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'The glideosome': a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii

OPITZ, Corinna, SOLDATI-FAVRE, Dominique

Abstract

Motion is an intrinsic property of all living organisms, and each cell displays a variety of shapes and modes of locomotion. How structural proteins support cellular movement and how cytoskeletal dynamics and motor proteins are harnessed to generate order and movement are among the fundamental and not fully resolved questions in biology today. Protozoan parasites belonging to the Apicomplexa are of enormous medical and veterinary significance, being responsible for a wide variety of diseases in human and animals, including malaria, toxoplasmosis, coccidiosis and cryptosporidiosis. These obligate intracellular parasites exhibit a unique form of actin-based gliding motility, which is essential for host cell invasion and spreading of parasites throughout the infected hosts. A motor complex composed of a small myosin of class XIV associated with a myosin light chain and a plasma membrane-docking protein is present beneath the parasite's plasma membrane. According to the capping model, this complex is connected directly or indirectly to transmembrane adhesin complexes, which are delivered to the parasite surface upon microneme [...]

OPITZ, Corinna, SOLDATI-FAVRE, Dominique. 'The glideosome': a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii. Molecular Microbiology , 2002, vol. 45, no. 3, p. 597-604

PMID : 12139608

DOI : 10.1046/j.1365-2958.2002.03056.x

Available at:

http://archive-ouverte.unige.ch/unige:78031

Disclaimer: layout of this document may differ from the published version.

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MicroReview

‘The glideosome’: a dynamic complex powering gliding motion and host cell invasion by Toxoplasma gondii

apical structures, including the conoid, the polar ring complex, subpellicular microtubules and secretory organelles including micronemes, rhoptries and dense granules (Morrissette and Sibley, 2002). The pellicle of the invasive forms of these parasites consists of the plasma membrane and the two membranes of the inner mem- brane complex (IMC), a continuous layer of flattened vesi- cles underlying the plasma membrane (Fig. 1). This apical complex and the IMC actively contribute to parasite motion, a process that appears to a large extent to be mechanistically conserved across the phylum. In the absence of locomotive organelles such as cilia or flagella, the invasive forms of these parasites have an unusual form of substrate-dependent motility that is essential for host cell invasion (Sibley et al., 1998). This latter process involves the invagination of the host cell plasma mem- brane to form a parasitophorous vacuole around the parasite. The parasite maintains a fixed shape and is pro- pelled forwards by a spiral movement. Early studies report the movement of proteins on the parasite surface, sug- gesting that a ‘capping model’ (Dubremetz and Ferreira, 1978; Russell and Sinden, 1981) would be best suited to explain the mechanism of gliding motility and host cell invasion by Apicomplexa. According to this model, para- site motility is driven by the redistribution of transmem- brane proteins from the apical pole along the subcortical actomyosin complex towards the posterior end of the par- asite. A recent very comprehensive review recapitulates the history of this model and reports the most recent progress in understanding the mechanism of invasion (Menard, 2001). At least three distinct classes of factors (cytoskeletal structures, ligands and regulatory mole- cules) contribute to the formation and dynamics of the gliding complex.

Toxoplasma gondiicytoskeleton

Although the integrity of the subpellicular microtubules is essential for invasion (Morrissette and Sibley, 2002), the basic engine for gliding locomotion is the parasite’s actin cytoskeleton (Dobrowolski and Sibley, 1996), which involves myosin(s) to generate the mechanochemical force along the actin filaments (Dobrowolski et al., 1997).

Actin filaments are not detectable under physiological Corinna Opitz1and Dominique Soldati2*

1Robert Koch-Institut, 13353 Berlin, Germany.

2Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.

Summary

Motion is an intrinsic property of all living organisms, and each cell displays a variety of shapes and modes of locomotion. How structural proteins support cellular movement and how cytoskeletal dynamics and motor proteins are harnessed to generate order and movement are among the fundamental and not fully resolved questions in biology today. Protozoan parasites belonging to the Apicomplexa are of enor- mous medical and veterinary significance, being responsible for a wide variety of diseases in human and animals, including malaria, toxoplasmosis, coc- cidiosis and cryptosporidiosis. These obligate intra- cellular parasites exhibit a unique form of actin-based gliding motility, which is essential for host cell inva- sion and spreading of parasites throughout the in- fected hosts. A motor complex composed of a small myosin of class XIV associated with a myosin light chain and a plasma membrane-docking protein is present beneath the parasite’s plasma membrane.

According to the capping model, this complex is connected directly or indirectly to transmembrane adhesin complexes, which are delivered to the para- site surface upon microneme secretion. Together with F-actin and as yet unknown bridging molecules and proteases, these complexes are among the structural and functional components of the ‘glideosome’.

‘A capping model’ to accommodate motility and host cell invasion

Members of the Apicomplexa are obligate intracellular parasites, morphologically unified by a common set of

Accepted 29 April, 2002. *For correspondence. E-mail d.soldati@

ic.ac.uk; Tel. (+44) 20 759 45342; Fax (+44) 20 758 42056.

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conditions, and the artificial formation of acrosome-like structures has only been visualized after treatment with jasplakinoide (Shaw and Tilney, 1999). At lower doses, this drug appears to change the velocity and orientation of parasite movement, suggesting that actin polymeriza- tion is a rate-limiting factor for gliding motion (Morrissette and Sibley, 2002). The dynamic process of both actin polymerization and rapid depolymerization is important for gliding motility. The abundant actin-depolymerizing factor (ADF) is localized to the cytoplasm predominantly beneath the plasma membrane and most likely causes destabilization of actin filaments (Allen et al., 1997). Its role might be reinforced by the action of toxofilin, a molecule sequestering G-actin and capping filament ends (Poupel et al., 2000) (Fig. 2C).

Using time-lapse video microscopy, the motility of Toxoplasmacould be divided into three different modes:

circular and helical gliding for forward movement and clockwise spinning at a fixed point (Hakansson et al., 1999). The directionality and helical nature of the motility strongly suggest a connection between the actomyosin system and the helical organization of the subpellicular microtubules. This connection possibly occurs indirectly

via the linear arrays of intramembranous particles (IMPs) present in the outer face of the IMC. The IMPs are indeed arranged in double rows matching the underlying subpel- licular microtubules (Morrissette et al., 1997).

Myosin A: a molecular motor powering motility Myosins are actin-based motors forming a very large superfamily of proteins. Besides the well-characterized, conventional myosins-II, 15 classes of unconventional myosins have been described (Mermall et al., 1998).

Five T. gondiiand four Plasmodium myosins have been identified so far. Except for the two large Plasmodium falciparum myosins, PfMyoC and PfMyoD, these small unconventional myosins are the founders of a novel phylo- genetic and structural class of myosin (XIV) restricted to the Apicomplexa (Heintzelman and Schwartzman, 1997).

Toxoplasma gondiiMyoA homologues are also expressed in other Apicomplexa, including Neospora caninum, Cryp- tosporidium parvum and Eimeria tenella. MyoA and its closest homologues in Plasmodium species have been localized beneath the plasma membrane of the parasites, a suitable localization to sustain gliding motility (Pinder

© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 597–604 598 C. Opitz and D. Soldati

Fig. 1. Electron micrograph of an intracellular tachyzoite of T. gondiishowing the pellicle with the inner membrane complex (IMC) and plasma membrane (PM). The secretory organelles include the rhoptries (R), the micronemes (M) and the dense granules (DG). The conoid is positioned at the apical pole (C). This figure is reprinted from J. F.

Dubremetz. Host cell invasion by Toxoplasma gondii. Trends Microbiol 6: 27–30. Copyright (1998), with permission from Elsevier Science.

Fig. 2. Model for gliding motility and the formation and dissolution of the ‘glideosome’.

A. The motor complex is composed of myosin A (MyoA), the myosin light chain 1 (MLC1) and the MyoA docking protein (MADP) and is permanently anchored in the plasma membrane. The microneme protein protease 1 (MPP1) activity appears to be constitutively present on the parasite surface. Actin monomers (globular actin, G-actin) are abundant.

B. Upon external stimulation, microneme protein complexes composed of adhesins and escorters are secreted and redistributed to the surface of the parasites. They are presumed to establish a dual interaction with receptors and structures on the surface of the host cell via their adhesive domains and with the parasite actomyosin via the cytoplasmic tail of transmembrane microneme proteins. The current model predicts the existence of an as yet unknown (?) bridging molecule (factor X) that links the two complexes. Actin polymerization and myosin A crawling on filamentous actin scaffold (F-actin) contribute to the creation of movement. The actomyosin system needs to be anchored in the inner membrane complex and connected to the subpellicular microtubules possibly by the intermediate of the intramembranous particles (IMPs).

C. Actin depolymerization occurs fast under the action of the abundant actin-depolymerizing factor (ADF) and possibly toxofilin (T). The microneme protein proteases 1 and 2 (MPP1, MPP2) dissolve the complexes by proteolytic cleavage of microneme proteins and release them from the parasite surface.

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Plasma membrane

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et al., 1998; Hettmann et al., 2000; Margos, 2000;

Matuschewski et al., 2001). The functional MyoA motor complex was purified from exchange recombinant par- asites to recombinant T. gondii to demonstrate that the biochemical and biophysical properties of this motor are compatible with parasites gliding at 3–5mm s-1(Herm-Götz et al., 2002). Transient kinetic analysis showed that ATP rapidly dissociates MyoA–actin complexes. This motor shows a low affinity for ADP, a property diagnostic of a fast myosin. Motility and microneedle/laser trap assays estab- lished that MyoA moves in unitary steps of 5.3 nm with a velocity of 5.2mm s-1and towards the plus (barbed) end of actin filaments. MyoA is the first example of a fast, single- headed myosin. Disruption of the MyoA gene was achieved recently using a tet-inducible system to generate a conditional knock-out for this gene. Depletion in MyoA impairs both host cell invasion and parasite egress (M.

Meissner, D. Schlüter and D. Soldati, submitted). As the inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime) disrupted the three forms of T. gondiigliding motility (Hakansson et al., 1999), parasite movement might involve more than one myosin.

Two other T. gondiimyosins, MyoD and E, are very similar to MyoA, with MyoD having very similar transient kinetics of a fast-moving motor. MyoD and MyoE are predominantly expressed in bradyzoites and might fulfil a similar role to MyoA in other life stages. Unlike MyoA, MyoB and MyoC are larger myosins encoded by two alternatively spliced mRNA variants that appear to play a role in plasma mem- brane integrity during cell division (Delbac et al., 2001). In P. falciparum, two small myosins, PfMyoA and PfMyoB, are similar to the TgMyoA. Although PfMyoA is submembra- nous and expressed in the different invasive life stages, no data are available for PfMyoB.

The MyoA motor complex is anchored in the pellicle

MyoA is not recruited at the plasma membrane at the time of invasion, but is permanently and tightly associated with the plasma membrane, ready to come into action at any time. The last few amino acids within the MyoA tail, includ- ing two basic residues, are essential for its membrane localization (Hettmann et al., 2000). A recent study reported the characterization of a coiled-coil protein serving as a docking protein for MyoA in the pellicle (MADP) (C. Beckers, International Congress on Toxo- plasmosis, Freising, Germany, 2001). MADP is most probably anchored in the plasma membrane by N- terminal myristoylation (C. Beckers, personal communi- cation). The topology of actin and myosin at the plasma membrane and with respect to the inner membrane complex was examined previously by immunoelectron microscopy (Dobrowolski et al., 1997). A double immuno- fluorescence using specific antibodies recognizing MyoA and the inner membrane protein IMC1 (Mann and Beckers, 2001) provided additional evidence for anchor- ing of MyoA in the plasma membrane (Fig. 3). The overlay clearly shows that the staining for MyoA covers the entire surface of the parasite, including the poles, whereas the staining for the IMC is interrupted. In addition to MADP, a calmodulin-like myosin light chain of 30 kDa, MLC1, was recently shown to be associated with MyoA. These three proteins belong to the functional motor complex that can be purified from the parasites (Herm-Götz et al., 2002) (Fig. 2A). Like MyoA, MADP and MLC1 are extremely conserved throughout the Apicomplexa. In contrast to several unconventional myosins from other organisms, MyoA does not bind to calmodulin (CaM) as a light chain.

The deletion of the last 53 residues in the tail of MyoA

© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 597–604 600 C. Opitz and D. Soldati

Fig. 3. Topology of TgMyoA beneath the plasma membrane. Double immunofluorescence by confocal microscopy of intracellular T. gondii tachyzoites using antibodies recognizing MyoA and the inner membrane complex associated protein, IMC1. The parasites inside the parasitophorous vacuole are arranged in rosettes. The anterior and posterior poles of the parasites are only stained for MyoA (arrows in the overlay panel indicate the apical pole of the parasites), whereas the IMC staining is interrupted at the level of the conoid.

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eliminates interaction with the MLC1 and MADP proteins (Herm-Götz et al., 2002). As the only very degenerated IQ motif present in the last 53 amino acids of MyoA over- laps with the determinant for plasma membrane localiza- tion, the precise topology within the complex remains to be determined.

Discharge of adhesin complexes

By successive regulated exocytosis, micronemes and rhoptries release their contents as part of the invasion process (Carruthers and Sibley, 1997). Micronemal pro- teins are involved in host cell recognition, binding and, possibly, motility, whereas the contents of the rhoptries participate in parasitophorous vacuole formation (Hakansson et al., 2001) and association with host cell organelles (Sinai and Joiner, 2001). In T. gondii, microneme discharge can be stimulated in vitro by calcium ionophore or ethanol (Carruthers and Sibley, 1999; Carruthers et al., 1999a). Proteins secreted by micronemes share extensive domains of homology between members of the Apicomplexa, supporting the hypothesis of a common molecular mechanism for host recognition, attachment and invasion (Tomley and Soldati, 2001). Currently, a repertoire of 14 microneme proteins has been identified in T. gondii.Some of these proteins (MIC1, MIC3 and MIC4) possess extracellular adhesive domains but lack a transmembrane domain. It has been shown that they bind to host cells, probably to receptors or structures at the surface of the cell that remain to be identified (Fourmaux et al., 1996; Brecht et al., 2000;

Cérède et al., 2002). In contrast, MIC2 and the members of a novel family of proteins, MIC6–9, containing multiple epidermal growth factor (EGF)-like motifs have a trans- membrane-spanning domain and a short cytoplasmic tail (Meissner et al., 2002). MIC6 and MIC8 are expressed in the tachyzoites, whereas MIC7 and MIC9 genes are predominantly expressed in bradyzoites (Meissner et al., 2002). The analysis of mic6 knock-out parasites uncov- ered the particular role of MIC6 as an escorter for the two soluble adhesins MIC1 and MIC4 (Reiss et al., 2001).

More recently, the existence of two additional complexes secreted by the micronemes has confirmed that this is a general phenomenon. MIC8 serves as an escorter for the soluble adhesin MIC3 (Meissner et al., 2002), and the adhesin MIC2 is bound to M2AP (MIC2-associated protein) (Rabenau et al., 2001). The disruption of MIC2 by double homologous recombination caused the mis- sorting of M2AP to the dense granules and para- sitophorous vacuole (C. Opitz, V. Carruthers and D.

Soldati, unpublished). The existence of stable complexes between microneme proteins provides an attractive expla- nation as to how soluble, regulated secretory proteins can be sorted and how soluble adhesins could contribute to

the invasion process (Soldati et al., 2001; Carruthers, 2002) (Fig. 2B).

Bridging molecules between adhesins and motor complexes and IMC

At the point of invasion, a moving junction is formed between the parasite and the host cell plasma membrane.

At this site, proteins are segregated depending on their anchoring in the membrane, leading to the exclusion of host cell transmembrane proteins and not GPI-anchored proteins from the forming parasitophorous vacuole (Mordue et al., 1999). During invasion, the complex of MIC2 and M2AP moves together with the moving junction towards the posterior pole (Carruthers et al., 1999b; Car- ruthers, 2002). Additionally, MIC3, which is escorted by the transmembrane MIC8, was also documented to be released from the micronemes at the apical pole and to redistribute towards the posterior pole. These observa- tions suggest, but do not yet prove, the existence of an interaction with the actomyosin system, conforming to the capping model (Garcia-Reguet et al., 2000). Members of the TRAP (thrombospondin-related anonymous protein) family are, by virtue of their transmembrane domains, likely candidates to be involved in the capping process, and previous studies have provided substantial evidence supporting such a role in Plasmodium (Sultan et al., 1997). The short cytoplasmic tail of TRAP is likely to establish a connection with the actomyosin system, enabling the parasite to glide. Mutagenesis studies of the cytoplasmic tail of TRAP showed that this domain is essential for gliding motility and interchangeable between homologous proteins of T. gondii and Plasmodium berghei (Kappe et al., 1999). How this domain is con- nected to the actomyosin system remains to be eluci- dated. A direct interaction between microneme protein tails and myosin A could not be demonstrated, suggest- ing that a bridging molecule named ‘factor X’, might be involved (Fig. 2B). A second connection needs to be established with the IMC and subpellicular microtubules.

It is conceivable that actin filaments or, again, another bridging molecule links the IMPs to the actomyosin system and thus confers directionality to the helical gliding movement.

Dissolution of the glideosome

In contrast to rhoptry proteins, all the microneme proteins analysed so far are strictly excluded from the forming vacuole, and most are subject to proteolytic processing on several occasions during their lifetime. In addition to processing taking place during their transport to the organelle, extensive cleavages also occur after release on the parasite surface. For some of these proteins, the

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exact cleavage site was mapped using sequencing of processed forms, e.g. for MIC3 and MIC4 (Brecht et al., 2000; Garcia-Reguet et al., 2000), as well as mass spectrometry analysis, e.g. MIC6 (Meissner et al., 2002).

Two distinct parasite-derived protease activities have been reported to act on MIC2 after discharge from the micronemes. MPP1 (microneme protein protease 1) cleaves C-terminally and MPP2 (microneme protein protease 2) cleaves N-terminally (Carruthers et al., 2000) causing the loss of adhesive properties of MIC2 and its release from the parasite surface (Carruthers et al., 1999b) (Fig. 2C). The processing ensures dissociation of the tight interactions between parasite and host cells and is an important step in the accomplishment of invasion.

From the size of the products analysed by Western blot, the cleavage site of MPP1 was predicted to be close to the transmembrane domain. This assumption was further strengthened by the fact that SAG1 (major surface antigen 1 of T. gondii) fused to the transmembrane and cytoplasmic domain of MIC2 (SAG1TMCDmic2) is not only accurately sorted to the micronemes (Di Cristina et al., 2000) but also secreted and processed, like MIC2.

This result excluded any significant role for the ectodomain in the processing event (Opitz et al., 2002).

Alignments of the transmembrane domains of microneme proteins from different species revealed an unusually high conservation at the amino acid level. Site-directed muta- genesis in the intramembrane region of MIC2, MIC6 and MIC12 confirmed that few conserved residues are essential for the C-terminal processing of these mole- cules. Expression of an unprocessed MIC2 mutant appeared to be toxic for the parasites suggesting that this cleavage is prerequisite for the successful accomplish- ment of the penetration process. The precise site of cleav- age by MPP1 (IA*GG) was confirmed by mass spectrometry analysis of the secreted form of MIC6 (Opitz et al., 2002). The strict conservation at the cleavage site suggests that MPP1 is evolutionary and functionally con- served among the Apicomplexa, but the nature and iden- tity of this protease is still unknown.

The protease behind the action

Little is known about proteases in T. gondii, and the fact that MPP1 is insensitive to known protease inhibitors (Carruthers et al., 2000) does not facilitate its identifica- tion. A study based on protease inhibitors showed that two serine protease inhibitors altered T. gondii growth and blocked host cell invasion, whereas aspartic and cysteine protease inhibitors had no effect (Conseil et al., 1999). A recent report describes a subtilisin-like serine-protease (SUB1) in T. gondii (Miller et al., 2001). Although SUB1 localizes to the micronemes and is thus a candidate enzyme for the processing of microneme proteins, it is

unlikely to correspond with MPP1, which appears to be a constitutively active, resident surface protein (Opitz et al., 2002) (Fig. 2A and C).

A signalling cascade that brings parasites into motion

Microneme secretion, host cell attachment and invasion are accompanied by an increase in cytoplasmic calcium of the parasite, suggesting the existence of a number of calcium-sensitive effector systems (Carruthers and Sibley, 1999; Vieira and Moreno, 2000). T. gondii pos- sesses the genes for at least two different calmodulin-like domain protein kinases (CDPKs), CDPK1 and CDPK2, that seem to be expressed at different stages of the par- asite (Kieschnick et al., 2001). The initial attachment of tachyzoites requires activation of CDPK1 and leads to the phosphorylation of three unknown parasite proteins.

KT5926, an inhibitor of calcium-dependent protein kinases, blocks CDPK1 and has been shown before to reduce the amount of MIC2 released and to inhibit para- site motility (Dobrowolski et al., 1997).

Although no moving junction and vacuole are formed during parasite egress, this process requires, as does the invasion, the actin cytoskeleton and activation of the Ca2+- dependent protein kinase CDPK1, indicating that part of the motility machinery for moving in or out of cells is the same (Moudy et al., 2001). For egress-related motility, the Ca2+ concentration in the medium surrounding the infected cells has no effect, whereas the reduction in host cell cytoplasmic [K+] works as a signal and leads to an increased concentration in parasitic cytoplasmic [Ca2+] (Moudy et al., 2001). In T. gondii, the generation of egress mutants resistant to Ca2+ionophore established that host cell permeabilization is a critical part of the signalling pathway leading to parasite egress (Black et al., 2000). A recent report shows that thiols may play a role in egress (Stommel et al., 2001).

Summary

Several components of the invasion machinery have been identified recently in Apicomplexa. Actin, a polymerizing motor, and myosin(s) propel the parasites into the host cells. MyoA and its light chain are constitutively present in the pellicle, associated with MADP. A transient physical link must exist between the transmembrane adhesin–host receptor complexes and the actomyosin system in order to transmit the mechanical force across the plasma membrane. Additional unknown bridging molecules must also connect the actomyosin system to the subpellicular microtubules, probably via the IMC. Finally, regulatory molecules including kinase(s), myosin light chain(s) and protease(s) initiate, stabilize and then dislocate the gliding

© 2002 Blackwell Science Ltd, Molecular Microbiology, 45, 597–604 602 C. Opitz and D. Soldati

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complex by mobilizing, modulating and modifying the diverse components of the complex.

The availability of an inducible system conditionally to disrupt essential genes in T. gondii should assist in assessing the importance of these factors in the host cell penetration process. When all the components of the gliding complex have been identified, it will be funda- mental to unravel how parasites respond so rapidly and precisely to environmental conditions by elucidating the mechanisms governing the formation, activation, regula- tion and dissolution of the gliding complex.

Acknowledgements

We thank Dr Gary Ward for kindly providing us with the mono- clonal anti-IMC1 antibodies. We are grateful to Angelika Herm-Götz for generating the IFA data, Dr J. F. Dubremetz for his permission to include Fig. 1, and M. Meissner and R.

Werner-Meier for their helpful comments on the manuscript.

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