International Journal for Parasitology 17 "0887# 0996Ð0902
Invited review
Apical organelles and host!cell invasion by Apicomplexa
J[F[ Dubremetz\ Nathalie Garcia!Reguet\ Valerie Conseil\
Marie Noelle Fourmaux
Unite 31 INSERM\ 258 rue J Guesde\ 48544 Villeneuve d|Ascq\ France
Received 04 December 0886^ accepted 09 March 0887
Abstract
Host!cell invasion by apicomplexan parasites involves the successive exocytosis of three di}erent secretory organelles^
namely micronemes\ rhoptries and dense granules[ The _ndings of recent studies have extended the structural homologies
of each set of organelles between most members of the phylum and suggest shared functions for each set[ Micronemes
are apparently used for host!cell recognition\ binding\ and possibly motility^ rhoptries for parasitophorous vacuole
formation^ and dense granules for remodelling the vacuole into a metabolically active compartment[ In addition\ gene
cloning and sequencing have demonstrated conserved domains\ which are likely to serve similar functions in the invasion
process[ This is especially true for microneme proteins containing thrombospondin!like domains\ which are likely to be
involved in binding to sulphated glycoconjugates[ One such protein was recently shown to be required for the motility
of Plasmodium sporozoites[ These molecules have been shown to be shed on the parasite and:or cell surfaces during the
invasion process in Plasmodium\ Toxoplasma and Eimeria[ For rhoptries and dense granules\ the association between
exocytosed proteins and the parasitophorous vacuole membrane has been analysed extensively in Toxoplasma\ as these
proteins are likely to play a crucial role in metabolic interactions between the parasites and their host cells[ The
development of parasite transformation by gene transfection has provided powerful tools to analyse the fate and
function"s# of the corresponding proteins[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science
Ltd[
Keywords] Invasion^ Apicomplexa^ Exocytosis^ Organelles^ Motility^ Adhesion
0[ Introduction
The electron!dense organelles of the apical com!
plex of apicomplexan parasites have aroused a great
deal of interest from investigators since they were
_rst described during EM studies of these organ!
isms[ However\ their function in the biology of the
parasite has remained unclear for a long time\ apart
from the suggestion that as they were found in
invasive stages they were therefore likely to serve in
host!cell invasion by these obligatory intracellular
parasites[ Recent studies have shed some light on
the possible role of these structures[
1[ The invasion process
Corresponding author[ Tel[] 22 219 760 062^ fax] 22 219 760
047^ e!mail] JFDubremetzÝcompuserve[com[
Internalisation of apicomplexan zoites in host
cells follows a very conserved scheme that can be
S9919!6408:87 ,08[99¦9[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ Printed in Great Britain
PII] S9919!6408"87#99965!8
0997
J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902
summarised as follows] an initial contact between
zoite and putative host cell must trigger a recog!
nition event that starts the entire process*which is
sequential[ Host!cell entry is initiated by contact
between the apex of the parasite and the cell surface
and is immediately followed by progressive intern!
alisation at the site of apical contact\ which pro!
ceeds from anterior to posterior\ eventually closing
the vacuole behind the parasite[ Entry usually takes
about 4Ð09 s[ Once inside the cell\ the parasite no
longer moves[ At the EM level\ internalisation
occurs in a vacuole that is always continuous with
the plasmalemma of the host cell\ although it is
separated from it by the moving junction\ which is
a very close apposition of the two plasmalemmas
located at the site of entry[ The moving junction
has a peculiar appearance in freezeÐfracture\ and
this technique also shows that the developing vacu!
ole has almost no intramembrane particles\ show!
ing a dramatic di}erence between host!cell
plasmalemma and parasitophorous vacuole during
the invasion process\ despite the fact that the lipid
bilayer is continuous between both ð0\ 1Ł[
Exocytosis of rhoptries is suggested during
invasion\ as proteins from the rhoptries are found
in the vacuole membrane early during the process
ð2Ð4Ł[ Dense granules are exocytosed in the vacuole
within minutes after it closes and their contents
become associated with the vacuole contents and
membrane ð5Ł[ As invasion is fully driven by the
parasite\ it is entirely dependent on the gliding
motility of the zoite[ This motility is known to be
dependent on actin and myosin present in the pel!
licle ð6Ð8Ł\ but how gliding motility of the Api!
complexa works is entirely unknown[ Recent data
suggest that microneme contents may be involved
in motility[
2[ Micronemes and host!cell recognition\ binding
and motility
Early data concerning the involvement of mic!
ronemes in host!cell invasion derive from the dis!
covery of adhesive proteins in Plasmodium knowlesi
and Plasmodium falciparum ð09Ł that were later
localised in micronemes ð00Ł[ It was then shown
that despite di}erences in ligand speci_city\ these
parasite proteins belong to a conserved family in the
Plasmodium genus[ Subsequently\ another family of
microneme proteins\ highly conserved in Apic!
omplexa\ has been identi_ed progressively\ at _rst
as SSP1!TRAP ð01Ł in Plasmodium spp[\ then in
Eimeria ð02Ł and Toxoplasma gondii ð03Ł[ All these
proteins contain conserved domains\ especially
thrombospondin!like motifs[ In Plasmodium spp[
sporozoites\ these proteins are involved in parasite
binding to hepatocytes ð04Ł[ Their function is
unknown in other genera[ Thrombospondin
domains usually bind to sulphated glycoconjugates
found on cell surfaces\ which are highly variable\
allowing for host!cell speci_city of binding[ A very
exciting recent _nding is that TRAP may also be
involved in motility\ as the knock!out of TRAP in
Plasmodium berghei leads to non!motile sporozoites
ð05Ł[
Other molecules containing putative adhesive
domains have also been found in micronemes of
Eimeria ð06Ł\ Toxoplasma ð07Ł and Sarcocystis ð08Ł[
These results show that many putative ligandÐ
receptor interactions can originate from microneme
contents and are therefore likely to be responsible
for the early interactions between zoites and sub!
strate and host cells\ either in binding alone\ or
in binding and motility\ including the invasion[ In
support of this hypothesis\ exocytosis of microneme
proteins during invasion has been reported in
several Apicomplexa] Sarcocystis ð19Ł\ Eimeria ð06Ł\
Plasmodium ð10Ł and T[ gondii ð4Ł[
Gliding motility may work through trans!
membrane molecules that would be responsible for
binding a substrate in the outer surface\ and would
be moved along the body of the zoite through inter!
action with a motor located in the deeper layers of
the pellicle\ such as the inner membrane complex
where organised arrays of structures likely to be
involved are present ð11Ł[ The di.culty with this
hypothesis is that no transmembrane surface pro!
tein is known in apicomplexan zoites^ all surface
proteins known so far in these organisms are GPI
anchored\ and have no cytoplasmic domain[ There!
fore\ no surface protein seems to ful_l the properties
required for contributing to gliding motility[ An
attractive hypothesis is being proposed that derives
from sequence information on microneme proteins\
and the observation of microneme proteins being
)
J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902
relocated to the parasite surface during invasion\
and the recent discovery that a microneme protein
knock!out in P[ berghei sporozoites abolishes
motility ð05\ 12Ł[ In this model "Fig[ 0#\ microneme
0998
proteins which have a putative transmembrane
sequence close to their C!terminal end would be
relocated in the zoite plasmalemma "the hypo!
thetical translocation of proteins from the lumen of
the organelle to the parasite plasmalemma remains
a mystery for the moment#\ could act as ligands
on the outer domain and could be carried by the
pellicular motor on the inner side[ This remains to
be demonstrated\ but provides an attractive
hypothesis[ As suggested by some data showing
inhibition of invasion by anti!surface protein anti!
bodies ð13\ 14Ł\ GPI anchored surface proteins may
also be involved in ligand binding and motility] this
may require that they interact with transmembrane
microneme proteins likely to transduce the mech!
anical forces[ Whether microneme proteins are
components of the moving junction remains to be
elucidated[
3[ Rhoptries and vacuole formation
Rhoptries discharge their contents during the
internalisation of the parasite and rhoptry proteins
are then found in the parasitophorous vacuole
membrane^ no clue to their function in the process
has been obtained so far\ although the most likely
hypothesis is that they are crucial in the building of
this vacuole[
The origin of the vacuole membrane is still an
unsolved matter\ although considerable progress
has been made[ During r[b[c[ invasion by Plas!
modium spp[\ vacuole membrane lipids seem to be
derived almost exclusively from the r[b[c[ mem!
brane ð15Ł[ How this can happen is not understood\
as the r[b[c[ is thought not to be able to increase its
surface lipid content[ Indeed\ as zoites appear to
enter the cell by forcing the development of a new
vacuole that is continuous with the plasmalemma\
one would expect an increase in the total surface
area of the host cell plus the vacuole[ Very elegant
work by G[ Ward and co!workers ð16Ł on T[ gondii
has suggested that this may not actually be true[
These authors recorded electrically "by patch clam!
ping# variation in host!cell capacitance while video!
recording simultaneously the invasion process\ and
their measurements have shown no signi_cant
increase of total capacitance during the formation
Fig[ 0[ A hypothetical motor for Apicomplexa gliding motility[
"0# The motor is associated with the inner membrane complex
"IMC#\ oriented by the subpellicular microtubules "T#\ but plas!
malemma "P# GPI anchored molecules cannot interact with it[
"1# A microneme molecule "MIC# exocytosed as a soluble protein
changes conformation and becomes inserted in the plas!
malemma[ "2# The MIC protein is carried along the inner com!
plex motor and interacts with the substrate "S#\ or associates
with a GPI anchored protein that interacts with the substrate\
resulting in gliding motility[
CMYK Page 0998
)
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J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902
of the vacuole\ and a decrease at vacuole closure[ As
cell capacitance is correlated directly to cell surface\
this suggests that the vacuole internalises part of
the cell surface[ These data are puzzling\ especially
as they do not show any signi_cant return to the
initial cell!surface area after closure\ suggesting that
the cell does not regulate rapidly the obvious "but
small# increase in total volume by restoring the
corresponding surface area value[ Indeed\ either the
bilayer must be able to withstand some stretching
"a few percent#\ or the parasite must add molecules
that do not restore capacitance\ but do increase the
surface[ This enigmatic aspect of invasion remains
to be studied[ At the least\ these experiments suggest
that\ as shown for Plasmodium spp[\ most of the
lipids of the T[ gondii vacuole are derived from the
host!cell plasmalemma[ Contrasting with this\ it
seems that no proteins from this membrane are
integrated in the vacuole\ as if they are restricted
from crossing the moving junction zone[
The capacitance measurement results still allow
approximately 19) of the surface increase to be
contributed by the parasite^ rhoptries have also
been suggested to contribute lipids to the vacuole
membrane\ and a phospholipase A1 activity that
may induce some local surface increase has been
shown to be associated with invasion ð17Ł[ This
parasite contribution needs to be further inves!
tigated[ What is known in T[ gondii and in Eimeria
nieschulzi is that the vacuole membrane is enriched
with rhoptry proteins during invasion ð2Ð4\ 18\ 29Ł[
Some of these proteins are supposed to be periph!
eral\ whereas others are likely to be transmembrane[
Integration of rhoptry proteins in the vacuole mem!
brane seems to occur by insertion in a trans!
membrane fashion as exempli_ed by the ROP1
family in T[ gondii^ in this case\ again\ how these
proteins are translocated from an organelle lumen
to a transmembrane location raises unsolved prob!
lems of proteinÐmembrane interaction[ The func!
tions of these proteins are not yet known\ although
two major hypotheses suggest that they either con!
tribute the pores that allow entry of small molecules
in the vacuole ð20\ 21Ł or they are involved in the
association of host!cell endoplasmic reticulum or
mitochondria to the outer side of the vacuole mem!
brane ð22Ł[
An interesting feature of rhoptry proteins is their
synthesis as preproteins ð23Ł\ which may mean that
the parasite synthesises these proteins in an inactive
form\ and then packages them into the active form[
Protein processing is likely to occur in rhoptry pre!
cursors\ known as non!pedunculated!condensing
vesicles found in developing zoites[ Therefore\ pro!
teolytic activities must be present in the organelles
which are activated upon condensing of the
organelle contents[
4[ Dense granules
This third type of exocytic organelles has been
studied extensively in T[ gondii\ although exocytosis
was _rst suggested in P[ knowlesi ð24Ł and dem!
onstrated in Sarcocystis muris ð25Ł[ The dense!gran!
ule proteins associate with vacuole membrane or
intravacuolar structures after their exocytosis\
which occurs after internalisation of the zoite in the
vacuole[ In the case of T[ gondii\ the function of one
dense!granule protein is known\ as it is an NTPase\
likely to salvage purines from the host cell ð26\
27Ł[ Caution must be exercised when comparing T[
gondii and other Apicomplexa with regard to dense
granules\ as this parasite unusually retains its zoite
morphology during all intracellular stages of devel!
opment in the intermediate host[ Indeed\ T[ gondii
releases dense granules during all intracellular
development\ whereas most other Apicomplexa
release their dense granules after invasion\ but then
dedi}erentiate into schizogonic stages that grow in
the vacuole before redi}erentiating into invasive
stages at the end of the schizogonous development[
One must therefore bear in mind that what is
described for T[ gondii may not fully apply to other
Apicomplexa\ and that protein tra.cking from
parasite to vacuole or host cell cannot be restricted
to this type of organelle[
A major question that concerns proteins con!
tained in all of these organelles is that many of them
contain putative transmembrane sequences and are
found associated with membranes after exocytosis
"either in parasite or vacuolar membranes#\ and for
some their transmembrane topology is has indeed
been demonstrated ð18Ł[ How these molecules can
be found in the lumen of a vesicle and then become
integrated as transmembrane is yet unexplained[
)
J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902
0900
Fig[ 1[ Schematic drawing of the successive steps of Apicomplexa invasion[ "0# A zoite comes in contact with a host cell surface^ a
signal is transduced from the surface "star\ arrow# to the apex[ "1# The signal induces re!orientation\ microneme exocytosis\ apical
binding to the host cell\ formation of the moving junction[ "2# Rhoptries are exocytosed\ while the moving junction glides backward
and the parasitophorous vacuole starts expanding[ Rhoptry material is integrated in the vacuole membrane[ The exocytosed micronemal
material expands on the zoite surface and is capped behind the moving junction[ "3# The vacuole continues to expand\ getting most of
its lipids from the host cell plasmalemma[ The junction reaches the posterior end of the parasite and eventually seals the vacuole[ Dense
granules are exocytosed in the vacuolar space[
A summary of the
schematised in Fig[ 1[
invasion
process
characteristics\ and therefore speed up the identi!
_cation of major functions in invasion[ Thus\ future
research should concentrate on the search for hom!
ologies at the molecular level between organelles\
and the manipulation of their expression through
the new possibilities o}ered by parasite trans!
formation[ The path opened in this direction by the
pioneering work of Boothroyd and collaborators
ð28\ 39Ł that has recently led to a spectacular dem!
onstration of a phenotype for a TRAP knock!out
by Sultan et al[ ð05Ł\ is likely to bring further exciting
results in the near future[
is
5[ Conclusion
Host!cell invasion by Apicomplexa is a unique
way of cell entry[ This is due mainly to the develop!
ment of a sophisticated invasion apparatus that has
no counterpart in other models[ As the mechanisms
involved are likely to be similar throughout the
phylum\ as exempli_ed by the conservation of a
family of microneme proteins containing throm!
bospondin!related domains\ investigating invasion
in any genera will help to understand the shared
general features of the process[ In addition\ com!
paring organelle molecules will also help to separate
important basic functional domains of the speci_c
CMYK Page 0900
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