the parasite upon stimmulation
• How does this help moving and how are they linked
  to the internal actin-based engine?

Sultan et al, Cell 90 511-522
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The conveyor-belt model of gliding motility

• Gregarines are a group of primitive
  apicomplexans which parasitize invertebrates
• In comparison ot Toxoplasma or the malaria
  parasite these are fairly large cells which makes
  them easier to study

Movies by Dr. C. King (University College, London)
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The conveyor-belt model of gliding motility

• Beads attached to the surface of gregarines are
  treadmilled to the end of the parasite cells
• This suggests that the gliding machine moves
  microneme proteins over the surface from the
  apical to the basal end of the parasite

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Circles, helices twirls
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Gliding parasites deposit proteins and lipid
trails

• Similar to a slug the parasite leaves behind a
  trail of surface proteins and lipids
• A protease cleaves microneme proteins at the end
  of the cell to detach and allow for propulsion
  (sheddase)
• What is the force driving microneme proteins --
  actin polymerisation or an actin/myosin motor?

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A special myosin is required to move microneme
proteins over the surface
Normal myosin

• Toxoplasma and other Apicomplexa have a parasite
  specific myosin (MyoA)
• This myosin is localized right under the surface
  membrane of the parasite
• Using genetic engineering a mutant parasite was
  constructed in which this myosin can be
  suppressed
• Suppression of myosin result in loss of parasite
  motility (as seen for actin loss of motility also
  causes loss of host cell invasion)
• What are the gears that connect this motor to
  the Mic tires and which part is anchored in the
  parasite?

Suppressed myosin
Meissner et al., Science. 298837-40
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The gliding machinery is anchored in the inner
membrane complex
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The gliding machinery is anchored in the inner
membrane complex

• Biochemical studies aimed at identifying pellicle
  proteins let to the discovery of GAP45 a protein
  associated with outer face of the IMC
• GAP45 was associated with the IMC despite the
  fact that is primary sequence suggested that this
  should be a soluble protein

http//www.jcb.org/cgi/content/full/165/3/383
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GAP45 is part of a complex including GAP50, MyoA
MLC
http//www.jcb.org/cgi/content/full/165/3/383
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The apicomplexan engine - the glideosome
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The treadmilling model of apicomplexan gliding
motility
Soldati et al., Trends in Parasitology 20
567-574
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The conveyor-belt model

• Motility depends of parasite actin/myosin (MyoA)
• Myosin is anchored into the outer IMC membrane
  (GAP45/50, MyoA, MLC)
• Short actin filaments form and are moved towards
  the posterior end of the parasite by the myosin
  power stroke
• The short actin filaments are linked to microneme
  proteins by an adaptor (aldolase) -- movement of
  actin filaments results in movement of microneme
  proteins
• Microneme proteins are shed at the back end
  (rhomboid proteases are the best candidates for
  this activity)
• The parasite glides over the substrate

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Formation of the PV moving junction
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Formation of the PV moving junction
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Invasion of the red blood cell by the malaria
parasite

• Although there are significant differences in the
  host cells which are invaded by different
  apicomplexa the mechanism seems conserved
• Here the Plasmodium merozoite/red blood cell
  example

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Invasion of the red blood cell by the malaria
parasite

• A moving junction is formed tightly opposing
  parasite and host cell membrane and separating
  parasitophorous vacuole lumen and outside medium

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Invasion of the red blood cell by the malaria
parasite
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Secretion of the rhoptries is associated with PV
formation

• Rhoptries (Rh) secretion of rhoptries is
  required for the formation of the parasitophorous
  vacuole
• Like micronemes rhoptries are secreted at the
  apical tip of the parasite
• Some rhoptry proteins make up (part) of the
  moving junction (they are stored in the neck
  portion of the organelles and called RONs)
• Other rhoptry proteins a found throughout the
  membrane of the parasitophorous vacuole after
  secretion and are stored in the bulbous part
  (ROPs)
• A third group of very interesting rhoptry
  proteins is injected into the host cell and
  manipulates gene expression in the host nucleus
  -- kiss and spit (check out review on the class
  web site)

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Kiss spit (thank John Boothroyd for the term)

• It appears that a variety of rhoptry proteins are
  directly injected into the host cell and that
  this is involved in the formation of the
  parasitophorous vacuole
• Note that staining for the rhoptry protein ROP1
  highlights the parasitophorous vacuole (arrow) as
  well as numerous vesicles (arrowheads) formed at
  the invasion site within the host cell cytoplasm
• Does the parasite inject the PV membrane?

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Where does the membrane for the parasitophorous
vacuole come from?

• The PV membrane is derived from the host cell
  plasma membrane
• The PV is provided by the parasite (e.g. by
  secretion from the rhoptries)
• Both contribute to the PV

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Where does the membrane for the PV come from?

• Patch clamp cells and follow invasion by video
  microscopy
• Certain electric properties of the cell (their
  capacitance) can be used as a measure of their
  total surface membrane
• If membrane is parasite derived the surface area
  should grow during invasion if it is derived from
  the host cell surface the area should stay
  constant

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There is no significant change of host cell
surface during invasion
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Host cell surface area decreases after PV pinches
off

• Cell surface area remains constant over invasion
• When the parasitophorous vacuole pinches off the
  cell surface area drops
• The parasitophorous vacuole is derived from the
  membrane of the host cell

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The moving junction
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Rhoptry proteins organize the moving junction

• The parasite protein Ron4 (red) localizes exactly
  to the moving junction during invasion
• Note that other parasite surface proteins (green)
  do not enter the parasitophorous vacuole but are
  shed at the moving junction
• In non invading parasites Ron4 is stored in the
  rhoptry (necks, green, compare to micronemes red)

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Rhoptry proteins have multiple functions in
vacuole formation

• Several RON proteins (2/4/5) assemble into a
  complex in the rhoptry neck
• The complex is secreted and inserts into the host
  membrane (this is not fully defined yet but might
  parallel E. colis insertion of its own receptor)
• AMA1 is a special microneme protein that engages
  the RON complex and serves as a specialized
  invasion ligant
• ROPs are secreted into the cytoplasm and fuse
  back to the PV

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The moving junction skims proteins out of the
membrane as the PV forms

• The surface membrane of the host cell was labeled
  for protein (green) and lipid (red) prior to
  infection with Toxoplasma (blue)
• Note that while the lipids are clearly visible in
  the vacuole the proteins are excluded

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Rhoptry proteins modify the function of the host
cell nucleus

• Several rhoptry proteins are injected into the
  host cell cytoplasm during invasion
• They accumulate in the host cell nucleus
• Interestingly, many of them are enzymes capable
  of changing the phosphorylation state of proteins
  (kinases phosphatases)
• Their precise function remains to be determined
  but it appears that they modulate gene expression
  in the host cell and that their activity is
  required for rapid growth and the ability to
  cause disease (virulence)

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Rhoptry proteins have multiple functions in
vacuole formation host manipulation
Bradley Sibley, Current Opinion in
Microbiology 10 582-587
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Apicomplexan host cell invasion
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Invasion depends on sequential protein secretion
from three organelles

• Dense granules (DG) secretion of dense granules
  occurs after the vacuole is fully formed and
  continues throughout the intracellular growth of
  the parasites
• Dense granules are secreted from the basal end of
  the parasites
• Dense granule proteins likely play a role in
  modification of the vacuole into an environment
  supportive of parasite growth

39
Dense granule proteins are secreted into the PV

• Certain dense granule proteins are soluble in the
  lumen of the PV others integrate into the
  membrane
• These proteins are probably involved in modifying
  the vacuole

40
The PV is highly modified to suite the parasites
needs

• Tubular network increases surface (dense granule)
• Sieving pores give access to small nutrient
  molecules in the host cell cytoplasm (probably
  dense granule)
• Specific host cell organelles are recruited close
  to the PV membrane (rhoptry)

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Dense granules are involved in establishing the
intravacuolar network
42
the parasitophorous vacuole contains a sieving
pore
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Host cell mitochondria and ER are recruited to
the PVM
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Apicomplexan invasion

• Active, parasite driven process
• Depends on parasite actin/myosin motility
  (conveyor belt model)
• Involves secretion of micronemes (attachment,
  motility), rhoptries (PV MJ formation) and
  dense granules (makes PV into a suitable home)
• Sets up a parasitophorous vacuole which initially
  is derived from the host cell cell-membrane
• A moving junction is formed which screens out
  host membrane proteins from the PV, the PV is
  fusion incompetent and the parasite protected

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• Some additional detail on the gliding machine

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Microneme protein complexes interact with the the
host cell

• Micronemes contain a large set of proteins
  containing protein and carbohydrate binding and
  interaction domains
• MIC proteins associate into complexes in the ER
  and this association is critical for protein
  targeting
• Proteolytic processing is critical for complex
  association, maturation and finally shedding

Dowse T et al. Host cell invasion by the
api...PMID 15358257
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Microneme protein complexes interact with the the
host cell

• The different roles of individual microneme
  proteins has been studied using conditional KOs
  in Toxoplasma (also see TRAP study by Sultan in
  Plasmodium)
• MIC 2 is required for gliding and invasion (no
  full block of invasion)
• AMA1 is critical for invasion yet dispensable for
  gliding motility
• This could suggest different sets of tires for
  locomotory motility and invasion motility -- both
  depending on the actin/myosin engine

http//www.molbiolcell.org/cgi/content/full/16/9/4
341 doi10.1371/journal.ppat.0020084
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How to let go - rhomboid proteases in Toxoplasma
Plasmodium

• Sequencing of shed soluble adhesins suggest
  cleavage within the transmembrane region
• Most characterized apicomplexan adhesins show
  conserved amino acids in this region which are
  similar to those demonstrated to be critical for
  rhomboid proteases in Drosophila
• Rhomboids are transmembrane proteins and act as
  proteases cleaving the target with in the TM
  region

http//www.pnas.org/cgi/content/full/102/11/4146 h
ttp//www.jcb.org/cgi/content/full/174/7/1023 Dows
e TJ et al. Rhomboid-like proteins in
Api...PMID 15922242
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How to let go - rhomboid proteases in Toxoplasma
Plasmodium

• Several rhomboids are encoded by apicomplexan
  genomes and they are differentially expressed
  over the life cycle
• TgROM5 in T. gondii localizes to the surface,
  its distribution is patchy and often at the
  posterior in gliding parasites.
• TgROM5 (but not other ROMs) is capable of
  shedding MIC2
• Similar work in Plasmodium confirms the rhomboid
  model

http//www.pnas.org/cgi/content/full/102/11/4146 h
ttp//www.jcb.org/cgi/content/full/174/7/1023 Dows
e TJ et al. Rhomboid-like proteins in
Api...PMID 15922242
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The constriction is a result of the moving
junction