International Journal for Parasitology 17 "0887# 0996Ð0902

Invited review Apical and !cell invasion by J[F[ Dubremetz \ Nathalie Garcia!Reguet\ Valerie Conseil\ Marie NoeÃlle 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 \ 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 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 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 sporozoites[ These molecules have been shown to be shed on the parasite and:or cell surfaces during the invasion process in Plasmodium\ Toxoplasma and [ For rhoptries and dense granules\ the association between exocytosed proteins and the 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 from the suggestion that as they were found in invasive stages they were therefore likely to serve in The electron!dense organelles of the apical com! host!cell invasion by these obligatory intracellular plex of apicomplexan parasites have aroused a great parasites[ Recent studies have shed some light on deal of interest from investigators since they were the possible role of these structures[ _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 1[ The invasion process

Corresponding author[ Tel[] 22 219 760 062^ fax] 22 219 760 Internalisation of apicomplexan zoites in host 047^ e!mail] JFDubremetzÝcompuserve[com[ 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 parasite proteins belong to a conserved family in the zoite and putative host cell must trigger a recog! Plasmodium genus[ Subsequently\ another family of nition event that starts the entire process*which is microneme proteins\ highly conserved in Apic! sequential[ Host!cell entry is initiated by contact omplexa\ has been identi_ed progressively\ at _rst between the apex of the parasite and the cell surface as SSP1!TRAP ð01Ł in Plasmodium spp[\ then in and is immediately followed by progressive intern! Eimeria ð02Ł and ð03Ł[ All these alisation at the site of apical contact\ which pro! proteins contain conserved domains\ especially ceeds from anterior to posterior\ eventually closing thrombospondin!like motifs[ In Plasmodium spp[ the vacuole behind the parasite[ Entry usually takes sporozoites\ these proteins are involved in parasite about 4Ð09 s[ Once inside the cell\ the parasite no binding to hepatocytes ð04Ł[ Their function is longer moves[ At the EM level\ internalisation unknown in other genera[ Thrombospondin occurs in a vacuole that is always continuous with domains usually bind to sulphated glycoconjugates the plasmalemma of the host cell\ although it is found on cell surfaces\ which are highly variable\ separated from it by the moving junction\ which is allowing for host!cell speci_city of binding[ A very a very close apposition of the two plasmalemmas exciting recent _nding is that TRAP may also be located at the site of entry[ The moving junction involved in motility\ as the knock!out of TRAP in has a peculiar appearance in freezeÐfracture\ and Plasmodium berghei leads to non!motile sporozoites this technique also shows that the developing vacu! ð05Ł[ ole has almost no intramembrane particles\ show! Other molecules containing putative adhesive ing a dramatic di}erence between host!cell domains have also been found in micronemes of plasmalemma and parasitophorous vacuole during Eimeria ð06Ł\ Toxoplasma ð07Ł and Sarcocystis ð08Ł[ the invasion process\ despite the fact that the lipid These results show that many putative ligandÐ bilayer is continuous between both ð0\ 1Ł[ receptor interactions can originate from microneme Exocytosis of rhoptries is suggested during contents and are therefore likely to be responsible invasion\ as proteins from the rhoptries are found for the early interactions between zoites and sub! in the vacuole membrane early during the process strate and host cells\ either in binding alone\ or ð2Ð4Ł[ Dense granules are exocytosed in the vacuole in binding and motility\ including the invasion[ In within minutes after it closes and their contents support of this hypothesis\ exocytosis of microneme become associated with the vacuole contents and proteins during invasion has been reported in membrane ð5Ł[ As invasion is fully driven by the several Apicomplexa] Sarcocystis ð19Ł\ Eimeria ð06Ł\ parasite\ it is entirely dependent on the gliding Plasmodium ð10Ł and T[ gondii ð4Ł[ motility of the zoite[ This motility is known to be Gliding motility may work through trans! dependent on actin and myosin present in the pel! membrane molecules that would be responsible for licle ð6Ð8Ł\ but how gliding motility of the Api! binding a substrate in the outer surface\ and would complexa works is entirely unknown[ Recent data be moved along the body of the zoite through inter! suggest that microneme contents may be involved action with a motor located in the deeper layers of in motility[ 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 2[ Micronemes and host!cell recognition\ binding hypothesis is that no transmembrane surface pro! and motility tein is known in apicomplexan zoites^ all surface proteins known so far in these organisms are GPI Early data concerning the involvement of mic! anchored\ and have no cytoplasmic domain[ There! ronemes in host!cell invasion derive from the dis! fore\ no surface protein seems to ful_l the properties covery of adhesive proteins in required for contributing to gliding motility[ An and ð09Ł that were later attractive hypothesis is being proposed that derives localised in micronemes ð00Ł[ It was then shown from sequence information on microneme proteins\ that despite di}erences in ligand speci_city\ these and the observation of microneme proteins being )

J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902 0998 relocated to the parasite surface during invasion\ proteins which have a putative transmembrane and the recent discovery that a microneme protein sequence close to their C!terminal end would be knock!out in P[ berghei sporozoites abolishes relocated in the zoite plasmalemma "the hypo! motility ð05\ 12Ł[ In this model "Fig[ 0#\ microneme thetical translocation of proteins from the lumen of the 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 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 Fig[ 0[ A hypothetical motor for Apicomplexa gliding motility[ "0# The motor is associated with the inner membrane complex area of the host cell plus the vacuole[ Very elegant "IMC#\ oriented by the subpellicular microtubules "T#\ but plas! work by G[ Ward and co!workers ð16Ł on T[ gondii malemma "P# GPI anchored molecules cannot interact with it[ has suggested that this may not actually be true[ "1# A microneme molecule "MIC# exocytosed as a soluble protein These authors recorded electrically "by patch clam! changes conformation and becomes inserted in the plas! ping# variation in host!cell capacitance while video! malemma[ "2# The MIC protein is carried along the inner com! plex motor and interacts with the substrate "S#\ or associates recording simultaneously the invasion process\ and with a GPI anchored protein that interacts with the substrate\ their measurements have shown no signi_cant resulting in gliding motility[ increase of total capacitance during the formation

CMYK Page 0998 ) 0909 J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902 of the vacuole\ and a decrease at vacuole closure[ As synthesis as preproteins ð23Ł\ which may mean that cell capacitance is correlated directly to cell surface\ the parasite synthesises these proteins in an inactive this suggests that the vacuole internalises part of form\ and then packages them into the active form[ the cell surface[ These data are puzzling\ especially Protein processing is likely to occur in rhoptry pre! as they do not show any signi_cant return to the cursors\ known as non!pedunculated!condensing initial cell!surface area after closure\ suggesting that vesicles found in developing zoites[ Therefore\ pro! the cell does not regulate rapidly the obvious "but teolytic activities must be present in the organelles small# increase in total volume by restoring the which are activated upon condensing of the corresponding surface area value[ Indeed\ either the organelle contents[ 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 4[ Dense granules surface[ This enigmatic aspect of invasion remains to be studied[ At the least\ these experiments suggest This third type of exocytic organelles has been that\ as shown for Plasmodium spp[\ most of the studied extensively in T[ gondii\ although exocytosis lipids of the T[ gondii vacuole are derived from the was _rst suggested in P[ knowlesi ð24Ł and dem! host!cell plasmalemma[ Contrasting with this\ it onstrated in Sarcocystis muris ð25Ł[ The dense!gran! seems that no proteins from this membrane are ule proteins associate with vacuole membrane or integrated in the vacuole\ as if they are restricted intravacuolar structures after their exocytosis\ from crossing the moving junction zone[ which occurs after internalisation of the zoite in the The capacitance measurement results still allow vacuole[ In the case of T[ gondii\ the function of one approximately 19) of the surface increase to be dense!granule protein is known\ as it is an NTPase\ contributed by the parasite^ rhoptries have also likely to salvage purines from the host cell ð26\ been suggested to contribute lipids to the vacuole 27Ł[ Caution must be exercised when comparing T[ membrane\ and a phospholipase A1 activity that gondii and other Apicomplexa with regard to dense may induce some local surface increase has been granules\ as this parasite unusually retains its zoite shown to be associated with invasion ð17Ł[ This morphology during all intracellular stages of devel! parasite contribution needs to be further inves! opment in the intermediate host[ Indeed\ T[ gondii tigated[ What is known in T[ gondii and in Eimeria releases dense granules during all intracellular nieschulzi is that the vacuole membrane is enriched development\ whereas most other Apicomplexa with rhoptry proteins during invasion ð2Ð4\ 18\ 29Ł[ release their dense granules after invasion\ but then Some of these proteins are supposed to be periph! dedi}erentiate into schizogonic stages that grow in eral\ whereas others are likely to be transmembrane[ the vacuole before redi}erentiating into invasive Integration of rhoptry proteins in the vacuole mem! stages at the end of the schizogonous development[ brane seems to occur by insertion in a trans! One must therefore bear in mind that what is membrane fashion as exempli_ed by the ROP1 described for T[ gondii may not fully apply to other family in T[ gondii^ in this case\ again\ how these Apicomplexa\ and that protein tra.cking from proteins are translocated from an organelle lumen parasite to vacuole or host cell cannot be restricted to a transmembrane location raises unsolved prob! to this type of organelle[ lems of proteinÐmembrane interaction[ The func! A major question that concerns proteins con! tions of these proteins are not yet known\ although tained in all of these organelles is that many of them two major hypotheses suggest that they either con! contain putative transmembrane sequences and are tribute the pores that allow entry of small molecules found associated with membranes after exocytosis in the vacuole ð20\ 21Ł or they are involved in the "either in parasite or vacuolar membranes#\ and for association of host!cell endoplasmic reticulum or some their transmembrane topology is has indeed mitochondria to the outer side of the vacuole mem! been demonstrated ð18Ł[ How these molecules can brane ð22Ł[ be found in the lumen of a vesicle and then become An interesting feature of rhoptry proteins is their 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 invasion process is characteristics\ and therefore speed up the identi! schematised in Fig[ 1[ _cation of major functions in invasion[ Thus\ future research should concentrate on the search for hom! ologies at the molecular level between organelles\ 5[ Conclusion and the manipulation of their expression through the new possibilities o}ered by parasite trans! Host!cell invasion by Apicomplexa is a unique formation[ The path opened in this direction by the way of cell entry[ This is due mainly to the develop! pioneering work of Boothroyd and collaborators ment of a sophisticated invasion apparatus that has ð28\ 39Ł that has recently led to a spectacular dem! no counterpart in other models[ As the mechanisms onstration of a phenotype for a TRAP knock!out involved are likely to be similar throughout the by Sultan et al[ ð05Ł\ is likely to bring further exciting phylum\ as exempli_ed by the conservation of a results in the near future[ family of microneme proteins containing throm! bospondin!related domains\ investigating invasion References in any genera will help to understand the shared general features of the process[ In addition\ com! ð0Ł Aikawa M\ Miller LH\ Rabbege J\ Epstein N[ FreezeÐ paring organelle molecules will also help to separate fracture study of the erythrocyte membrane during malarial important basic functional domains of the speci_c parasite invasion[ J Cell Biol 0870^81]44Ð51[

CMYK Page 0900 ) 0901 J[F[ Dubremetz et al[ : International Journal for Parasitolo`y 17 "0887# 0996Ð0902

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