Invasion factors are coupled to key signalling events leading to the establishment of infection in...

Microreview

Invasion factors are coupled to key signalling eventsleading to the establishment of infection inapicomplexan parasites

Joana M. Santos and Dominique Soldati-Favre*Department of Microbiology, Faculty of Medicine,University of Geneva, 1 rue-Michel Servet, 1211Geneva 4, Switzerland.

Summary

Invasion of host cells by apicomplexan parasites isinitiated when specialized secretory organellescalled micronemes discharge protein complexesonto the parasite surface in response to a rise inparasite intracellular calcium levels. The micron-eme proteins establish interactions with host cellreceptors, engaging the parasite with the host cellsurface, and signal for the immediate exocytosisof another set of secretory organelles namedthe rhoptries. The rhoptry proteins reprogram theinvaded host cell and participate in the formationof the parasitophorous vacuole in which the intra-cellular parasite resides and replicates. Disen-gagement of the invading parasite from the hostcell receptors involves the action of at least oneparasite plasma membrane rhomboid protease,which is concomitantly implicated in a checkpointthat signals the parasite to switch from an invasiveto a replicative mode.

Introduction

The phylum Apicomplexa groups unicellular eukaryoticpathogens, including Toxoplasma gondii and Plasmo-dium, the causative agents of toxoplasmosis and malariarespectively. Host cell invasion by these parasites isan active process, which critically relies on the parasiteactomyosin system (glideosome) and the sequentialsecretion of proteins from two types of specialized apicalorganelles, micronemes and rhoptries (Carruthers and

Sibley, 1997). The microneme proteins (MICs) are dis-charged onto the parasite surface upon the release ofcalcium from intracellular stores (Carruthers and Sibley,1999; Lovett et al., 2002; Lovett and Sibley, 2003) andform tight interactions with host cell receptors. Assemblyof these complexes is essential for glideosome function(Huynh et al., 2003) and formation of the moving junction(MJ), an electron dense constriction produced at the pointof apposition between the parasite and the host cell mem-branes (Aikawa et al., 1978; Michel et al., 1980). Duringinvasion, the MIC-receptors complexes are re-distributedtowards the posterior end of the parasite powered by theglideosome, and as a result the parasite is propelled intothe host cell. Concomitantly, migration of the MJ results inthe formation of a specialized vacuole called the parasi-tophorous vacuole (PV) (Mordue et al., 1999). Recentstudies have indicated that the binding of the MICs to hostcell receptors triggers exocytosis of the rhoptry compo-nents (Singh et al., 2010), which in turn contribute to theformation of the MJ, participate in formation of the PV andmodify the invaded host cell [reviewed in (Boothroyd andDubremetz, 2008)]. At the end of the invasion process, theMIC adhesins are released from the parasite surface byproteolytic cleavage and there is sealing of the PV, result-ing in parasite disengagement from the host cell mem-brane. Once secluded inside the PV, the parasite caninitiate replication [reviewed in (Carruthers and Boo-throyd, 2007)]. Most intracellular stages of the life cycleare not infectious, and therefore, cell division has to beprecisely timed in order to ensure that the new daughterzoites are fully formed and invasive at the time of host cellegress. In this review, we will summarize the most recentadvances in the study of the mechanisms that lead tothe establishment of infection by Plasmodium and Toxo-plasma and pinpoint the similarities and differencesbetween the two parasites.cmi_1585 787..796

MICs assemble into multi-protein complexes

The majority of MICs identified in T. gondii form com-plexes, which preassemble in the endoplasmic reticulum

Received 4 December, 2010; revised 21 January, 2011; accepted 27January, 2011. *For correspondence. E-mail [email protected]; Tel. (+41) 223795672; Fax (+41) 223795702.

Cellular Microbiology (2011) 13(6), 787–796 doi:10.1111/j.1462-5822.2011.01585.xFirst published online 22 February 2011

© 2011 Blackwell Publishing Ltd

cellular microbiology

prior to transit to the micronemes. Four complexes havebeen functionally characterized. Micronemal protein 2(TgMIC2), a member of the conserved thrombospondin-related adhesive protein family, is found in a heterohex-americ complex with MIC2-associated protein (TgM2AP)and plays a fundamental role in gliding motility, host cellattachment and invasion (Huynh and Carruthers, 2006).TgMIC6 forms a complex with two adhesins (TgMIC1 andTgMIC4) and contributes to invasion in vitro and virulencein vivo (Cerede et al., 2005; Blumenschein et al., 2007;Sawmynaden et al., 2008). TgMIC8 assembles with alectin (TgMIC3) and is essential for rhoptry secretion andinvasion (Kessler et al., 2008). The fourth complex isunusual in that it assembles into its final, functional formonly at the MJ following exocytosis (Alexander et al.,2005; Straub et al., 2009; Besteiro et al., 2009), bringingtogether the MIC apical membrane antigen 1 (TgAMA1)and several preassembled rhoptry neck proteins (RONs)(TgRON2-RON4-RON5-RON8). Parasites deficient inTgAMA1 still attach to host cells but are defective inrhoptry secretion and hence fail to form the MJ and invadehost cells (Mital et al., 2005). Unlike the complexes men-tioned above that are specific to T. gondii and Neosporacaninum, the AMA1-RONs complex appears to beconserved across the phylum, with the exception ofCryptosporidium (Cao et al., 2009; Collins et al., 2009;Straub et al., 2009).

The transmembrane MICs present in each complex –TgMIC2, TgMIC6, TgMIC8 and TgAMA1 – (Fig. 1A) allportray a modular structure comprising an ectodomain, amembrane-spanning domain and a short cytoplasmic tail.While the ectodomains establish connections with hostreceptors and hence engage the parasite with the hostcell surface (Fig. 1B), the C-terminal domains can some-times escort the complex to the micronemes and mayassociate with the glideosome via binding to aldolase.While TgMIC2, TgMIC6 and TgAMA1 have all beenshown to bind to aldolase, at least in vitro (Jewettand Sibley, 2003; Zheng et al., 2009; Sheiner et al.,2010), only TgMIC2 and TgMIC6 are proven to function asescorters (Di Cristina et al., 2000; Reiss et al., 2001; Opitzet al., 2002).

Signalling events leading to microneme secretion

Regulated secretion of the micronemes depends onsignal transduction cascades involving calcium as a keyintracellular messenger (Carruthers et al., 1999a; Chenet al., 2004). Organelle discharge can be induced in extra-cellular parasites by pharmacological agents such asethanol and calcium ionophores (Carruthers and Sibley,1999; Carruthers et al., 1999b; Lovett et al., 2002; Naga-mune et al., 2007) and it is inhibited by treatment withintracellular calcium chelators (Carruthers et al., 1999a).

Elevation of the parasite intracellular free calcium levelsoccurs upon exocytosis of ER calcium stores after bothcADPR and IP3 stimulus (Carruthers et al., 1999b; Lovettet al., 2002; Lovett and Sibley, 2003; Chini et al., 2005;Nagamune and Sibley, 2006; Nagamune et al., 2007).Once penetration of the host cell is completed, the intra-cellular calcium levels drop rapidly as a result of negativefeedback, and secretion events in intracellular parasitesare undetectable (Carruthers et al., 1999a; Lovett andSibley, 2003; Singh et al., 2010).

Egress from host cells also involves microneme dis-charge and the concerted action of the MICs in Toxo-plasma (Kafsack et al., 2009). Disruption by homologousrecombination of the gene coding for TgMIC perforin-likeprotein 1 (TgPLP1) leads to a marked impairment incalcium ionophore-induced egress. This defect is notcaused by interference with parasite motility but due tothe lack of rupture of the PV membrane. Interestingly,TgPLP1 can act in trans as tgplp1ko parasites areable to egress from host cells co-infected with wild-typeparasites.

Calcium-mediated signalling pathways in T. gondii andPlasmodium have been consistently linked to the action ofcalcium-dependent protein kinases (CDPKs) [reviewed in(Nagamune et al., 2008)], which are otherwise only foundin plants. Phylogenetic analysis indicates that T. gondiiand P. falciparum encode 11 and 8 CDPK-like genes,respectively (Lourido et al., 2010), and crystal structurestudies revealed a striking conservation between the plantand apicomplexan CDPKs (Wernimont et al., 2010). TheCDPKs possess a Ser/Thr kinase domain highly homolo-gous to that of vertebrate calmodulin-dependent kinases(CaMKs) fused to four EF-hand domains (Nagamune andSibley, 2006). Enzymatic activation depends on binding ofcalcium to the calmodulin-like domain, which induces adramatic structural rearrangement in the kinase domain,releasing it from an inactive conformation (Harper andHarmon, 2005; Wernimont et al., 2010).

A recent description of the conditional knockdown ofTgCDPK1 has validated this kinase as a key mediator ofthe signalling cascade leading to microneme secretion(Lourido et al., 2010). Because of a severe defect inmicroneme secretion, tgcdpk1iko parasites fail to attach,invade or egress host cells. Moreover, the fact that theseparasites are unable to egress when treated with calciumionophores indicates that TgCDPK1 acts downstream ofthe calcium signal that regulates microneme exocytosis(Lourido et al., 2010). TgCDPK1 substrates remainunknown for the moment. Microneme exocytosis is alsoregulated by the cyclic guanosine 3′, 5′-cyclic monophos-phate (cGMP) pathway. Inhibition of cGMP-dependentprotein kinase (PKG) with a selective inhibitor impairsP. berghei ookinete gliding motility (Moon et al., 2009) andT. gondii attachment, invasion and gliding (Wiersma et al.,

788 J. M. Santos and D. Soldati-Favre

© 2011 Blackwell Publishing Ltd, Cellular Microbiology, 13, 787–796

2004). Intriguingly, PKG activity is independent of CDPK1function in T. gondii (Wiersma et al., 2004; Lourido et al.,2010) but in P. berghei the cGMP- and calcium-dependentpathways of microneme exocytosis converge. Indeed,deletion of a cGMP-degrading enzyme (phosphodi-esterase) in the strain deleted for PbCDPK3, which isaffected in ookinete movement (Siden-Kiamos et al.,

2006), restored a normal motility phenotype (Moon et al.,2009). PKG has also been implicated in secretion of Plas-modium exonemes (Dvorin et al., 2010a), which dis-charges a protease required for parasite egress from thehost erythrocyte (Yeoh et al., 2007). PfCDPK5 is alsoinvolved in egress but appears to act downstream in thesignalling cascade (Dvorin et al., 2010a). The cyclic

Fig. 1. Regulated secreted proteins and proteases: key players in the mechanism of cell invasion.A. The rhomboid proteases TgROM4 and TgROM5 are expressed at the parasite’s plasma membrane but whereas TgROM4 is ubiquitouslydistributed (light blue), TgROM5 concentrates at the posterior end of the parasite (purple). The transmembrane micronemal proteins TgMIC2(red), TgMIC6 (orange) and TgMIC8 (violet) are stored in the micronemes (red) already assembled in complexes with other micronemalproteins. TgAMA1 (pink) is also stored in the micronemes. In the rhoptries are stored the TgRONs, at the rhoptry necks (dark green), and theTgROPs, at the rhoptry bulbs (light green). The parasite nucleus, ER and Golgi (in dark blue), the conoid (yellow) and the inner membranecomplex (dark grey) are also shown.B. Upon a calcium signal, the TgMICs are discharged from the micronemes in the form of complexes (not shown), onto the parasite surface.At the time of invasion, they form complexes with host cell receptors via binding with their ectodomains.C. Formation of the complexes TgMICs-host cell receptors signals for exocytosis of the rhoptries. While the TgROPs migrate to either the PV(in yellow) or to the host cell nucleus (blue), the TgRONs are discharged onto the parasite surface and, in some cases, anchor to the host cellmembrane. The TgRONs associate with TgAMA1 and form the MJ complex. The TgMICs are shed from the parasite surface viaTgROM4-mediated proteolytic cleavage within the transmembrane domain. Cleavage promotes host cell penetration and the formation of agradient of adhesins from the anterior to the posterior end of the parasite.D. At the end of the penetration process, TgROM5 cleaves the remaining surface localized TgMICs and allows for PV closure anddisengagement of the parasite from the host cell surface. TgROM4-mediated cleavage of TgAMA1 releases the TgAMA1 cytosolic domain,which hypothetically interacts with an unknown associated protein and promotes transcription of genes implicated in parasite replication.

Regulated secretion and signalling events 789


adenosine 3′, 5′-cyclic monophosphate (cAMP) pathwayis also involved in organelle exocytosis in Plasmodiumsporozoites and merozoites by a mechanism dependenton protein kinase A (PKA) (Beraldo et al., 2005; Onoet al., 2008; Leykauf et al., 2010). The mobilization ofcalcium induced by cAMP is also seen upon stimulation ofinositol 1,4,5-triphosphate production via the phospholi-pase C (PLC) pathway, suggesting that the cAMP andPLC pathways are linked (Beraldo et al., 2005).

The RONs and rhoptry bulb proteins: two differentfamilies of rhoptry proteins

The rhoptry organelles store lipids and proteins that canbe found either at the neck (RONs) or at the bulb of theorganelle (rhoptry bulb proteins, ROPs) (Fig. 1A). TheROPs and RONs can be distinguished not only in terms oftheir localization in the organelle but also by the timing ofdischarge and their ultimate destination and function.

The RONs have been associated so far with the forma-tion of the MJ (Fig. 1) together with AMA1, both in T.gondii tachyzoites (Alexander et al., 2005) and in P. falci-parum merozoites (Cao et al., 2009; Collins et al., 2009).In Toxoplasma the MJ complex is composed of TgRON2,TgRON4, TgRON5 and TgRON8 (Alexander et al., 2005;Besteiro et al., 2009; Collins et al., 2009). In Plasmodium,RON8 is absent and PfRON2, PfRON4 and PfRON5 forma pre-complex in the rhoptries, which is then deliveredonto the parasite’s surface (Collins et al., 2009) where itbinds to AMA1 via RON2 (Alexander et al., 2005; Besteiroet al., 2009; Collins et al., 2009; Tyler and Boothroyd,2011). Only a minority of AMA1 present on the parasitesurface associates with the RONs at the MJ (Alexanderet al., 2005; Collins et al., 2009; Tyler and Boothroyd,2011). The exact function of the MJ complex is still amatter of debate but it is known to be required only afterparasite reorientation and establishment of the initial tightjunction (Richard et al., 2010). In T. gondii the RONscomplex is targeted to the host cell membrane duringinvasion and one hypothesis is that it effectively serves asthe parasite’s own receptor at the host cell surface,perhaps contributing to the ability of T. gondii to invade awide range of cell types (Besteiro et al., 2009). It shouldbe noted however that Plasmodium, which is capable ofinvading a much more restricted host cell repertoire, alsopossesses the AMA1-RONs complex (Cao et al., 2009;Collins et al., 2009).

Unlike the known RONs, which are restricted to the MJ,the ROPs are delivered either to the PV or into the hostcell (Fig. 1C) (Carruthers and Sibley, 1997; Alexanderet al., 2005). The largest family of Toxoplasma ROPgenes encodes at least 44 kinases and pseudokinases,including 16 predicted to be active (Peixoto et al., 2010),and some of them have been shown to be involved in

post-invasion manipulation of host cell signalling[reviewed in (Pollard et al., 2009)]. The rhoptries alsorelease phosphatases, proteases and other proteinsof unknown function [reviewed in (Boothroyd andDubremetz, 2008)]. Additionally the dense granules alsocontribute to the discharge of proteins capable of actingas effectors and subverting host functions. A recentexample is TgGRA15, which activates the NF-kB pathway(Rosowski et al., 2011).

Signalling for rhoptry secretion is coupled toMIC function

In Plasmodium, recent studies have indicated that signal-ling for rhoptry secretion is mediated by binding of theparasite micronemal erythrocyte binding antigen-175(PfEBA-175) or -140 (PfEBA-140) to their respective hostreceptors (Singh et al., 2010) (Fig. 1C). The current modelfor rhoptry secretion advocates that adhesin-receptorbinding restores the basal calcium levels, which wereelevated to induce microneme discharge, through a signaltransmitted by the C-terminal tails of the PfEBA familymembers (Singh et al., 2010). The exact signal transduc-tion mechanism remains to be discovered.

In Toxoplasma both TgAMA1 (Mital et al., 2005) andTgMIC8 (Kessler et al., 2008) are involved in rhoptrysecretion. In parasites conditionally depleted in TgAMA1there is still secretion of the TgRONs (Alexander et al.,2005) but release of the TgROPs is impaired (Mital et al.,2005). In contrast, a complete abrogation of rhoptrysecretion is observed in the absence of TgMIC8 (Kessleret al., 2008). Curiously, inhibition of T. gondii phospholi-pase A2 blocks rhoptry secretion in a similar manner toTgMIC8 absence (Ravindran et al., 2009). The exact sig-nalling mechanism remains unknown but it is plausiblethat the binding of TgMIC8 to an unknown host cell recep-tor signals for secretion of the TgRONs and, subsequentlyTgAMA1 association with the TgRON complex triggersTgROP secretion. Such a model implies physical discon-tinuity between the necks and the bulbs of the rhoptries,which remains to be assessed, and interference with theTgAMA1 role during invasion does not impair TgROPinjection into the host cell (Tyler and Boothroyd, 2011).

MICs are proteolytically processed during invasion

The TgMICs undergo extensive proteolytic remodelling.The first cleavage event removes the signal peptide andsubsequently a series of other cleavages occur along thesecretory pathway (Dowse and Soldati, 2004; Carruthers,2006). Some TgMICs also contain propeptides that areremoved by a cathepsin-like protease (Parussini et al.,2010). Further proteolytic processing events occur postexocytosis at the parasite surface involving three distinct



enzymatic activities referred to as micronemal proteinproteases -1, -2 and -3 (MPP1, MPP2 and MPP3)(Carruthers et al., 2000; Zhou et al., 2004). While MPP2and MPP3 perform the so-called surface trimming, medi-ated by the micronemal subtilisin-like protease TgSUB1(Lagal et al., 2010), MPP1 mediates shedding ofthe transmembrane TgMICs from the parasite surface(Fig. 1C).

MPP1 is a constitutively active, resident plasma mem-brane protease (Opitz et al., 2002), which activity appearsto be controlled by compartmentalization of the substratesin the micronemes. MPP1 is an essential and likely con-served proteolytic activity across the Apicomplexa phylum(Opitz et al., 2002) but its biological function remains to beelucidated. It has been suggested that MPP1: (i) shedsMICs from the parasite surface so that they cannotbe targeted by neutralizing antibodies (Carruthers andBoothroyd, 2007); (ii) creates a gradient of MICs from theanterior to the posterior end of the parasite, contributing toparasite reorientation (Buguliskis et al., 2010); (iii) disen-gages the parasite from the host cell at the end of theinvasion process; or (iv) assures proper function of theglideosome by releasing the MIC-host cell receptor com-plexes during movement (Carruthers and Boothroyd,2007).

MPP1 was shown to cleave TgMIC2, TgMIC6, TgMIC12and TgAMA1 within their transmembrane domain (Opitzet al., 2002; Brossier et al., 2003; Urban and Freeman,2003; Zhou et al., 2004; Howell et al., 2005) at aminoacid motifs reminiscent of those targeted by rhomboid-like serine proteases (Dowse et al., 2005; Amarneh andRawson, 2009; Strisovsky et al., 2009). While T. gondiiexpresses two rhomboid-like proteases (TgROM4 andTgROM5) at the plasma membrane (Fig. 1A), the malariaparasite only possesses a ROM4 homologue (Brossieret al., 2005; Dowse et al., 2005; O’Donnell et al., 2006;Sheiner et al., 2008). Cell-based cleavage assays havevalidated TgROM5 (Brossier et al., 2005; Dowse et al.,2005) and PfROM4 (Baker et al., 2006; O’Donnell et al.,2006) as active enzymes with broad substrate specificity(Baker et al., 2006) but TgROM4 was either inactive ornot expressed in this context (Brossier et al., 2005; Dowseet al., 2005). However, the recent conditional knockdownof TgROM4 demonstrated that this protease is active onthe parasite surface and likely cleaves TgMIC2, TgAMA1and TgMIC8 (Buguliskis et al., 2010). TgROM4 deple-tion caused ª 50% reduction in invasion and did notaffect TgMIC6 shedding, despite the sequence similaritybetween TgMIC2 and TgMIC6 rhomboid cleavage motifs.These results suggest that the MPP1 activity in Toxo-plasma may be mediated by two rhomboid proteases withdistinct substrate specificities (Buguliskis et al., 2010). Anew model for MPP1 activity can thus be envisioned inwhich TgROM4 and TgROM5 both perform important and

not necessarily overlapping functions. As TgROM4 isevenly distributed over the parasite surface, it might con-tribute to the creation of the gradient of adhesins and toglideosome function (Fig. 1C), as recapitulated by thephenotype of the tgrom4iko parasites (Buguliskis et al.,2010). At the end of penetration, TgROM5, which prefer-entially localizes to the posterior end of the parasite, mightexecute the final pinching activity during closure of the PVand disengagement of the parasite from the host cell(Fig. 1D) (Brossier et al., 2005; Dowse et al., 2005). Vali-dation of TgROM5 as mediator of some aspects of MPP1activity awaits further investigation. In Plasmodium, onlyPfROM4 is expressed at the parasite surface (O’Donnellet al., 2006) but the parasite also possesses a subtilisin-like enzyme named PfSUB2, which acts as a surfacesheddase during invasion (Howell et al., 2005).

Coupled functions of TgROM4 and TgAMA1 ininvasion and replication

In parallel to the study of TgROM4 with a conditionalknockout (Buguliskis et al., 2010), TgROM4 functionwas investigated by generating of transgenic parasitesexpressing an inducible dominant negative TgROM4form. The protease carrying a mutation in the catalyticserine (ddROM4S-A) was predicted to sequester its sub-strate(s) from the active endogenous TgROM4 copy(Santos et al., 2010) (Fig. 2A). Expression of ddROM4S-A

was highly deleterious to parasite survival but surprisingly,it did not inhibit invasion but severely blocked para-site replication (Santos et al., 2010). Such effect couldbe specifically reverted by expression of the predictedTgAMA1 rhomboid cleavage product (TgAMA1-tail), sug-gesting that TgROM4-mediated cleavage of TgAMA1 isnot essential for invasion but switches instead the para-site from an invasive to a replicative mode (Santos et al.,2010). Rhomboid proteases have been previously asso-ciated with intercellular signal transduction events butthis is the first time that a rhomboid protease has beenshown to contribute to an intracellular signalling pathway[reviewed in (Urban, 2006)]. The TgAMA1-tail is alsoessential for invasion (Treeck et al., 2009; Leykauf et al.,2010), possibly by linking the MJ complex to the glideo-some via binding to aldolase (Sheiner et al., 2010), butthe amino acid motif implicated in this function is notrequired for replication, indicating that the two functionsare physically separated (Santos et al., 2010). TgROM4-mediated cleavage of TgMIC2 is essential for glidingmotility (Huynh and Carruthers, 2006; Buguliskis et al.,2010), but expression of TgMIC2-tail did not trans-complement the phenotype of ddROM4S-A parasites(Santos et al., 2010). Noteworthy, unlike TgMIC2 and theother MICs, TgAMA1 is present on the surface of invadedparasites (Howell et al., 2005). These observations are



Fig. 2. AMA1 and ROM4: coupled roles in invasion and replication.A. Scheme representing the hypothetical mode of action of the TgROM4 dominant negative form ddROM4S-A. In the presence of Shld-1,ddROM4S-A (yellow) is stabilized and recognizes and binds to ROM4 substrates (red) but it is unable to cleave them, because of the Alareplacement at the catalytic Ser residue (orange), leading to substrate sequestration from the active endogenous TgROM4 protease (blue)and to a dominant negative effect.B. ROM4-mediated cleavage of AMA1, at or following invasion, releases the AMA1 cytosolic C-terminal fragment (AMA1-tail), triggeringparasite replication. In Toxoplasma, further cleavage of AMA1 or another ROM4 substrate are needed in order to stimulate each new cycle ofreplication by endodyogeny.C. In wild-type extracellular parasites (RH), the protein levels of AMA1 (A) and ROM4 (R) are balanced and ROM4-mediated cleavage ofAMA1 and other ROM4 substrates promotes host cell invasion and triggers parasite replication. In intracellular parasites, there is very littleAMA1 expressed and ROM4 is in excess. In parasites expressing an inducible copy of wild-type ROM4 (ddROM4), ROM4 is in excess in bothextracellular and intracellular parasites because there is expression of endogenous (black R) and inducible ROM4 (green R). In parasitesexpressing an inducible mutant copy of ROM4 (ddROM4S-A), expression of the mutant copy (red R) in extracellular parasites is not sufficient toabrogate cleavage of all expressed AMA1 and the parasites can efficiently invade host cells. In intracellular parasites, on the other hand, thereis very little AMA1 expressed and mutant ROM4 can efficiently compete with the endogenous ROM4 for substrate binding, blocking parasitereplication. In parasites knockdown for ROM4 (rom4-iKO), there is very little ROM4 expressed in both intracellular and extracellular parasites.The reduced amount of ROM4 substrates shedding impairs host cell invasion but still releases enough AMA1-tail to trigger parasite replication.In parasites knockdown for AMA1 (ama1-iKO), there is very little AMA1 expressed in both intracellular and extracellular parasites. The reducedamount of AMA1 protein expressed in extracellular parasites dramatically impairs invasion but, as expression is not completely shut down,parasites are only modestly affected in replication.D. In Plasmodium, a single ROM4-mediated cleavage of AMA1 during invasion assures triggering of replication by schizogony.



especially relevant in the context of parasite migration/traversal of tissues as replication should only be triggeredpost invasion and not during pre-invasion motility.TgAMA1 appears well suited to participate in post-invasion events along with other integrated signals gen-erated exclusively after invasion to trigger replication andguarantee the necessary discrimination between migra-tion and invasion.

The dominant negative effect exerted by ddROM4S-A isreversible and can be uncoupled from invasion, suggest-ing that each cycle of replication requires a new boost ofsignalling engendered by substrate cleavage (Fig. 2B).Such a phenomenon appears to be tailored to the lyticcycle of T. gondii, which involves several cycles ofparasite division by endodyogeny. At the end of eachreplication cycle, the newly produced parasites are fullycompetent for egress and invasion and therefore a deci-sion regarding whether it should be initiated egress or anew round of replication needs to be made. At present, itremains unclear if TgAMA1 cleavage is indeed requiredfor all subsequent cycles of replication or whether there isinvolvement of other TgROM4 substrates. In this context,the presence in the T. gondii genome of two additionalgenes related to TgAMA1 and described as putativeapical membrane antigens in ToxoDB (TGME49-115730and TGME49-100130) might be of relevance.

How TgAMA1 cleavage may trigger replication remainsunknown. Regulated intramembrane proteolysis is asimple and powerful strategy for signal transduction inwhich the substrates are membrane-anchored proteinsand inactive in their membrane-tethered form. Activationoccurs upon cleavage within the transmembrane domain,through release of their cytosolic or extracellular domains(Brown, 2000; Urban et al., 2002; Erez and Bibi, 2009;Freeman, 2009). It is tempting to speculate that TgROM4is responsible for a mechanism resembling regu-lated intramembrane proteolysis acting on TgAMA1 thatreleases its cytosolic ‘tail’ to initiate a program of signaltransduction (Fig. 1D), involving unknown proteins onlypresent in intracellular parasites, that ultimately leads toexpression of genes involved in parasite replication. Forthe moment this hypothesis is only speculative but in itssupport, it was recently shown that an invasion-relatedevent triggers an immediate and sharp change in geneexpression in Toxoplasma (Gaji et al., 2010).

A role for TgROM4 and TgAMA1 during replication wasnot observed in parasites conditionally depleted for eitherthe protease or the MIC (Mital et al., 2005; Buguliskis et al.,2010) likely because parasites grown under repressiveconditions still express background levels of TgROM4 andTgAMA1 respectively (Fig. 2C). The use of alternativeexperimental approaches was thus instrumental for theidentification of the dual functions of TgROM4 andTgAMA1 in invasion and parasite replication. The tet-

inducible system established that TgROM4 and TgAMA1are important players during motility and invasion (Mitalet al., 2005; Buguliskis et al., 2010). However, althoughthe knockdown strategy efficiently reduced expressionof TgROM4 at the surface of extracellular parasitesand consequently the shedding of the TgMICs, includingTgAMA1, during invasion, it failed to completely blockcleavage in intracellular parasites and subsequently repli-cation (Buguliskis et al., 2010). We hypothesize that theseapparent discrepancies may be explained by the possibilitythat expression of ddROM4S-A is insufficient to sequesterall the substrates released from the micronemes duringinvasion and therefore to produce a strong invasionphenotype. Expression of ddROM4S-A is also insufficientto block the first round of division following invasion, butit effectively neutralizes the limited amount of sub-strate delivered by intracellular parasites and thus blockssubsequent cycles of replication (Santos et al., 2010).

Although the P. falciparum AMA1-tail trans-complements the division defect imposed by expressionof ddROM4S-A, it is premature to conclude that a similarmechanism dictates initiation of the replication program inmalaria parasites. Unlike TgAMA1, which is exclusivelycleaved by a rhomboid-like activity (Howell et al., 2005),PfAMA1 is primarily shed from the merozoite surface byjuxtamembrane cleavage by the subtilisin-like proteasePfSUB2 (Howell et al., 2005). Indeed PfAMA1 mutationsrendering it uncleavable by PfROM4 has no effect onparasite viability (C. Collins and M. Blackman, pers.comm.). As the malaria parasite replicates by schizogony,a process by which cytokinesis to produce all daughtercells occurs only once in each blood-stage cycle, it mightbe speculated that the cleavage of PfAMA1 by PfSUB2during invasion may be sufficient to trigger the entirereplication cycle (Fig. 2D). This idea is supported by dataindicating that stable parasite lines carrying PfAMA1forms resistant to PfSUB2 cleavage cannot be obtainedeven if these forms are functional in invasion (C. Collinsand M. Blackman, pers. comm.). The cleaved form ofPfAMA1 is phosphorylated at a Ser residue by PKA(Leykauf et al., 2010) and this modification is important forPfAMA1 function during invasion (Treeck et al., 2009).However, the PfAMA1-tail carried into the newly invadedring stages appears not to be phosphorylated (Leykaufet al., 2010) raising the possibility that phosphorylationcould distinguish between function during invasion andreplication. Nevertheless, expression of the PfAMA1 tailmutated at the phosphorylated Ser is still able to restorereplication of ddROM4S-A parasites (Santos et al., 2010).

Conclusion

Recent years have brought a new light into the mecha-nisms implicated in the establishment of infection by both



Toxoplasma and Plasmodium. While some aspects areconserved, others are specific to each parasite. The sig-nalling pathways leading to micronemes, and most likelyto rhoptries discharge, as well as function of conservedMICs appear to be conserved. Several studies have high-lighted how the modular structure of the MICs allows themto perform multiple tasks. The ectodomains interact withhost receptors and contribute to host cell recognition andinvasion, while the cytosolic tails may carry traffickinginformation and link the host cell surface to the parasiteglideosome or are implicated in downstream signallingevents. For the moment, such dual functions have onlybeen demonstrated for TgAMA1 and TgMIC8 in T. gondii(Mital et al., 2005; Kessler et al., 2008; Santos et al.,2010) and PfEBA-140 and PfEBA-175 in P. falciparum(Singh et al., 2010), but recent studies indicate that otherMICs may mediate similar functions (Dvorin et al., 2010b).The function of ROPs in modulating the host cell responsehas only been demonstrated for Toxoplasma but the pres-ence of the RONs at the MJ is well characterized in bothToxoplasma and Plasmodium. Proteolytic processing ofthe MICs, while involving different players, is also largelyconserved in both parasites. The new findings discussedhere add a novel layer of complexity to the mechanismsleading to the establishment of infection by apicomplexanparasites and reinforce the notion of a concerted action ofinvasion proteins, which is tightly controlled in time andspace and ultimately governs replication.

Acknowledgements

We are most grateful to Dr Michael J. Blackman stimulatingdiscussions and for critical reading of the manuscript. JMS waspreviously funded by MalParTraining FP6-Marie Curie ITN(MEST-CT-2005-020492) and currently by the Swiss NationalFoundation (FN3100A0-116722).

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