Apicomplexa host cell invasion
Extracellular
Host-Cell
membrane
Host Cytosol
PV
Toxoplasma gondii - overview from
last lecture
Sexual reproduction in the cat, very wide range
of intermediate hosts
Two asexual form, tachyzoites & bradyzoites ,
responsible for acute and chronic stages,
infection is for life.
Toxoplasma is an opportunistic pathogen
(severe pathology in immuno-compromised
individuals)
T. gondii is an obligate intracellular parasite
T. gondii host cell invasion
Host cell invasion can be divided to
different elements
Gliding
Attachments &
PV formation
(Kiss and spit)
Penetration
through the
moving
junction
PV
modifications
Host cell invasion can be divided to
different elements
Gliding
Attachments &
PV formation
(Kiss and spit)
Penetration
through the
moving
junction
PV
modifications
Everyone has their own move
Flagella
Amoeboid movement
Toxoplasma - gliding motility
Different parasites use different
mechanisms to invade cells
 Trypansoma cruzi -- recruites
lysosomes, then escape to replicate
in the cytoplasm.
 Leishmania -- invades macropahges
by phagocytosis and is adjusted to
extreme conditions of
phagolysosome
 Mycobacterium tuberculosis, -- enter
by phagocytosis and blocks
lysosomal maturation
 Toxoplasma -- actively penetrate
host and resides it own a specialized
parasitophorous vacuole.
Invasion depends on parasite not host
cell actin
Salmonella
Toxoplasma
 Cytochalasin D (CytD), a drug that prevents
actin polymerization
 Host cell invasion by Salmonella (which is
taken up by phagocytosis) is inhibited by
CytD.
 Same goes for Toxoplasma.
 In Salmonella, invasion can be rescued by
using a drug resistant host cell mutant
(Cyt1).
 Toxoplasma invasion remains inhibited in the
drug resistant host cell.
 Generating a drug resistant parasite rescues
invasion
Motility element alone is dependent on
actin polymerization
 CytD treatment does not 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
A special myosin is required for T.
gondii motility
Normal myosin
Suppressed 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)
The conveyor-belt model of gliding
motility
 Gregarines are a group of
‘primitive’ apicomplexans
which parasitize
invertebrates
 In comparison to
Toxoplasma or the
malaria parasite these are
fairly large cells, which
makes them good
subjects for microscopy
studies
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
motility machinery moves
little “hands” over their
surface from the apical to the
basal end of the parasite
 Traction
Gliding parasites deposit proteins and
lipid trails
 Like a slug the parasite leaves behind a trail of surface proteins
and lipids
 A protease cleaves the little hands to disengage the parasite from
the surface and allow for propulsion (sheddase)
Invasion depends on sequential protein
secretion from three organelles
Micronemes (Mn)
Rhoptries (Rh)
Dense granuoles (DGs)
Are micronemes the little hands acting
during invasion?
 Microneme proteins (MICs) can bind to a
variety of carbohydrates found on the
surface of cells
 Secretion of micronemes brings MICs
proteins to the parasite surface
 Micronemes are secreted at the apical tip
of the parasite and move to the posterior.
This movement is blocked by cytD!
 Are MICs the little hands?
MIC2 is required for T. gondii motility and
invasion
Using genetic engineering a mutant parasite was constructed in which the
expression of a microneme protein can be suppressed
Several molecules act together to create
the gliding machinery - the glideosome
?
?
? ?
? ?
The gliding machinery is anchored in
the inner membrane complex
PM
IMC
MT
The conveyor-belt model
Motility depends of parasite actin/myosin
Myosin is anchored into the inner membrane
complex membrane
Short actin filaments form and are moved towards
the posterior end of the parasite by the myosin
power
The short actin filaments are linked to microneme
proteins by an adaptor -- movement of actin
filaments results in movement of microneme proteins
holding host cell receptors
The parasite glides over the substrate
Microneme proteins are shed at the back end
Host cell invasion can be
divided to different elements
Gliding
Attachments &
PV formation
(Kiss and spit)
Penetration
through the
moving
junction
PV
modifications
Rhoptry proteins are involved at various
points of the invasion process
 Secretion of rhoptries is required for
the formation of the parasitophorous
vacuole
 Like micronemes rhoptries are
secreted at the apical tip of the parasite
 One group of very interesting rhoptry
proteins is injected into the host cell
and manipulates gene expression in
the host nucleus as well as start-up the
PV -- ‘kiss and spit’
 Some rhoptry proteins make up (part)
of the moving junction
 Other rhoptry proteins are found
throughout the membrane of the
parasitophorous vacuole (PVM)
Rhoptry release upon attachment kiss and spit
 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
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, some 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)
Second role of Rhoptry proteins during
invasion - The moving junction
Rhoptry proteins organize the moving
junction
?
Extracellular
Host-Cell
membrane
Host Cytosol
? ?PV
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
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
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)
Extracellular
Both contribute to the PV
Host-Cell
membrane
Host Cytosol
Rhoptry proteins are responsible for host cell
mitochondria and ER recruitement to the PVM
Host cell invasion can be divided to
different elements
Gliding
Attachments &
PV formation
(Kiss and spit)
Penetration
through the
moving
junction
PV
modifications
Dense granules proteins modify the PV
environment - Toxo makes a home
 Secretion of dense granules occurs after the
vacuole is fully formed and continues
throughout the intracellular growth of the
parasites
 Dense granule proteins likely play a role in
modification of the vacuole into an
environment supportive of parasite growth
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
Dense granules are involved in
establishing the intravacuolar network
Summary
Apicomplexan invasion
Active, parasite driven process
Depends on parasite actin/myosin motility (conveyor
belt model)
Involves secretion of three major groups of proteins:
MICs from micronemes (motility, Moving-Junction).
ROPs and RONs from rhoptries (spit, parasitoforouse
vacuole & moving Junction).
DGs from dense granules (makes PV into a suitable home).