The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa

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The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa

KEELEY, Anthony, SOLDATI-FAVRE, Dominique

Abstract

The apicomplexans are obligate intracellular protozoan parasites that rely on gliding motility for their migration across biological barriers and for host-cell invasion and egress. This unusual form of substrate-dependent motility is powered by the "glideosome", a macromolecular complex consisting of adhesive proteins that are released apically and translocated to the posterior pole of the parasite by the action of an actomyosin system anchored in the inner membrane complex of the parasite. Recent studies have revealed new insights into the composition and biogenesis of Toxoplasma gondii myosin-A motor complex and have identified an exciting set of small molecules that can interfere with different aspects of glideosome function.

KEELEY, Anthony, SOLDATI-FAVRE, Dominique. The glideosome: a molecular machine

powering motility and host-cell invasion by Apicomplexa. Trends in Cell Biology , 2004, vol. 14, no. 10, p. 528-532

DOI : 10.1016/j.tcb.2004.08.002 PMID : 15450974

Available at:

http://archive-ouverte.unige.ch/unige:78033

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

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The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa

Anthony Keeley

¹

and Dominique Soldati

^1,2

1Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, UK, SW7 2AZ

2Department of Genetics and Microbiology, Faculty of Medicine University of Geneva CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland

The apicomplexans are obligate intracellular protozoan parasites that rely on gliding motility for their migration across biological barriers and for host-cell invasion and egress. This unusual form of substrate-dependent motility is powered by the ‘glideosome’, a macromolecular complex consisting of adhesive proteins that are released apically and translocated to the posterior pole of the parasite by the action of an actomyosin system anchored in the inner membrane complex of the parasite. Recent studies have revealed new insights into the composition and biogenesis of Toxoplasma gondii myosin-A motor complex and have identified an exciting set of small molecules that can interfere with different aspects of glideosome function.

As the causative agent for the deadliest form of malaria in humans, Plasmodium falciparum is the most notorious member of the Apicomplexa. This phylum also includes opportunistic pathogens such asToxoplasma gondii and Cryptosporidia, and animal pathogens such asEimeria, which is responsible for coccidiosis in chickens. In the absence of pseudopodia, cilia or flagella, the invasive apicomplexan parasites use an unusual mode of substrate- dependent motility to gain entry into their host cells – a

mechanism that is distinct from phagocytosis and that avoids lysosomal destruction. Gliding motility is also necessary for migration to the appropriate location in the host organism to initiate the next stage of life-cycle differentiation.

In Plasmodium, the ookinetes cross the mosquito midgut epithelium [1], whereas the sporozoites migrate through the haemolymph to the mosquito salivary glands [2]and cross the sinusoidal cell layer to reach hepatocytes in the human liver[3]. Parasites that can disseminate to areas of immune privilege – for example, by crossing the placenta or blood brain barrier – are more life-threaten- ing, and recent evidence suggests that the transmigration of T. gondii is linked to motility and is associated with acute virulence[4].

After entry into host cells, many apicomplexan parasites create a unique vacuole, termed the parasitophorous vacuole, by invagination of the host-cell plasma membrane and by the delivery of membranous material from theRHOPTRIES(see Glossary). The newly formed parasitophorous vacuole is devoid of host-cell integral membrane proteins. In particular, the absence of host soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins is probably responsible for the resistance of this vacuole to lysosomal fusion and acidification, which assures intracellular parasite survival. Formation of this unique compartment occurs at the

Corresponding author:Dominique Soldati (dominique.soldati-favre@medecine.unige.ch).

Available online 11 September 2004

www.sciencedirect.com

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moving junction that develops between the parasite and the host-cell plasma membrane (Figure 1). This point of contact between the parasite and host was first described during red blood cell invasion byP. falciparummerozoites [5]. From within their vacuole, the parasite replicates safely and, in the case ofT. gondiitachyzoites, exert some degree of control over a few host-cell organelles[6]. After

several rounds of replication, the parasites egress from the host cell in response to the same signalling cascade that they used to promote host-cell invasion [7,8]. MICRONEME

secretion and gliding motility are required for this egress, as they are for host-cell invasion.

Although our understanding of this unusual mode of cell entry has immediate relevance, in particular for malaria research, most of the ultrastructural information was obtained more than 30 years ago from electron microscopic studies onEimeriaandGregarines. Progress has been recently made, however, in identifying and characterizing the molecular components of the gliding machinery in Plasmodium and Toxoplasma. Here, we describe the unique ultrastructure and the molecular machine (glideosome) that enable apicomplexans to invade host cells. In this context, we discuss two recent studies that have revealed new insights into the composition and mode of action of this elaborate machine.

The invasion apparatus: an elaborate cytoskeleton and unique organelles

In apicomplexans, the pellicle is composed of an outer plasma membrane and anINNER MEMBRANE COMPLEX(IMC), which is formed by flattened cisternae joined by longi- tudinal and traverse sutures. Freeze-fracture electron microscopy of the IMC has revealed extensive linear arrays of intramembranous particles arranged in single

TRENDS in Cell Biology IMC Rhoptries Micronemes

Conoid

Host-cell plasma membrane

Moving junction

Nascent parasitophorous

vacuole Parasitophorous

vacuole

Daughter IMC

Premature rhoptries Daughter conoid

Figure 1.Host-cell invasion byToxoplasma gondiiis an active process that depends on the ability of the parasite to glide, and leads to the formation of a non-fusiogenic parasitophorous vacuole. Invasion involves two specialized secretory organelles (the rhoptries and the micronemes), protrusion of the conoid, and formation of a moving junction at the point of contact between the parasite and host-cell plasma membranes. Inside the vacuole, the intracellular parasite divides by endodyogeny, a process that is initiated by the appearance of the conoid and subpellicular microtubules and the concomitant formation of the inner membrane complex (IMC) of the daughter cell.

Glossary

Apicoplast:an essential organelle of secondary endosymbiotic origin that is surrounded by four membranes and uses a bipartite leader sequence to import proteins encoded in the nuclear genome [33].

Conoid:a tubulin-based structure of unknown function that extends during invasion and forms a thimble shape [34].

Dense granules:organelles that constitutively secrete proteins in the parasitophorous vacuole space and membrane [30].

Endodyogeny:a specialized form of division in which two daughter cells are formed within an intact polarized mother cell. The mature daughter parasites bud from the mother and acquire her plasma membrane. (Plasmodiumspecies divide by schizogeny, in which 16–32 merozoites bud out of the maternal cell).

Inner membrane complex:a set of cisternae below the plasma membrane containing a network of intramembranous particles that form a two-dimensional particle lattice attached to the underlying subpellicular microtubules [11].

Microneme:a small oval organelle located in the apex of the parasite that secretes proteins onto the parasite surface. Several micronemal proteins contain adhesive domains and can bind to the surface of the host cell [35].

Polar ring:thickening of the IMC, which acts as a supporting structure for the subpellicular microtubules at the anterior pole below the conoid.

Rhoptries: club-shaped regulated secretory organelles that contribute to vacuole formation [14]. The stimuli that trigger secretion from the rhoptries are unknown.

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and double rows running throughout the IMC [9–11]

(Figure 2a). The double rows overlay the subpellicular microtubules and are interspersed with several parallel single rows of particles. In T. gondii, the cell shape is maintained by 22 subpellicular microtubules, which form a scaffold together with the tri-membranous pellicle. The lattice of intramembranous particles is maintained along the whole length of the parasite, whereas the subpellicular microtubules terminate at two-thirds of the length of the parasite. These microtubules radiate from the apicalPOLAR RING, which functions as a microtubule-organizing centre.

A recent study has described the existence of a filamentous network that associates with the cytoplasmic face of the IMC along the length of the parasite[12]. The filaments, which are 8–10 nm in length, comprise the complexes TgIMC1 and TgIMC2 and seem to act as a membrane skeleton that possibly contributes to maintenance of the lattice organization, mechanical strength and the determination of cell shape. In Toxoplasma and Eimeria, the cytoskeleton also includes the CONOID, a cone-like structure of spiral fibres composed of atypical microtubules that is absent in Plasmodium and Crytosporidium.

In T. gondii, the process of invasion involves two Ca^2C-dependent events: protrusion of the conoid and the induced secretion of adhesive complexes from the micronemes. The microneme proteins are discharged at the parasite surface, translocated via an actin-based movement from the anterior to the posterior, and eventually cleaved off, forming visible trails behind the gliding

parasite. A second set of specialized organelles, the rhoptries, release membranous and proteinacious material during invasion and contribute to formation of the parasitophorous vacuole[13,14].

Composition and formation of the glideosome

Until recently, the only known components of the glideosome were the motor complex comprising myosin A (MyoA), and the myosin light chain (MLC) anchored to an unidentified receptor in the parasite pellicle and linked via unknown factors to adhesive microneme proteins.

Plasmodium studies were the first to demonstrate the crucial role played by the microneme protein TRAP in sporozoite gliding motilityin vitroand in infection of the mosquito salivary glands and the rat liverin vivo[15,16].

In T. gondii the homologue of TRAP, TgMIC2, also has an essential role during invasion[17], and is produced as a hexameric complex of TgMIC2 and MIC2-associated protein (MIC2AP)[18](Figure 2b).

MyoA belongs to the family of unconventional class XIV myosins, which is restricted to the phylum of Apicomplexa and was initially described inT. gondiiandP. falciparum [19,20]. TgMyoA is associated with TgMLC [21]and has been recently demonstrated to be essential for motility, invasion and egress [22]. A recent study has shed new light on the topology of the motor in the pellicle of Plasmodium berghei. Indeed, a complex of PbMyoA and the MLC (known as MTIP) specifically localizes to the IMC and not to the plasma membrane, as was previously anticipated[23].

TRENDS in Cell Biology

TgMIC2–TgM2AP

Aldolase

Host-cell membrane

F-actin

TgMLC

TgGAP50 IMPs

TgGAP45 Plasma membr

ane

Outer IMC membr ane

Inner IMC membr ane

(a) (b)

Microtub ules

Membr ane sk

eleton

Parasite plasma membrane

TgMyoA

IMC outer membrane IMC inner membrane Membrane

skeleton TgIMC1–TgIMC2

Subpellicular microtubule

Figure 2.The cytoskeleton, the organelles and the proteins of the invasion apparatus.(a)The pellicle of apicomplexans contains an outer plasma membrane and an inner membrane complex (IMC) that is composed of flattened vesicles containing a two-dimensional lattice of intramembranous particles (IMPs) that have been observed by cryoelectron microscopy[9–11]. It is not known whether the IMPs of the outer and inner membranes of the IMC are aligned and connected. InToxoplasma gondii, a membrane skeleton composed of TgIMC1 and TgIMC2 is present on the cytoplasmic face of the IMC inner membrane and is likely to interact directly with the IMPs[12].

(b)The glideosome is the molecular machine that promotes gliding motility and, inT. gondii, comprises the motor complex [myosin A (TgMyoA), the myosin light chain (TgMLC), TgGAP45 and TgGAP50], which is anchored in the outer membrane of the IMC and connected via filamentous actin (F-actin) and aldolase to the hexameric microneme protein complex (TgMIC2–TgM2AP), which, in turn, interacts with host-cell receptors. The domains involved in the association of TgMyoA–TgMLC with TgGAP45 and, ultimately, with TgGAP50 have not been elucidated. TgGAP45 is potentiallyN-myristoylated, whereas TgGAP50 is an integral membrane protein.

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Gaskinset al.[24]have now identified and character- ized two new components of the glideosome: the gliding associated proteins TgGAP45 and TgGAP50 (Figure 2b).

The integral membrane glycoprotein TgGAP50 functions as a receptor for TgMyoA–TgMLC in the IMC [24].

Confirmation of the immobilization of TgMyoA in the plane of the IMC is in good agreement with recent studies showing that the cytoplasmic tail of TgMIC2 and TRAP indirectly associate with actin via aldolase, a protein that binds filamentous actin (F-actin) [25,26]. Elucidation of the topology of the actomyosin system in the pellicle is crucial to our understanding of how force generated by the myosin motor can be transduced to the underlying cytoskeleton and hence can move the parasite body forward.

TgMyoA translocates the parasite body relative to the actin filaments, which are anchored to micronemal protein–host receptor complexes, and the helical movement of the parasite is probably dictated by the organized lattice of intramembranous particles (Figure 2).

Notably, the IMC is derived from the Golgi apparatus and forms early during parasite division, which in T. gondii occurs by packaging two daughters within a single mother during mitosis, as illustrated inFigure 1, in a process termed ENDODYOGENY. The two-dimensional lattice formed by the intramembranous particles is possibly oriented along the newly formed basket of the subpellicular microtubules of the nascent daughter cells. On the basis of pulse-chase labelling experiments, Gaskins et al. [24]

concluded that formation of the glideosome is probably a two-stage process, in which TgMyoA, TgMLC and TgGAP45 initially assemble in the cytosol and then associate with TgGAP50 at the mature IMC. Their study also raises the issue of how these proteins are targeted to the IMC, which adds only further complexity to an already extremely elaborate secretory apparatus that is intimately linked to organelle biogenesis and parasite division.

Indeed, as many as six distinct destinations have been identified so far in the secretory pathway ofT. gondiiand, furthermore,Plasmodiumspecies export proteins beyond the parasitophorous vacuole membrane to the cytosol and plasma membrane of the infected red blood cells. The mechanisms that dictate accurate sorting have been unravelled for several of these destinations, including a tyrosine-based sorting signal for rhoptries [27] and micronemes [28], a bipartite signal for the APICOPLAST

[29], and a default pathway routing to theDENSE GRANULES

for soluble proteins and to the plasma membrane for lipid- anchored proteins[30]. In this context, biogenesis of the IMC and the sorting of proteins to this compartment present an exciting new challenge for research (Figure 3).

Given that the glideosome is composed of proteins emerging from the early secretory pathway (TgGAP50), from induced exocytosis (TgMIC2) and from the cytosol (TgMyoA–MLC–GAP45 and aldolase–F-actin), the con- struction of this sophisticated architecture must be precisely orchestrated in time and space.

Concluding remarks

The newly identified TgGAP45 and TgGAP50, as well as all of the other components previously known to constitute the glideosome, are essential for parasite survival, as well

as strikingly unique and highly conserved among the apicomplexans, suggesting that they might constitute useful targets for drug design. Indeed, a recent in vivo screening based on a high-throughput invasion assay has identified a collection of small molecules that show non- cytotoxic effects onT. gondii invasion[31]. These inhibitors of invasion have been dissected for their effect on motility, conoid extrusion, constitutive and induced microneme secretion, and the results have revealed surprising new findings.

An inhibitor that irreversibly blocks conoid extrusion provides the first evidence that this structure has a crucial role during invasion by T. gondii. Another intriguing group of inhibitors can enhance motility by mechanisms independent of actin polymerization, in contrast to previous results suggesting that actin polymerization is the rate-limiting step in parasite motility[32]. In addition, two groups of inhibitors are capable of pharmacologically uncoupling constitutive and Ca^2C-induced microneme secretion, suggesting that secretion either is controlled by two distinct pathways or involves two distinct pools of organelles.

These inhibitors are providing invaluable tools with which to identify new components of the glideosome and to unravel its mode of action. The continuous emergence of drug resistance in the malaria parasites poses a consider- able threat to human health, urging the development of novel therapies. Identifying the molecular targets of these invasion inhibitors will no doubt enlighten our understanding of the biology of apicomplexans, but will also potentially lead to the discovery of new candidates for drug intervention.

Acknowledgements

We thank the members of the laboratory for critically reading the manuscript and Jean-Francois Dubremetz for helpful discussions. We apologize to those authors of early work and reviews not cited. A.K. is funded by the Wellcome Trust and D.S. is a Howard Hughes Medical Institutes International Scholar.

TRENDS in Cell Biology

Plasma membrane Micronemes

Apicoplast

IMC

Rhoptries Dense granules

(i) (ii)

(iii) (iv) (v) (vi) Nucleus

Figure 3.‘Traffic jam’ in the secretory pathway ofToxoplasma gondii: the presence of several specialized organelles linked to the secretory pathway poses a unique challenge for the apicomplexans to sort out protein trafficking efficiently and accurately. Proteins that carry a signal peptide can be routed to no fewer than six distinct destinations: (i) the plasma membrane; (ii) the micronemes; (iii)the vestigial plastid organelle, the apicoplast;(iv)the rhoptries;(v)the dense granules, and subsequently to either the parasitophorous vacuole space or the parasitophorous vacuole membrane; and(iv)as now shown by Gaskinset al.[24], the inner membrane complex (IMC).

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doi:10.1016/j.tcb.2004.08.002

Bacterial cell polarity: a ‘swarmer–stalked’ tale of actin

Rong Li and Stephanie C. Wai

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

The actin cytoskeleton is important for cell polarity and morphogenesis in eukaryotic organisms. A recent article describes an unexpected requirement for the actin-like

protein MreB in the polarization of the bacterium Caulobacter crescentus. More surprisingly, the formation of a filamentous MreB structure that traverses the length of the cell is sufficient for randomized polar localization of cell-fate proteins. In this article, we

Corresponding author:Rong Li (rong_li@hms.harvard.edu).

Available online 11 September 2004