Transcript Apicomplexan host cell invasion

Apicomplexan host cell
invasion II
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?
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 249:641-6
Invasion depends on
parasite not host cell actin
Salmonella
Toxoplasma
 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
Dobrowolski JM, Sibley LD. Cell. 1996 84(6):933-9.
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.
Apicomplexan host cell
invasion
Movies Dr. Gary Ward, Univ. of Vermont
http://www.uvm.edu/~mmg1/videos_ward.php?id=23
Apicomplexan host cell
invasion
Invasion depends on sequential protein
secretion from three organelles
Sultan et al, Cell 90: 511-522
 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?
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)
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
Circles, helices & twirls
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?
A special myosin is required to move
microneme proteins over the surface
Normal myosin
Suppressed myosin
Meissner et al., Science. 298:837-40
 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?
The gliding machinery is anchored
in the inner membrane complex
PM
IMC
MT
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
GAP45 is part of a complex
including GAP50, MyoA & MLC
http://www.jcb.org/cgi/content/full/165/3/383
The apicomplexan engine - the
glideosome
The treadmilling model of
apicomplexan gliding motility
Soldati et al., Trends in Parasitology 20: 567-574
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
Formation of the PV &
moving junction
Formation of the PV & moving junction
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
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
Invasion of the red blood cell
by the malaria parasite
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)
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?
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
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
There is no significant change of
host cell surface during invasion
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
The moving junction
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)
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. coli’s 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
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
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)
Rhoptry proteins have multiple functions
in vacuole formation & host manipulation
Bradley & Sibley, Current Opinion in Microbiology
10: 582-587
Apicomplexan host cell
invasion
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
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
The PV is highly modified to
suite the parasite’s 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)
Dense granules are involved in
establishing the intravacuolar network
the parasitophorous vacuole
contains a sieving pore
Host cell mitochondria and ER
are recruited to the PVM
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 cellmembrane
A moving junction is formed which screens out
host membrane proteins from the PV, the PV is
Some additional detail on the gliding
machine
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]
Microneme protein complexes
interact with the the host cell
http://www.molbiolcell.org/cgi/content/full/16/9/4341
doi:10.1371/journal.ppat.0020084
 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
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
http://www.jcb.org/cgi/content/full/174/7/1023
Dowse TJ et al. Rhomboid-like proteins in Api...[PMID: 15922242]
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, it’s 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
http://www.jcb.org/cgi/content/full/174/7/1023
Dowse TJ et al. Rhomboid-like proteins in Api...[PMID: 15922242]
The constriction is a result
of the moving junction