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Role of the parasite and host cytoskeleton in apicomplexa parasitism
FRENAL, Karine, SOLDATI-FAVRE, Dominique
Abstract
The phylum Apicomplexa includes a large and diverse group of obligate intracellular parasites that rely on actomyosin-based motility to migrate, enter host cells, and egress from infected cells. To ensure their intracellular survival and replication, the apicomplexans have evolved sophisticated strategies for subversion of the host cytoskeleton. Given the properties in common between the host and parasite cytoskeleton, dissecting their individual contribution to the establishment of parasitic infection has been challenging. Nevertheless, recent studies have provided new insights into the mechanisms by which parasites subvert the dynamic properties of host actin and tubulin to promote their entry, development, and egress.
FRENAL, Karine, SOLDATI-FAVRE, Dominique. Role of the parasite and host cytoskeleton in apicomplexa parasitism. Cell Host & Microbe , 2009, vol. 5, no. 6, p. 602-11
DOI : 10.1016/j.chom.2009.05.013 PMID : 19527887
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http://archive-ouverte.unige.ch/unige:3973
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Role of the Parasite and Host Cytoskeleton in Apicomplexa Parasitism
Karine Fre´nal1,*and Dominique Soldati-Favre1
1Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
*Correspondence:karine.frenal@unige.ch DOI 10.1016/j.chom.2009.05.013
The phylum Apicomplexa includes a large and diverse group of obligate intracellular parasites that rely on actomyosin-based motility to migrate, enter host cells, and egress from infected cells. To ensure their intra- cellular survival and replication, the apicomplexans have evolved sophisticated strategies for subversion of the host cytoskeleton. Given the properties in common between the host and parasite cytoskeleton, dissect- ing their individual contribution to the establishment of parasitic infection has been challenging. Neverthe- less, recent studies have provided new insights into the mechanisms by which parasites subvert the dynamic properties of host actin and tubulin to promote their entry, development, and egress.
Introduction
An intracellular lifestyle is a necessity for viruses and is also one of the most effective strategies adopted by other microorganisms, both to evade the host cell defense mechanisms and to access host metabolites. Many bacteria and viruses are internalized into host cells by phagocytosis or pinocytosis. During these processes, they extensively manipulate the host cytoskeleton, remodeling actin filaments and microtubules by releasing effector molecules that hijack the host regulatory networks (Bhavsar et al., 2007; Radtke et al., 2006). The phylum Apicom- plexa contains important protozoan parasites responsible for severe diseases in humans and animals. Members of this phylum are auxotrophic for multiple classes of compounds and are unable to survive and replicate outside host cells. Access to the host cell is therefore of crucial importance to ensure the survival of these pathogens and the establishment of infection and relies upon the active process of parasite gliding motility. This unique mode of substrate-dependent motility is powered by the parasite actomyosin system and leads to the formation of a unique compartment named the parasitophorous vacuole (PV) that provides a safe environment in which the parasite resides and replicates. The lytic cycle begins with parasite egress from the infected cell, hence contributing to the dissemination of infection.
The cytoskeletal architecture and the endomembrane system of the host cells are known to critically contribute to establishment of infection by pathogens. In the case of eukary- otic pathogens such as the apicomplexans, it is challenging to experimentally discriminate between the roles of the parasite cytoskeleton versus that of the host cell during invasion.
Both organisms share similar cytoskeletal components; it is therefore difficult to interfere with one without affecting the other.
In the present review, we will highlight some recent findings that illustrate how the cytoskeleton of apicomplexans and the cytoskeleton of the host cell participate in the establishment of the infection.
Apicomplexans’ Diversity and Their Journey through Cells
The phylum Apicomplexa contains several thousand species of obligate intracellular parasites. We will focus on the four best- studied organisms that are of medical and veterinary significance and are responsible for severe diseases:Plasmodium(malaria), Toxoplasma (toxoplasmosis), Cryptosporidium (enteritis), and Theileria(theileriosis). These protozoan parasites exhibit com- plex life cycles that take place in multiple hosts and diverse cell types. The invasive stages are motile and named zoites.
Toxoplasma gondiiis globally spread, affecting up to one-third of the human population and virtually all warm-blooded animals.
In healthy individuals, toxoplasmosis is usually asymptomatic and self-limiting, with the parasite remaining encysted (brady- zoite stage) during the whole lifetime of the host. However, it can have serious or even fatal consequences in the case of primary infection during pregnancy or immunodeficiency, due to the uncontrolled proliferation of the rapidly dividing stage (tachyzoite). T. gondiiis able to invade any type of nucleated cell, including dendritic cells (DCs) and macrophages, where it can replicate inside a nonfusogenic PV (Dubey, 2008).
Plasmodium falciparum, one of the causative agents of human malaria, is estimated to be responsible for 230 million new cases every year, mainly in sub-Saharan Africa. The sporozoite stage is transmitted to humans by the bite of infected femaleAnopheles mosquitoes. Once in the bloodstream, the parasite reaches the liver, where it multiplies, liberating hundreds of merozoites.
These parasites then infect red blood cells (RBCs), where they proliferate, leading to the egress of dozens of new merozoites that can invade other erythrocytes, contributing to the spreading of the infection (Cowman and Crabb, 2006; Vaughan et al., 2008).
Cryptosporidiumspecies cause severe diarrheal illness in both humans and animals, but the disease is self-limiting in immuno- competent persons. Given the fact that this parasite relies prefer- entially on host metabolic pathways, there is no effective therapy against cryptosporidiosis. Once ingested, Cryptosporidium oocysts release motile sporozoites that rapidly infect epithelial
cells lining the intestines. After attachment to the apical surface of the host cell, the sporozoite induces host cell membrane protru- sions that encapsulate it into a unique PV that is intracellular but nevertheless extracytoplasmic and separated from the host cell by an electron-dense plaque (Thompson et al., 2005).
Theileria species are of great veterinary importance. They infect domestic livestock, mostly ruminants but also sheep and goats, and are responsible for economic losses principally in tropical and subtropical regions of the world. They are tick-trans- mitted parasites that invade lymphoid cells, but soon after entry, the PV dissolves, releasing sporozoites that differentiate and replicate freely in the host cytoplasm. Theileria induces a cancer-like phenotype in the infected host cells, causing uncon- trolled proliferation. This parasite exploits host cell transforma- tion for its rapid spreading into host daughter cells, avoiding extracellular exposure. Its tight association with the host cell mitotic apparatus assures an equal distribution of progeny into the daughter host cells (Bishop et al., 2004).
The apicomplexans share unusual features regarding their cyto- skeletal elements that ensure a structural framework necessary for maintenance of cell shape and integrity and a level of deformability during infection. The motile stages are surrounded by the pellicle, a three-layered structure composed of the plasma membrane and the inner and outer membranes of the inner membrane complex
(IMC). The IMC is formed by one or several flattened vesicles underlying the plasma membrane (Figure 1A). A network of sub- pellicular microtubules associated with the cytoplasmic face of the IMC originates from the apical polar ring (constituting a micro- tubule organization center [MTOC]) and runs in a spiral along two- thirds of the parasite length (Morrissette and Sibley, 2002a).
Several proteins shown to be associated with the IMC are part of the alveolin family, defined within the group of alveolates, including ciliates, dinoflagellates, and apicomplexans. A large repertoire of 12 and 7 genes coding for putative alveolins are found inT. gondiiandP. falciparum, respectively (Gould et al., 2008). One of them, TgIMC1, is involved in the maintenance of cell shape;
introducing mutations altered the stability and rigidity of the daughter cells’ subpellicular network (Mann et al., 2002). The or- thologs inP. berghei, PbIMC1a and PbIMC1b, are predominantly expressed during sporogony and in ookinetes, respectively. Para- sites lackingPbIMC1aorPbIMCbgenes display abnormal cell shape, reduced speed of motility, decreased mechanical resis- tance, and in consequence, reduced infectivity (Khater et al., 2004; Tremp et al., 2008). These phenotypes are in agreement with a structural role of these proteins. The organization of the cholesterol-rich IMC and the subpellicular microtubules defines and maintains the shape of the parasite. Drug-induced disruption of these microtubules leads to loss of both cell shape and apical Figure 1. Cytoskeletal Elements of an Apicomplexan and a Host Cell and the Disrupting Drugs
(A) Model of a typical apicomplexan parasite, highlighting the elements of the cytoskeleton.
(B) Scheme of the actomyosin system involved in gliding motility.
(C) Illustration of the effects of the drugs discussed in this review on actin and microtubule cytoskeletons. BDM, 2,3-butanedione monoxime; FH, formin homology domain.
(D) Effects of the drugs on the host cell (left) and on the parasite (right).
polarity (Morrissette and Roos, 1998). The pellicle and microtubule network provide at the same time a relative rigidity for the motor powering motility to function underneath the plasma membrane and a relative flexibility to enter into the host cell, as illustrated by the characteristic constriction displayed by the parasite upon invasion (Johnson et al., 2007; Sanders et al., 2007).
Cryptosporidium appears to lack subpellicular microtubules but presents two central microtubules and many longitudinal ridges along the parasite that could provide mechanical support in the absence of a subpellicular network (Matsubayashi et al., 2008). Some apicomplexans possess an additional apical cyto- skeletal structure, the conoid, which is composed of spirally arranged fibers ofa- andb-tubulins forming a new type of polymer (Hu et al., 2002). The motile conoid protrudes and retracts in a calcium-dependent fashion during invasion. However, the func- tion of this structure and the identity of the motor involved in its motion remain to be established.
Host and Parasite Cytoskeleton Contributions to Invasion
Contribution of the Parasite Cytoskeleton to Active Host Cell Entry
The apicomplexans rely on a common active entry process that leads in most cases to the formation of a unique PV, inside which the parasite replicates safely. Despite the absence of cilia or flagella, the zoites display a unique form of gliding motility pow- ered by the parasite actomyosin system (Figure 1B), which enables them to cross nonpermissive biological barriers and drives entry to and exit from diverse cell types (Barragan and Sibley, 2003; Kappe et al., 2004; Keeley and Soldati, 2004; Soldati et al., 2004). The contribution of the parasite actin and myosin in motility was suggested as early as the 1970s and 1980s (King, 1988; Vanderberg, 1974). It was later confirmed by the incapacity of the parasites to glide and/or to invade in the presence of drugs that interfere with either actin dynamics (cytochalasin D [CytD], jasplakinolide [JAS], and latrunculin B) or myosin ATPase function (2,3-butanedione monoxime [BDM]) (Dobrowolski et al., 1997a;
Poupel and Tardieux, 1999; Russell and Sinden, 1981; Shaw and Tilney, 1999; Siden-Kiamos et al., 2006; Wetzel et al., 2003) (Figure 1D). Although gliding motility ofT. gondiiandC. parvum can be blocked by treatment with CytD, attachment was unaltered (Dobrowolski and Sibley, 1996; Wetzel et al., 2005). The genera- tion of aToxoplasmamutant line resistant to CytD demonstrated that the invasion process relies essentially on the parasite actomy- osin cytoskeleton and not on the host actin (Dobrowolski and Sibley, 1996). However, the contribution of host actin has recently been revised and is discussed below (Gonzalez et al., 2009).
The motor responsible for the gliding motility is a short myosin of class XIV. The vital role ofToxoplasmamyosin A in motility, invasion, and egress was demonstrated with the assistance of an inducible expression system designed to study the function of essential genes (Meissner et al., 2002). Besides the actin and the myosin motor, the other components of the ‘‘glideosome,’’
the multiprotein complex responsible for generation of move- ment, were sequentially characterized (Keeley and Soldati, 2004). The current model proposes that the glideosome is firmly anchored in the parasite pellicle, indirectly connected to the sub- pellicular microtubules (Figure 1B). Although the details of this interaction are currently unknown, the anchoring of the motor
to the IMC involves two glideosome-associated proteins (GAPs): GAP45, inserted in the outer leaflet, and GAP50, an inte- gral membrane protein of the IMC (Gaskins et al., 2004). The tight interaction with GAP50 and an enrichment in cholesterol have been reported to contribute to the firm immobilization of myosin A in the IMC (Johnson et al., 2007).
Zoites are predominantly polarized cells with specialized secretory organelles named micronemes and rhoptries, whose contents are sequentially discharged during invasion, liberating proteins that critically contribute to motility, host cell recognition, and invasion. Micronemal proteins (e.g., TgMIC2 and TRAP) are translocated from the apical to the posterior pole by connection to the actin filaments via bridging with the glycolytic enzyme aldolase (Figure 1B) (Buscaglia et al., 2003; Jewett and Sibley, 2003; Keeley and Soldati, 2004). Aldolase and other enzymes of glycolysis that are cytosolic in intracellular parasites have recently been reported to relocalize at the pellicle of extracellular parasites (Pomel et al., 2008). The translocation of the glycolytic pathway in motile parasites could favor the local production of ATP to fuel the glideosome, without necessarily invalidating the bridging role of aldolase between actin and adhesive microneme proteins. In this context, a recent study aimed to dissect the dual role of aldolase (Starnes et al., 2009). An aldolase conditional knockout strain was generated and complemented by a class of aldolase mutations impaired in binding to the TgMIC2 tail or in enzymatic activity. As anticipated, impairment in energy production affects motility, invasion, and intracellular growth, whereas the mutant complemented with an aldolase defective in binding to TgMIC2 was impaired in invasion (Starnes et al., 2009). However, given the fact that this latter mutant was not impaired in gliding motility, the bridging role of aldolase to actin in vivo could not be formally established.
Despite the clear contribution of the actomyosin system in motility and invasion, parasite actin filaments have never been formally seen at the vicinity of the glideosome. Recent studies have highlighted that apicomplexan actin either directly extracted from parasites or produced in heterologous systems displays atypical biochemical properties by forming remarkably short filaments (about 100 nm in length) that are less stable than those formed by conventional actin (Sahoo et al., 2006;
Schmitz et al., 2005; Schu¨ler et al., 2005b). As a consequence, such short, thin, and unstable filaments failed to be visualized by indirect immunofluorescence or electron microscopy tech- niques. Although a large fraction of apicomplexan actin is found in its monomeric form (Dobrowolski et al., 1997b; Pinder et al., 1998), filaments can be stabilized by treatment with JAS, which induces acrosome-like structures at the apical pole ofT. gondii andP. falciparum(Shaw and Tilney, 1999).
According to searches of apicomplexan genomes for which significant data are available, the repertoire of actin-binding and actin-regulating proteins in these parasites appears unex- pectedly small compared to that of other eukaryotes (Baum et al., 2006; Schu¨ler and Matuschewski, 2006). This observation obviously does not exclude the possibility that the parasites have evolved novel proteins to perform the function of the ‘‘missing’’
ones. Most strikingly, apicomplexans lack the actin-related protein complex Arp2/3, one of the main machineries orches- trating actin nucleation in eukaryotes (Gordon and Sibley, 2005). In contrast, actin polymerization in these parasites
appears to involve profilin (PRF) and formins (FRMs). Three different apicomplexan PRFs are able, at steady state, to depoly- merize barbed end-capped filaments by sequestering rabbit skeletal muscle G-actin, while they stabilize filaments when barbed ends are free (Plattner et al., 2008). A conditional knockout of theTgPRFgene uncovered an essential function in gliding motility, invasion, and egress of host cells. Moreover, two recombinant formin homology 2 (FH2) domains ofP. falcipa- rumFRM1 and FRM2 have been demonstrated to act as potent nucleators of chicken skeletal muscle actin in vitro, by binding the barbed end of F-actin and remaining associated with the filament as it elongates (Baum et al., 2008). Based on their localization at the moving junction (MJ) during invasion, TgFRM1 and PfFRM1 have been postulated to be important actors in actin nucleation during host cell entry (Baum et al., 2008). The balance between G- and F-actin is likely to be drastically influenced by the activity of the FRMs and the abundantly expressed actin depoly- merizing factors (Allen et al., 1997; Schu¨ler et al., 2005a).
While most apicomplexans enter host cells by a fast and active process lasting less than 15 s,Theileriasporozoites are nonmotile and enter lymphocytes in any orientation by a progressive zipper- ing mechanism. This process is not dependent on the parasite actin cytoskeleton and is achieved within minutes. Moreover, invasion byTheileriasporozoites involves neither the formation of a MJ nor any significant remodeling of the host cell surface (Shaw, 2003).
Contribution of the Host Cytoskeleton to Parasite Entry The cortical host cell cytoskeleton represents a physical and mechanical barrier for parasite entry and egress. To study the contribution of the host cell cytoskeleton during a pathogen infection, drugs are frequently used to act specifically on myosins (BDM) or actin filaments (JAS as stabilizer or CytD as disrupter) (Figure 1C). However, the effects of these drugs on apicomplex- ans complicate an assessment of the host cell cytoskeleton’s contribution to invasion. A recent study has revisited the current model of invasion and suggested that de novo polymerization of host actin is indeed important for the entry ofToxoplasmatachy- zoites andPlasmodium sporozoites into host cells (Gonzalez et al., 2009). The formation of a host ring-shaped F-actin structure was detected at the point of apposition of the host and parasite plasma membranes, the MJ, and was shown to be stable during parasite entry. This accumulation of F-actin detected at the posterior end of the internalized parasite disappeared within 10 min postentry. The host Arp2/3 complex was present at the MJ together with cortactin, a nucleation-promoting factor. Based on RNA interference analyses, cortactin appears to play a role in Toxoplasmatachyzoite but not inPlasmodiumsporozoite inva- sion (Gonzalez et al., 2009) (Figure 2A). These data implicate for the first time the host actin in parasite entry, and the proposed model postulates that, at the zoite-cell junction, a bridge between parasite and host actins is built to provide a solid anchor for pulling the parasite inside the cell (Gonzalez et al., 2009). The recruitment of Arp2/3 and cortactin at the site of parasite entry is indicative of a de novo polymerization of the host actin induced by the parasite. In this context, a recent study demonstrated that interacting parasite components of the MJ can be found inserted on either side of the MJ, both at the host and parasite plasma membranes (Besteiro et al., 2009). A yet unexplored aspect of entry regards the sealing of the plasma membrane and the
detachment of the PV at the end of the invasion process. It is conceivable that the host actin cytoskeleton critically contributes to this event and involves either a mechanism related to phagocy- tosis or a plasma membrane repair process.
In the context of overcoming the resistance conferred by the host cortical actin, it is relevant to note thatT. gondiiexpresses a protein named toxofilin, which was previously shown to effi- ciently sequester host actin monomers and cap filament ends (Poupel et al., 2000). Given the fact that toxofilin is a rhoptry protein potentially secreted at the site of entry (Bradley et al., 2005), it is reasonable to speculate that this protein could act as an effector molecule, remodeling the host cytoskeleton and hence facilitating parasite internalization.
Host and Parasite Cytoskeleton Contributions to Intracellular Growth
Parasite Cytoskeleton Involvement in Intracellular Growth and Replication
Once established in the cell within a nonfusogenic PV,Toxo- plasma undergoes several rounds of multiplication before the parasites egress from the host cell to establish a new infection.
T. gondii replicates by endodyogeny, a process distinct from cell division of most other eukaryotes and during which the two daughter cells are formed by internal budding, incorporating most of the cytoplasm of the mother cell and involving a tempo- rally well-orchestrated organellar replication (Nishi et al., 2008).
The initiation of daughter-cell budding corresponds to the dupli- cation of centrioles near the spindle pole and the synthesis of the microtubular conoid and IMC structures (Hu, 2008). In contrast to the broadly active nocodazole or colchicine, dinitroa- nilines (oryzalin, trifluralin, ethalfluralin) selectively disrupt api- complexan microtubules without affecting host cells (Morrissette et al., 2004). Treatment ofT. gondiiwith the destabilizing oryzalin or the stabilizing taxol blocked replication and led to the formation of abnormal parasites (Shaw et al., 2000). Serial dilutions of the oryzalin herbicide allowed uncoupling of these two effects and revealed that nascent subpellicular microtubules were more sensitive to disruption than spindle microtubules (Morrissette and Sibley, 2002b). This finding illustrates that each microtubule population involves an individual MTOC; whereas the subpellicu- lar microtubules originate from the apical polar ring, the spindle microtubules nucleate from two centrioles. The nuclear division and budding can then be disconnected, leading, in drug-treated intracellular parasites, to the formation of both diploid and anucleated daughter cells (Morrissette and Sibley, 2002b). More recently, the identification of cell division markers allowed a better morphological description of the events leading to the formation of new parasites, e.g., DNA replication and chromosome segre- gation, nuclear division, and finally cytokinesis. For example, the characterization of the membrane occupation and recognition nexus 1 (MORN1) permitted tracking of spindle morphology and daughter cell budding (Gubbels et al., 2006). MORN1 localizes specifically to the anterior and posterior poles of mature and nascent parasites as well as to the interior side within the nucleus, corresponding to the centrocone that organizes the mitotic spindle. The distribution of MORN1 is highly dynamic during parasite division and colocalizes with the extremity of the growing daughter IMC (Gubbels et al., 2006). Moreover, colocal- ization of MORN1 withT. gondii myosin C suggests that this
actin-dependent motor, whose overexpression provokes a divi- sion defect (Delbac et al., 2001), could be associated with or could even power this highly dynamic movement of the IMC.
Several other proteins functioning in conjunction with the actin- or tubulin-based cytoskeleton have been identified by
purification of an enriched conoid and cytoskeletal fraction of T. gondii(Hu et al., 2006). A dynein light chain (TgDLC) that might be part of a microtubule-based motor complex and two calmod- ulins (TgCaM1 and TgCaM2) were found at the conoid of both mature and nascent parasites (Hu et al., 2006). Two centrins Figure 2. Involvement of Parasite and Host Cytoskeletons during Infection
(A) Invasion of the host cell byToxoplasmatachyzoite orPlasmodiumsporozoite involves the parasite actin-myosin A motor, formin 1 and profilin, host actin polymerization, and the recruitment of the Arp2/3 complex at the MJ.
(B) Division of theToxoplasmatachyzoite is driven by parasite microtubules, and access to nutrients involves the recruitment of host cell microtubules to deliver lysosomes to the parasitophorous vacuole (PV).
(C) Remodeling of the host actin cytoskeleton byCryptosporidiumin the apical region of the intestinal epithelial cell and remodeling of the RBC membrane cyto- skeleton byPlasmodium(knobs).
(D) Division and spreading ofTheileriaby interaction of the parasite with the host cell mitotic spindle.
(E) Egress from the host cell byToxoplasmarelies on parasite actin-based motility. FO, feeder organelle; TaSE,Theileria annulatasecreted protein; PfEMP,Plas- modium falciparumerythrocyte membrane protein; KAHRP, knob-associated histidine-rich protein; MESA, mature parasite-infected erythrocyte surface antigen;
MTOC, microtubule organization center.
were also of great use for following the cell division process, since Tgcentrin1 labeled the centrioles and Tgcentrin2 appeared to associate with preconoidal rings. All these proteins are excel- lent markers to study the biogenesis of the highly polarized budding daughter cells and the formation of subcompartments during cell division (Hu, 2008).
Quite surprisingly,T. gondiigrows normally in the presence of low concentration (<1mM) of the actin inhibitors CytD, JAS, or latrunculin, indicating that the parasite actin cytoskeleton is not essential for replication and budding of the daughter cells (Shaw et al., 2000). Given thatT. gondiipossesses 11 genes encoding putative myosins (Foth et al., 2006), the effects of CytD are unlikely to disrupt all the actomyosin-dependent events in this parasite. Intriguingly,T. gondiiand other apicomplexans possess groups of actin-like (ALPs) and actin-related (ARPs) proteins specific to this phylum, whose functions are likely to be resistant to classical actin-targeting drugs (Gordon and Sibley, 2005). Recently, TgALP1 has been shown to transiently colocalize with the nascent IMC ofT. gondiidaughter cells, and overexpression of the TgALP1 gene causes a defect in IMC formation and parasite growth (Gordon et al., 2008).
Hijacking of the Host Microtubules for Parasite Access to Nutrients
The apicomplexans have a high demand for nutrients and energy to sustain their rapid intracellular replication rate. Host mitochon- dria and endoplasmic reticulum (ER) are recruited around the PV, most likely to provide access to host metabolites (Goldszmid et al., 2009; Martin et al., 2007). Originally, theT. gondii-containing vacuole was thought to be isolated from the host endolysosomal compartments, but a detailed electron microscopy-based study showed that, in fact, the PV accumulates material derived from the host endolysosomal system via active recruitment of host microtubules (Coppens et al., 2006). By following host endocytic compartments, using different markers and microscopy tech- niques, it was shown that these organelles, concentrated in the perinuclear area in uninfected cells, progressively gathered around the PV inT. gondii-infected cells. At the same time, the host MTOC relocalized from the nuclear envelope to close proximity of the PV, and a reorganization of the host microtubules was initiated within 4–6 hr postinfection, coming in close apposition with the PV membrane (PVM) and culminating with the PV totally enclosed by a network of host microtubules 24 hr postinfection (Coppens et al., 2006; Walker et al., 2008). A similar reorganization from the host nuclear surface to close apposition of the PVM was previously observed for vimentin intermediate filaments during infection (Hal- onen and Weidner, 1994). Host microtubules were observed growing directly from a PV-associated focus ofg-tubulin (whose size is compatible with a MTOC) on one side of the vacuole (Walker et al., 2008) where they codistribute with vimentin around the PV, whereas no redistribution of the host actin filaments was observed.
Moreover, deep invaginations of the PVM into the lumen of the vacuole were formed, always containing a central host microtubule that might serve as conduits for the delivery of intact lysosomes (Coppens et al., 2006) (Figure 2B). The parasite protein GRA7 was found to coat the host microtubules, but its function remains unclear. Remodeling of host microtubules appears to occur specif- ically in response to infection byT. gondiiand is not observed with the closely related parasiteNeospora caninum(Coppens et al., 2006; Walker et al., 2008). Several questions remain to be resolved,
such as what are the signals inducing host microtubule reorganiza- tion and what is the mechanism circumventing the membranous barriers and leading to delivery of nutrients to the parasite. Very recently, a more intimate connection between the parasite and host cell organelles has been reported, involving a fusion between ER and PV membranes (Goldszmid et al., 2009).
Modification of the Host Cytoskeleton Remodeling of the Host Actin Cytoskeleton by Cryptosporidium
Following invasion byCryptosporidiumof epithelial cells lining the intestine, several elements of the host cell cytoskeleton appear to be disrupted or remodeled. The microvilli of the apical region of the cell become long and thick next to the area of a parasite vacuole, and an electron-dense plaque is formed at the PV-host cell interface (Figure 2C). Upon internalization,Cryptosporidium induces membrane protrusions from the host cell that encapsu- late the parasite in a PV at the apical region of the cell. The inter- nalized parasite resides in a compartment located between the cytoplasm and the plasma membrane and is therefore described morphologically as intracellular but extracytoplasmic (Borowski et al., 2008). A considerable accumulation of host actin filaments has been observed at the parasite-host interface, whereas no other changes in the organization of F-actin were noticed else- where in the cell. The presence of the actin crosslinking protein a-actinin is also detected in the plaque early in parasite develop- ment, but not during parasite replication (Elliott and Clark, 2000).
In addition to actin, the Arp2/3 complex actin nucleator, the vasodilator-stimulated phosphoprotein (VASP), and the neural Wiskott-Aldrich syndrome protein (N-WASP) were found to accumulate at the parasite-host interface, indicating thatCrypto- sporidiuminduced host cell actin polymerization (Chen et al., 2004a; Elliott et al., 2001). In cells expressing Scar-WA, a fragment of the Sca1 protein known to inhibit actin polymerization,Crypto- sporidiuminfection rate is reduced by 71%, reinforcing the idea that host cell actin polymerization is necessary for infection (Elliott et al., 2001). The infection rate is reduced by half in host cells expressing a dominant-negative form of N-WASP that is unable to bind to Arp2/3 complex (Elliott et al., 2001). Moreover, cells ex- pressing a dominant-negative form of the Rho GTPase, Cdc42, exhibited a decrease ofCryptosporidiuminvasion, implying that the actin remodeling is induced by the Arp2/3 complex and acti- vated by N-WASP via Cdc42 (Chen et al., 2004a). Lastly, the recruitment and activation of PI3K in host cells appears to activate the Cdc42 pathway and to be required forC. parvum-induced actin remodeling and invasion (Chen et al., 2004a, 2004b).
Although the molecular cascade driving host actin remodeling upon invasion by Cryptosporidium is well characterized, the functional significance of this actin rearrangement is still unclear.
Nevertheless, the parasite-induced plaque is suggested to play a structural role, maintaining the parasite in its unique extracyto- plasmic position at the apex of the cell.
Adding to its arsenal of subversion strategies, it has been shown thatCryptosporidiumhas the ability to manipulate host cell membrane protrusions by locally increasing cell volume via the recruitment of the host Na+/glucose cotransporter (SGLT1) and aquaporin 1 (AQP1) to the attachment site (Chen et al., 2005). This process is proposed to facilitate parasite invasion and PV formation.
Remodeling of the Erythrocyte Membrane Skeleton by Plasmodium
Invasion of RBCs byP. falciparumleads to the modification of the host membrane cytoskeleton at specific areas that appear elec- tron-dense and are named knobs (Figure 2C). These modifica- tions reduce the deformability of the erythrocyte membrane by making it more rigid and increase the adhesiveness of the cell, inducing an abnormal adherence to the vascular endothelium (Cooke et al., 2004). During the intracellular cycle, the parasite exports numerous proteins (up to 200 proteins, according to a proteomic study [van Ooij et al., 2008]), among which 5 were shown to interact specifically with the host cell spectrin network underlying the plasma membrane. These proteins induce changes in the mechanical properties of the RBC, leading to an altered circulation. The membrane cytoskeletal network is composed of tetramers ofa- andb-spectrin bound to actin fila- ments and some other junction proteins, such as protein 4.1R, p55, and glycophorin C. Remodeling of the RBC plasma membrane is initiated upon invasion with the discharge of the parasite apical organelles. The ring-infected erythrocyte surface antigen (RESA) is secreted during the invasion process and inter- acts with repeat 16 ofb-spectrin during the first 24 hr postinva- sion (Pei et al., 2007a). A functional analysis suggested that RESA might play a role in stabilizing the spectrin network of the infected RBC and therefore protect against febrile episodes (Silva et al., 2005). While RESA disappears from the cell late in parasite development, several other proteins, includingP. falci- parumerythrocyte membrane proteins 1 and 3 (PfEMP1 and 3), the mature parasite-infected erythrocyte surface antigen (MESA), and the knob-associated histidine-rich protein (KAHRP), are secreted and exported to the RBC plasma membrane. PfEMP1 is inserted into the erythrocytic membrane and is the major adhe- sion factor exposed at the surface of the erythrocyte (Cooke et al., 2004). Its cytoplasmic domain interacts with actin and spectrin as well as with KAHRP, which also associates with repeat 4 of a-spectrin. RBCs infected with parasites deficient for KAHRP exhibit less adhesion under flow conditions (Crabb et al., 1997).
MESA binds to the host cytoskeletal protein 4.1R in a region usually implicated in the formation of a ternary complex with p55 and glycophorin C. MESA is able to compete with p55 and therefore could modulate the interactions within the ternary complex and destabilize the RBC membrane cytoskeleton (Wal- ler et al., 2003). WhenP. falciparumwas cultivated in RBCs lack- ing 4.1R, MESA accumulated in the host cell cytoplasm and the parasites died. Even if the function of MESA remains unclear, its interaction with protein 4.1R appears to be essential for para- site survival (Magowan et al., 1995). PfEMP3 interacts with actin anda-spectrin at a site close to the junction between spectrin, actin, and protein 4.1R. A peptide corresponding to the interact- ing part of PfEMP3 destabilizes the junction and therefore the membrane (Pei et al., 2007b). Thus, PfEMP3 may weaken the host plasma membrane and facilitate the release of merozoites into the bloodstream (Pei et al., 2007b).
Contribution of Host and Parasite Cytoskeleton in Parasite Spreading
Dissemination by Exit from the Host Cells
Egress is an important phase of the life cycle of intracellular path- ogens. Once space has been filled and host cell nutrients
consumed, the newly formed daughter cells are programmed to egress and invade neighboring cells to ensure their survival, leading to the propagation of the infection (Figure 2E). Parasite exit requires lysis of the PVM and of the host cell plasma membrane. Several studies inT. gondiihave highlighted that, similar to invasion, egress occurs very quickly and relies on calcium signaling and gliding motility. It is possible to induce parasite egress with the calcium ionophore A23187 and to block it with CytD at a concentration known to inhibit gliding motility. In T. gondii, ionophore-induced egress provokes a discharge of microneme content and conoid extension and stimulates para- site motility (Black et al., 2000). A drop in the host cytoplasmic potassium concentration was shown to be responsible for the rise in parasite intracellular calcium concentration via activation of parasite phospholipase C (Moudy et al., 2001). The mecha- nisms by which T. gondii detects a drop in host intracellular potassium concentration and activates its phospholipase C are still unknown. The factor causing host plasma membrane perme- abilization is also not known, while permeabilization of the PVM has been shown to involve a pore-forming protein named TgPLP1. This perforin-like protein is secreted from the micro- nemes in a calcium-dependent manner. Deletion of TgPLP1 resulted in an egress defect characterized by entrapment of the PV in the host cells even after treatment with calcium ionophore and despite activation of parasite motility (Kafsack et al., 2009).
The expression of DsRed as a soluble marker in the PV unambig- uously established that TgPLP1 is essential for the permeabiliza- tion of the PVM, a process necessary for egress (Kafsack et al., 2009). This study reinforces the concept that egress is an active process of the parasite; however, egress could also occur without active motility of the parasite. Rupture of the host cell membrane would presumably be a consequence of mechanical forces applied on the host cell membrane as the volume of the PV increases upon parasite division (Lavine and Arrizabalaga, 2008). Nevertheless, as shown by the egress defect of the TgPLP1 knockout strain, this internal tension applied on the host cell membrane is not usually sufficient to release the para- sites but may be responsible for the residual egress observed for this mutant (Kafsack et al., 2009). One more player in parasite egress has recently been uncovered. The host cell calpain prote- ases appear to be hijacked byToxoplasmaandPlasmodiumfor efficient egress and could facilitate parasite exit by cleaving host cell cytoskeletal elements, given that calpains are involved in remodeling of the cytoskeleton and plasma membrane in mammalian cells (Chandramohanadas et al., 2009).
Dissemination by Induced Migration of the Host Cell Recent studies illustrate the vast potential ofT. gondiito hijack the migratory properties of immune DCs in order to promote rapid parasite dissemination in the host (Courret et al., 2006;
Lambert et al., 2006, 2009). Indeed,Toxoplasmawas shown to enter into DC11b and DC11c cells, where it mainly remains at the cell periphery without dividing. More interestingly,T. gondii is able to exploit the specific migratory mechanism used by these DCs to cross the blood-brain barrier (Courret et al., 2006). Such a strategy of using the DC as a shuttle enhances the ability of the parasite to migrate, since an inoculation ofToxo- plasma-infected DC leads to an earlier brain infection and increased lethality in mice when compared to an inoculation per- formed with free parasites (Lambert et al., 2006). The molecular
mechanism underpinning the subversion of the host cytoskel- eton and affecting host cell migration behavior remains to be determined.
Spreading Infection without Exiting from the Host Cell In contrast to most apicomplexans, entry into bovine lympho- cytes byTheileriasporozoites does not rely on the parasite acto- myosin motor or the formation of a MJ. The sporozoites can enter host cells in any orientation using a zippering mechanism with the host cell membrane. Nevertheless, this passive mode of infection requires an intact host actin cytoskeleton to maintain the integrity of the surface membrane, even if not to actively promote parasite internalization (Shaw, 2003). The parasite is fully internalized within 3 min at 37C, a time comparable with phagocytosis. Once inside, the membrane surrounding the para- site dissolves concomitantly with the discharge of secretory organelles. The parasite develops freely in the cytoplasm, inter- acting with the components of the host cell as illustrated by the array of host microtubules that are associated with the diffuse material secreted at the surface of the parasite (Shaw, 2003).
This association with host cell microtubules remains stable during the intracellular life of the parasite and facilitates the movement of the parasite to the perinuclear region of the host cell. Upon differentiation into a multinucleated schizont stage, the parasite hijacks the host nuclear factor kappaB (NF-kB) pathway, dramatically changing the fate of the host cell. This hijacking leads to protection against apoptosis and causes uncontrolled cell proliferation, ensuring a rapid clonal expansion of the parasitized cells (Dobbelaere and Ku¨enzi, 2004). Pictures from electron and confocal microscopy reveal that parasite multiplication is superbly synchronized with host cell division, implicating a tight association of the schizont with the host cell mitotic apparatus to ensure an equal partitioning of the schizont into host daughter cells (Figure 2D). In line with this prediction, theT. annulatasecreted protein TaSE has recently been identi- fied and detected in infected cells in association with the para- site, as well as in the host cell cytoplasm, where it colocalizes witha-tubulin (Schneider et al., 2007). Moreover, during mitosis and cytokinesis of the host cell, TaSE location is dynamic and colocalizes with components of the mitotic spindle, suggesting that this protein could be involved in the distribution of the schizont into the daughter cells (Schneider et al., 2007). In addi- tion, a GPI-anchoredTheileriaprotein (gp34) may be involved in the synchronization of parasite and host cell division via an inter- action with host Plk1.
Theileriaconstitutes a striking example of a pathogen capable of hijacking host signaling pathways to reprogram cell division and exploit the mitotic spindle to ensure its expansion. This strategy avoids direct exposure of the parasite to the host immune system and reduces the effort dedicated to host cell entry.
Conclusion and Perspectives
Viruses and bacteria gain access to their intracellular niche by inducing host-mediated phagocytosis, leading to their internali- zation (Bhavsar et al., 2007; Radtke et al., 2006). In contrast, eukaryotic parasites that are significantly larger in size would only be taken up by professional phagocytes such as macro- phages or DCs. To avoid such a limited range of host cell types, the apicomplexans have evolved a unique active process of host
cell penetration that gives them access to virtually all cell types.
Analogous to the numerous effector proteins injected by bacte- rial secretion systems, these parasites also dramatically remodel and hijack host cell processes by discharging parasite molecules from their specialized secretory organelles, rhoptries and dense granules. The intricate contributions of host and parasite cyto- skeletons in parasite invasion, intracellular replication, and dissemination have been elegantly and abundantly studied in the last few years. Despite the inherent complexities and similar- ities between the cytoskeleton and their accessory proteins of these eukaryotic pathogens and their hosts, a large body of infor- mation has been gathered. It is evident that the host-parasite interplay relies extensively on the manipulation of host cellular processes by the parasite, and in this respect, future discoveries are guaranteed to be equally exciting.
ACKNOWLEDGMENTS
The authors wish to thank all the colleagues who contributed to the work described in this review, and to apologize to the many scientists whose interesting studies could not be cited because of space constraints. The authors are grateful to Dr. Isabelle Tardieux for her critical reading of the manu- script and constructive comments and Louise Kemp, Nikolas Friedrich, and Dr. Thierry Soldati for their valuable suggestions. K.F. is supported by the Swiss National Foundation (DS 3100A0-116722), and D.S. is a HHMI Interna- tional Scholar.
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